Figure Progesterone Docked in the Active Site of P450 2C5. Two views of the position and orientation of progesterone are shown together with elements of secondary structure (cyan) and amino acid side chains that form the substrate binding cavity. The atom types are colored with the carbon atoms of the side chains colored gray and progesterone shown in yellow. The C21 carbon of the docked progesterone is sufficiently close to the heme iron for hydroxylation to occur. (A) The view is looking across SRS-5, which includes L358, L359 and L363, toward SRS-4 in helix I(H10), where D290, A294, and T298 reside. Three other SRS regions are depicted: SRS-1, the B–C loop containing L102, A113, and F114; SRS-2, helix F(H7) containing V205 and L208; and SRS-6, beta2 containing F473 and V474. SRS-3 located on helix G(H8) is not in close proximity to the docked progesterone. In the structure of P450 2C5, the contact residue Val205 in SRS-2 helix F(H7) corresponds to a residue that was shown to determine the difference in substrate specificity between P450s 2A4 and 2A5, which catalyze coumarin and steroid hydroxylations, respectively (Lindberg and Negishi 1989). Mutations of the corresponding residue in P450 2B enzymes alter the regioselectivity of steroid hydroxylations (Luo et al. 1994).
Leu102,Ala113,Phe114,Val205,Leu208,Asp290,Ala294,Thr298,Leu358,Leu359,Leu363,Phe473,Val474 off

      The membrane protein was modified for crystallization by replacement of the hydrophobic N-terminal transmembrane domain with a short hydrophilic sequence before residue 28. The structure of the native sequence is complete from residue 28 to the beginning of a C-terminal histidine tag used for purification. CYP2C8 is one of the principal hepatic drug-metabolizing enzymes that oxidizes therapeutic drugs such as taxol and cerivastatin and endobiotics such as retinoic acid and arachidonic acid. Consistent with the relatively large size of its preferred substrates, the active site volume is twice that observed for the structure of CYP2C5. The extended active site cavity is bounded by the β1 sheet and helix F′ that have not previously been implicated in substrate recognition by mammalian P450s.
     Xenobiotic metabolizing cytochrome P450 monooxygenases provide crucial protection from the harmful effects of exposure to a wide variety of chemicals, including environmental toxins and therapeutic drugs. In general, these microsomal enzymes determine the bioavailability of hydrophobic compounds by controlling the rate of conversion to more soluble, inactive products that are readily excreted. Different P450s can show overlapping substrate specificities, and individual enzymes can interact with numerous structurally diverse substrates. This broad catalytic activity usually serves a positive defensive role; however, in some cases, it can lead to adverse drug-drug interactions.
      P450s often possess enzyme-specific catalytic repertoires and can display exquisite catalytic selectivity for regio- and stereospecific reactions. This is particularly evident for the mammalian family 2C P450s, which exhibit extensive, independent evolution and functional divergence in mammals, leading to multiple enzymes in each species while retaining a high degree of amino acid identity (>70%). Thus, structural comparisons within this highly related but functionally diverse P450 subfamily are likely to be particularly revealing of mechanisms leading to the catalytic diversity of P450 enzymes.
1PQ2Marz,2c8 19-490 AA Cys435 off strands thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys435 in 2cC8. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).

49-62,79-88,90-95,100-108,116-131,140-158,166-184,191-209,212-217,221-225,226-253,
262-273,283-299,299-316,316-331,338-344,345-360,390-396,408-413,437-456 helixes
H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,     ,        F   F'  , G' ,   G,   H ,      I ,  I' , J ,    K ,                          L  
64-69,72-77,386-389,368-369 beta sheet 1-4
B1,B2,B3,B4 4(B4)
      The active site of P450 2C8 is much larger than that of 2C5. The active site volume of CYP2C8dH is estimated to be 1438 ų, whereas the active site volume of CYP2C5/3LVdH (PDB ID code 1N6B) is estimated to be 645 ų.
Active site reveals 23 additional residue positions that potentially contact substrates (boldface).
Conserved residues that reside in the active sites of both enzymes are underlined.
Ile50,Phe54;Phe69;Asn72,Ile74;Arg97,Gly98,Asn99;Ser100,Ile102,Ser103,Ile106,Thr107;Ile113,Ser114;Arg200,Phe201,Asn202,Asn204,Phe205,Leu208,Asn209;Ile213,Gln214,Asn217,Asn218;Val233,Asn236,Val237,Thr240,Arg241;Gly289,Ala292,Asp293,Val296,Ala297,Thr301;Leu361,Val362,Thr364,Gly365,Val366,Pro367;Thr386,Met388;Gly475,Ile476,Val477; In contact with substrate 23 amino acids:
Ile50,Phe54,Phe69,Asn72,Ile74,Arg97,Gly98,Asn99,Ser100,Arg200,Asn202,Asn209,Gln214,Asn217,Asn218,Asn236,Leu361,Thr364,Gly365,Pro367,Thr386,Met388,Gly475;
off and for all 30 contacts 23 and conserved 7 amino acids:
Ile50,Phe54,Phe69,Asn72,Ile74,Arg97,Gly98,Asn99,Ser100,Ile102,Arg200,Asn202,Asn204,Leu208,Asn209,Gln214,Asn217,Asn218,Asn236,Asp293,Ala297,Thr301,Leu361,Thr364,Gly365,Pro367,Thr386,Met388,Gly475,Val477;
off and for all 48 amino acid residues in active site:
Helix AH1,beta1=1,beta12,B=B'loop,Helix B'H4,B'=C loop,Helix FH8,Helix F'H9,Helix GH11,Helix IH13,K=β1=3 loop,beta1=4,Sheet beta4
Helix A(H1),   beta1=1,  beta12,             B=B'loop,      Helix B'(H4),                                                
Ile50,PheF54;Phe69;Asn72,Ile74;Arg97,Gly98,Asn99;Ser100,Ile102,Ser103,Ile106,Thr107;
B'=C loop,       Helix F(H8),                                                                             
Ile113,Ser114;Arg200,Phe201,Asn202,Asn204,Phe205,Leu208,Asn209;
Helix F'(H9),                                     Helix G(H11),
Ile213,Gln214,Asn217,Asn218;Val233,Asn236,Val237,Thr240,Arg241;
Helix I(H13),                                                                K=beta1=3 loop,
Gly289,Ala292,Asp293,Val296,Ala297,Thr301;Leu361,Val362,Thr364,Gly365,Val366,Pro367;
beta1=4,                 Sheet beta4
Thr386,Met388;Gly475,Ile476,Val477;
Ile50,Phe54,Phe69,Asn72,Ile74,Arg97,Gly98,Asn99,Ser100,Ile102,Ser103,Ile106,Thr107,Ile113,Ser114,Arg200,Phe201,Asn202,Asn204,Phe205,Leu208,Asn209,Ile213,Gln214,Asn217,Asn218,Val233,Asn236,Val237,Thr240,Arg241,Gly289,Ala292,Asp293,Val296,Ala297,Thr301,Leu361,Val362,Thr364,Gly365,Val366,Pro367,Thr386,Met388,Gly475,Ile476,Val477 off 1-2 off 3 off 4 off 5 off ;

Structure of cytochrome P450 4B1 (CYP4B1) complexed with octane:
An n-Alkane and fatty acid omega-hydroxylase with a covalently bound heme to Glu310

      P450 family 4 fatty acid omega-hydroxylases preferentially oxygenate primary C–H bonds over adjacent, energetically favored secondary C–H bonds. Substrate octane to define features of the active site thatcontribute to a preference for omega-hydroxylation. Octane is bound in a narrow active-site cavity that limits access of the secondary C–H bond to the reactive intermediate.
P450 4B1 exhibits structural adaptations for omega-hydroxylation that include changes in the conformation of the heme and changes in a highly conserved helix I H18 motif that is associated with selective oxygenation of unactivated primary C–H bonds.
      Potentially toxic non-esterified fatty acids resulting omega-alcohols are generally oxidized further to produce dicarboxylic acids. Urinary dicarboxylic fatty acids are elevated in humans by fasting or in uncontrolled diabetes, which are conditions where adipocyte lipolysis increases the availability of non-esterified fatty acids to fuel metabolism. It is estimated that roughly 15% of fatty acids undergo omega-hydroxylation during peak periods of fatty acid catabolism of non-esterified fatty acids at high concentrations. Collectively, these enzymes provide pathways for the metabolic clearance of branched chain fatty acids, eicosanoids, and xenobiotic substrates such as dietary phytanic acid, drugs, toxins, and vitamins E and K.
      A highly conserved sequence motif on helix I H18 contributes to positioning the terminal carbon of octane for omega-hydroxylation. Glu310 of this motif auto-catalytically forms an ester bond with the heme 5-methyl, and the immobilized Glu310 contributes to substrate positioning.
5T6QM1171Marz 4b1 30-510 Glu321,Cys457 off +9 AA thin off
29-48,60-66,69-81,100-109,115-120,121-123,135-146,150-155,156-177,184-201,
211-232,233-236,237-243,244-272,275-280,288-295,296-299,305-338,338-354,360-365,
367-381,413-417,430-435,452-456,459-477             jbc.M117.775494-2(30-510) helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23,H24,H25
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
75-79,85-89,399-403,377-381
84-88,94-98,408-412,386-390     beta sheet 1-4
B1,B2,B3,B4 4(B4)
5T6Q4b1Marz,4b1 6-20-501-506 AA HEM FeIII+ OCT Glu310,Cys448 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys448 in 4b1. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
20-39,52-57,60-72,91-100,106-111,112-114,126-137,141-146,147-168,175-192,
202-223,224-227,228-233,235-263,266-271,279-286,287-290,296-329,329-345,351-356,
358-372,404-408,421-426,443-447,450-468 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23,H24,H25
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
75-79,85-89,399-403,377-381 beta sheet 1-4
B1,B2,B3,B4 4(B4)
This reaction is generally considered to be the most difficult reaction catalyzed by cytochrome P450 monooxygenases because the enzyme needs to exclude access of neighboring and more reactive secondary C-H bonds to the iron-bound singlet oxygen
O-O reactive specy. The reactive intermediate is formed by the reduction of oxygen covalent three bounds of the triplet covalent bonding dioxygen
OO to the heme iron followed by protonation and heterolytic scission atom of the dioxygen
-O-O-Fe³⁺-S with elimination of the water molecule and formation of a highly electrophilic singlet oxygen atoms bound to the hypervalent (VI=coordination number) heme iron is transition state complex which promote production of C-O-H and releasing the water H-O-H:

              singlet oxygen           water disociated                      water molecule
2C-H + -O-O-Fe³⁺-S^-Cys +2(H⁺ +   O-H^- )=> 2C-O-H     + 2H-O-H + Fe³⁺-S^-Cys
omega              iron            proton   hydroxyl     omega hydroxyl  water      iron cysteine

triplet oxygen                          singlet oxygen adsorbed on heme iron(III)
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
C-H + -O-O-Fe³⁺-S^-Cys =>C⁺ + H-O-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- )=> C-O-H     + H⁺
H⁺ + H-O-O-Fe²⁺-S^-Cys => H-O-H + -O-Fe³⁺-S^-Cys
C-H + -O-Fe³⁺-S^-Cys =>C⁺    + H-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- ) => C-O-H     + H⁺
H⁺ + H-O-Fe²⁺-S^-Cys => H-O-H + Fe³⁺-S^-Cys
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
triplet                                        singlet oxygen adsorbed on heme iron(III)
Oxygenation of aliphatic C-H bonds is thought to proceed by abstraction of a hydrogen hydride H^- by the iron(III) singlet oxygene or mono oxigen intermediate formed iron(II) transiently iron-bound hydroxyl radical H-O-O-Fe²⁺-S^-Cys448 or H-O-Fe²⁺-S^-Cys448. Followed by recombination of the disociated water positive proton with the transiently iron-bound hydroxyl radical in new water molekule recombinant formation H-O-H.
     As the strength of primary C-H bonds is greater than that of secondary C-H bonds, oxygen addition at the omega-1 or omega-2 positions of fatty acids is typically seen for less specialized enzymes. These considerations suggest that the omega-hydroxylases have structural features that favor the approach to the reactive iron oxo species of the omega-carbon relative to the omega-1 carbon.
          Absorption band of CYP 4b1 red 422 nm

              Wavelength , nm; Octane shifts maximum to left from maximum of red 422 nm.
HEM FeIII+ OCT Glu310,Cys448 off thin off HOH703,HOH704,HOH705,HOH708 off
     off Lys105,Tyr110,Arg380,Ser442,Arg446 In contrast, the heme 7-propionate in structures of most mammalian microsomal P450s is oriented on the other side of the heme plane, where it interacts with basic residues corresponding to Arg-380 that resides on the strand of beta-sheet 1 nearest to the heme. In the structure, there is a bridging water molecule between the 7-propionate and Arg380. The water molecule exhibits additional hydrogen bonding interactions with the side-chain hydroxyl group of Ser442 and the carbonyl oxygen of Arg446. This arginine also exhibits hydrogen bond stabilized ionic interactions with the heme 6-propionate. The heme 7-propionate exhibits additional H-bonds with Lys105,Tyr110. Tyr110 together with the heme 7-propionate form part of the surface of the active-site cavity that contributes to positioning of the substrate relative to the heme iron (Figs. 3 and 4).
off Glu310,His312,Met308,Thr314,Gly311 Two of these residues compensate for hydrogen bonds that are lost due to extension of the turn in helix I adjacent to the Glu310. Conserved His312 donates an H-bond to the carbonyl of Met308. Similarly, the side-chain hydroxyl group of conserved Thr314 forms an H-bond with the Glu310 carbonyl oxygen in the peptide bond with Gly311.
off Phe309,Leu485 A third conserved residue, Phe309, is oriented to cap the height of the cavity above the heme iron and the terminal carbon of octane. The cap over the substrate-binding cavity is propagated by the close packing of the Leu485 on the turn in beta-sheet 4 of the C-terminal loop.
off Val375,Leu122 Another conserved residue in the human ω-hydroxylases, Val375, on the connector between helix K and beta-sheet 1-3 fills the space under the C-terminal loop and contacts Thr314 to form the side of the cavity opposite Glu310. An additional residue conserved in the human omega-hydroxylases, Leu122 on the beta-turn between helices B' and C, fills the space between Glu310 and Tyr110. Together, these highly conserved residues form the narrow cavity for the approach of octane to the heme iron .
off His88,Gln377,Tyr379,Arg96 The residues that form the distal portion of the substrate-binding cavity contacts both Phe309,Leu485 to cap the distal active site. The distal end of the cavity is delineated by Gln377,Tyr379. Accommodates longer fatty acids with the side chain of Arg96 positioned on the second strand of the beta-sheet 1 to facilitate the binding of lauric acid and longer fatty acids, such as palmitic and arachidonic acid, which are substrates for human CYP4A11. Arg96 is conserved at this alignment position in rabbit. Arginine that corresponds to His88.
      Twelve 12 of the 57 cytochrome P450 genes in the human genome encode family 4 P450s, which are assigned to six subfamilies in mammals designated by a letter 4B1,4V2,4X1,4A,4F.

        P450 46a1, the principal cholesterol hydroxylase in the brain.

CSO '. CSO

C3S CSO CHOLEST-5-EN-3-YL HYDROGEN SULFATE CHOLESTEROL-SULFATE
     By converting cholesterol to 24S-hydroxycholesterol, cytochrome P450 46A1 (CYP46A1) initiates the pathway step for removal from the brain at cholesterol homeostasis metabolism. CH-3S is bound in the productive orientation and occupies the entire length of the banana-shaped hydrophobic active-site cavity. A helix B'–C loop insertion (residues 116–120) contributes to positioning cholesterol for oxygenation catalyzed by CYP46A1. Substrate-free structure reveals substantial substrate-induced conformational changes in CYP46A1 and suggests that structurally distinct compounds could bind in the enzyme active site. The conversion of cholesterol to 24S-hydroxycholesterol (24 H-O-C ) is an important mechanism step that controls cholesterol turnover in the central nervous system binding on the HEME to iron(III) moiety active site:
              singlet oxygen           water disociated                      water molecule
2C-H + -O-O-Fe³⁺-S^-Cys +2(H⁺ +   O-H^- )=> 2C-O-H     + 2H-O-H + Fe³⁺-S^-Cys
omega              iron            proton   hydroxyl     omega hydroxyl  water      iron cysteine

triplet oxygen                          singlet oxygen adsorbed on heme iron(III)
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
C-H + -O-O-Fe³⁺-S^-Cys =>C⁺ + H-O-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- )=> C-O-H     + H⁺
H⁺ + H-O-O-Fe²⁺-S^-Cys => H-O-H + -O-Fe³⁺-S^-Cys
C-H + -O-Fe³⁺-S^-Cys =>C⁺    + H-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- ) => C-O-H     + H⁺
H⁺ + H-O-Fe²⁺-S^-Cys => H-O-H + Fe³⁺-S^-Cys
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
triplet                                        singlet oxygen adsorbed on heme iron(III)
Oxygenation of aliphatic 24C-H bond is thought to proceed by abstraction of a hydrogen hydride H^- by the iron(III) singlet oxygene or mono oxigen intermediate formed iron(II) transiently iron-bound hydroxyl radical H-O-O-Fe²⁺-S^-Cys437 or H-O-Fe²⁺-S^-Cys437. Followed by recombination of the disociated water positive proton with the transiently iron-bound hydroxyl radical in new water molekule recombinant formation H-O-H.
Cholesterol hydroxilated product at carbon 24 H-O-C can cross the membranes and is by lipokalin proteins delivered to the liver for further turn over to bile acids. Studies indicate the turnover of cerebral cholesterol via 24-hydroxylation are necessary for memory and learning.
Accumulating evidence indicates Alzheimer's disease (AD) is associated with disturbances in cholesterol homeostasis in the brain. For side-chain oxysterols, including 24 H-O-C, as endogenous ligands for liver X receptors, that regulate the expression of genes involved in fatty acid and cholesterol metabolism.
2Q9F46a1Marz(51-500 AA) cholesterol3sulfate, HEM FeIII+ CSO Cys437 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys437 in 46a1. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
59-72,89-98,105-113,130-142,143-145,146-153,153-170,180-198,202-206,208-225,
235-267,274-283,289-303,304-319,321-337,343-350,350-364,394-400,412-417,432-436,
439-457 2Q9F46a1(51-500)cholesterol3sulfate helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
74-77,84-87,389-393,370-374 beta sheet 1-4
B1,B2,B3,B4 4(B4)
      The sulfate group forms four hydrogen bonds, with His81 (beta1-1-beta1-2 loop),Arg110 (B' helix),Asn227 (F-G loop) of the enzyme (Fig. 1b).
off
The steroid nucleus interacts with Phe80 (beta1-1-beta1-2 loop) ,Met108,Tyr109,Ala111,Leu112 (B' helix),Ile222 (F helix),Trp368,Phe371 (beta1-4 strand),Ala474 (beta4 loop). Three of these residues, Ala111,Leu112,Ile222, contact the flat surface of the steroid nucleus; and three, Phe80,Trp368,Phe371 are on the opposite side, contacting the steroid axial methyl groups.
off Phe80,Met108,Tyr109,Ala111,Leu112,Ile222,Trp368,
Phe371,Ala474,Ala111,Leu112,Ile222,Phe80,Trp368,Phe371
Met108,Tyr109 restrain the steroid nucleus along one edge as does Ala474 at the edge of the C ring. A hydrogen bond between Trp368,Ala474.
    A network of hydrogen bonds involving Tyr109,Thr370,Phe371,Arg372, and a heme propionate position these active-site residues.
off
The aliphatic tail of CH-3S is surrounded by Phe121,Val126 (in a B'-C loop insertion, unique to CYP46A1),Ile301,Ala302,Thr306 (I H14 helix),Ala367 (beta4-1 strand),Thr475 (beta4 loop), which are located at a 3.7- to 4-Å distance and likely to limit its motion. The C24 and C25 atoms of CH-3S, the primary and secondary sites of hydroxylation by CYP46A1, respectively, are positioned at a 5.7 ± 0.05-Å distance from the heme iron (Fig. 1c), which is 1-1.5 Å greater than expected for hydroxylation by the oxyferryl intermediate during turnover (24). It is likely that CH-3S moves closer to the heme iron during subsequent steps in the catalytic cycle after reduction of the heme iron. The orientation and position of CH-3S suggest that cholesterol will have a similar overall mode of binding. A difference could be in contacts of the cholesterol 3beta hydroxyl with CYP46A1, and if so, in the depth of insertion in the active site. Residues that may be involved in recognition of the cholesterol 3beta hydroxyl are His81,Asn227.
off The volume from 309 ų in the ligand-free structure to 320 ų in the CH-3S-bound structure. Phe121,Val126,Ile301,Ala302,Thr306,Ala367,Thr475
     Water plays an essential role in P450 catalysis by providing disociation of hydroxyl negative ion and positive proton. In the ligand-free structure, the heme-coordinating water, W718 2Q9G.pdb, is located 2.35 Å from the Fe (Fig. S1). Binding of CH-3S displaces this water molecule 0.8 Å parallel to the heme plane to the position of W732. The distance to Fe increases to 2.87 Å, and W732 becomes hydrogen-bonded to Thr306 (2.9 Å),Ala302 (2.9 Å). The proximity of W732 to the heme iron could explain the partial low-to-high spin conversion in delta(2-50)CYP46A1dH when CH-3S binds. Thr306,Ala302,HOH732,HOH711,HOH726
off thin off HEM FeIII+ CSO Cys437
off Thr306,Ala302

        P450 11a1 cholesterol side chain cleaving enzyme for pregnenolone.

the principal enzyme cleaving the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and
20alpha,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the “soft” recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1–22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s. HCCCSO

HCC (3ALPHA,8ALPHA,22R)-CHOLEST-5-ENE-3,22-DIOL
     Steroid hormones are essential for life. In vertebrates, they are all synthesized from pregnenolone, which is in turn formed from cholesterol via a three-step process catalyzed by cytochrome P450 11A1 CYP11A1. During the first step, cholesterol is converted to 22R-hydroxycholesterol (22HC),3 the second step produces 20alpha,22R-dihydroxycholesterol (20,22DHC), and the third step involves the cleavage of the C20-C22 bond in 20,22DHC to yield pregnenolone. Sterol intermediates do not accumulate during the conversion of cholesterol to pregnenolone and bind much more tightly to CYP11A1 than cholesterol, suggesting that they remain in the active site binding on the HEMEHCC to iron(III) moiety active site until all three oxidative steps are completed (1, 3, 4). All enzymatic steps take place in the inner mitochondrial membrane of steroidogenic tissues and require a total of 3 mol of O₂, 3 mol of NADP⁺, and 3 mol of hydrogen hydride H^- with six electrons transferred separately to form reduced state intermediate 3 mol NADPH to adrenodoxin reductase, adrenodoxin (Adx), and CYP11A1. CYP11A1 is an important enzyme whose deficiency leads to lipoid congenital adrenal hyperplasia, a lethal disease if untreated. (22 H-O-C).
Oxygen molecule mono oxygenase CYT P450mechanism:
              singlet oxygen           water disociated                      water molecule
2C-H + -O-O-Fe³⁺-S^-Cys +2(H⁺ +   O-H^- )=> 2C-O-H     + 2H-O-H + Fe³⁺-S^-Cys
omega              iron            proton   hydroxyl     omega hydroxyl  water      iron cysteine

triplet oxygen                          singlet oxygen adsorbed on heme iron(III)
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
C-H + -O-O-Fe³⁺-S^-Cys =>C⁺ + H-O-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- )=> C-O-H     + H⁺
H⁺ + H-O-O-Fe²⁺-S^-Cys => H-O-H + -O-Fe³⁺-S^-Cys
C-H + -O-Fe³⁺-S^-Cys =>C⁺    + H-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- ) => C-O-H     + H⁺
H⁺ + H-O-Fe²⁺-S^-Cys => H-O-H + Fe³⁺-S^-Cys
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
triplet                                        singlet oxygen adsorbed on heme iron(III)
Oxygenation of aliphatic 22C-H bond is thought to proceed by abstraction of a hydrogen hydride H^- by the iron(III) singlet oxygene or mono oxigen intermediate formed iron(II) transiently iron-bound hydroxyl radical H-O-O-Fe²⁺-S^-Cys423 or H-O-Fe²⁺-S^-Cys423. Followed by recombination of the disociated water positive proton with the transiently iron-bound hydroxyl radical in new water molekule recombinant formation H-O-H. Cholesterol hydroxilated product at carbon 22 H-O-C.
3MZSMarz((6-41-475-520) 11a1 cholesterol 22OH, HEM FeIII+ HCC HOH518 off Cys423 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys423 in 46a1. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
9-13,19-31,32-34,35-48,65-75,85-96,106-121,129-152,160-178,190-205,
206-210,213-215,216-221,221-252,261-269,273-306,306-318,327-332,335-349,379-386,
397-403,418-422,425-442 3MZS(6-41-475-520) helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
50-52,61-63,376-378,355-357 3MZS(6-41-475-520) beta sheet 1-4
B1,B2,B3,B4 4(B4)
      Ile351 (beta1–4 strand region), Thr291 (I helix), Phe203 (F helix), and Met202 (F helix). The sterol alpha-face is surrounded by the side chains of Ile85 (B-B' loop), Leu460 (beta4-1/4-2 loop), Trp88 (B' helix), and the beta-face is defined by Gln356,Thr354, Val-353, and Ser-352 (all residues are from the β1–4 strand region). In addition, Phe-458 (β4–1 strand) restrains the edge of sterol ring A.
      Like the 3beta-hydroxyl in 22HC, the 3beta-hydroxyl in 20,22DHC has no direct contacts with the protein and is hydrogen-bonded to two H2O molecules, HOH42 and HOH599, which are water-bridged to the side chains of
His78,Tyr100,Gln416 as well Glu91 carbonyl, analogs of His40,Tyr62,Gln377,Glu53, which participate in the interactions with the 3beta-hydroxyl in 22HC. (The two CYP11A1 structures have different numbering of amino acid residues; numbering in 3NA0 is based on the sequence of the precursor protein, whereas that in 3MZS reflects the sequence of the mature protein.)
The Val96 carbonyl also participates in the interactions with the water array in 3NA0.
      Its analog in 3MZS does not have water contacts; instead, the Leu210,Asn211 carbonyls are involved. The 20alpha- and 22R-hydroxyls in 20,22DHC are at 3.3 and 3.6 Å, respectively, from the iron and, similar to 22R-hydroxyl in 22HC, are not at a hydrogen bond distance (2.8–3.2 Å) to any of the amino acid residues or waters in the active site 23 AA.
His40,Glu53,Tyr62,Arg82,Tyr83,Ile85,Trp88,Ala168,Met202,Phe203,
Leu210,Asn211,Gly288,Thr291,Ile351,Ser352,Val353,Thr354,Gln356,Gln377,
Val378,Glu432,Leu460
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516,518,521,522,523,525,526,532,549,599
40,53,62,82,83,85,88,168,202,203,210,211,288,291,351,352,353,354,356,377,378,432,460
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516,518,521,522,523,525,526,532,549,599
HOH42,HOH516,HOH518,HOH521,HOH522,HOH523,HOH525,HOH526,HOH532,HOH549,HOH59
A remarkable feature of the CYP11A1 co-complex is the nature of the interactions with the sterol 3beta-hydroxyl. Although this group has no direct contacts with the protein, it forms two hydrogen bonds with crystallographically observed waters HOH523 and HOH526, which are hydrogen-bonded to the side chain of Gln377 and HOH525, respectively.Gln-377 participates in an extensive hydrogen bond network with the Tyr62 hydroxyl, HOH549,HOH521,HOH522, Glu53, and the His40 imidazole, whereas Wat-525 interacts with the Leu-210 carbonyl and Tyr83 hydroxyl (waters are found in similar positions in all four monomers)
off HOH516,HOH518,HOH521,HOH522,HOH523,HOH525,HOH526,HOH532,HOH549,HOH599
     Consortium (University of Toronto). The structure of CYP11A1 bound to 22HC (code 3MZS) shows a root mean square deviation of 0.95 Å for all atoms of 462 aligned residues (3872 atoms of 3NA0 and 3904 atoms of 3MZS). The Cα atom traces of the two structures are very similar, as are the positions of the substrates and substrate contact residues. Similarities also extend to the shape and volume (624 and 625 ų in 3NA0 and 3MZS, respectively) of the active site and the presence and position of ordered water molecules at the entrance to the active site, six waters in 3NA0 and seven waters in 3MZS.
      Overall, there is excellent correlation between the membrane-interacting residues in CYP11A1 identified by Asn19,Trp21,Leu22,Tyr25,Leu216,Leu219,Phe220 and results of site-directed mutagenesis and mass spectrometry studies (33,-,35).
     One such interaction involves the invariant glutamic acid Glu383 in the K helix, which hydrogen bonds with the invariant tryptophan Trp440 in the K' helix in CYP24A1; in CYP11A1, this interaction exists as the Glu344,Trp401 hydrogen bond. In addition, Glu432 in CYP11A1 and its counterpart Gln471 in CYP24A1, at the N terminus of the L helix, are hydrogen-bonded to the spatially overlaid structural HOH532 and HOH516, respectively, which in turn interact with residues in the I Gly288, E Ala168 helices in CYP11A1 and with Ser211 in the E helix in CYP24A1.
     Two residues, Lys339,Lys343, from the K helix motif, have been unambiguously shown to interact with Adx in CYP11A1, and four residues, Lys378,Lys82 from the K helix and Arg465,Arg466 from the L helix, are predicted to bind Adx in CYP24A1. These conserved basic residues and their conserved secondary structural elements are positioned similarly in CYP11A1 and CYP24A1 because of conservation of the tertiary structure interactions. It is thus the conserved tertiary structure interactions that likely underlie the specificity of mitochondrial P450s for their shared redox partner, Adx.
in the active site 40 AA.

        Human P450 11a1 cholesterol side chain cleaving enzyme for pregnenolone.

     The precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C–C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information with high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s.
CLR,                       HCD22,                   HCD20,                       DHC,

CLR, CHOLESTEROL C27 H46 O;
HC9 HCD22 (3ALPHA,8ALPHA,22R)-CHOLEST-5-ENE-3,22-DIOL C27 H46 O2;
HCD20 (3ALPHA,8ALPHA)-CHOLEST-5-ENE-3,20-DIOL C27 H46 O2;
DHC ,2DC DDC (3ALPHA,8ALPHA,22R)-CHOLEST-5-ENE-3,20,22-TRIOL C27 H46 O3;
    Complex of human Adx and CYP11A1-the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 Å away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s. The cytochrome P450 enzymes comprise a superfamily of hemeproteins that participate in an array of metabolic processes. Members of this family are unified by a common fold and yet catalyze diverse reactions. In humans, there are at least 57 P450s that can be divided into classes based on their intracellular localization and requirement for redox partners, which provide electrons for the monooxygenase reaction. One class is localized in the inner mitochondrial membrane and receives electrons from adrenodoxin reductase (AdR), via the [2Fe-2S] ferredoxin, Adx. The molecular mechanism of complex formation and electron transport within this system have remained unclear: AdR, Adx, and P450 have been proposed to form 1:1:1 or 1:2:1 complexes, but Adx has also been suggested to act as a shuttle, sequentially transporting one electron at a time from AdR to P450. Both complex and shuttle models assume that interactions are mainly electrostatic.
     The first step in steroid hormone biosynthesis is the conversion of cholesterol to pregnenolone and isocaproic aldehyde by mitochondrial CYP11A1. The reaction occurs in three steps: two stereospecific hydroxylations, with formation of 22R-hydroxycholesterol (22-HC) and 20R,22R-dihydroxycholesterol (20, 22-DHC) followed by a C-C bond cleavage, which does not mechanistically typify a P450 reaction and so, is less understood. Contrary to drug-metabolizing but similarly to P450s involved in physiological functions, CYP11A1 has narrow substrate specificity-limited to cholesterol, 7-dehydrocholesterol, and vitamin D. Notably, CYP11A1 hydroxylates but does not cleave the side chain of vitamin D3 producing biologically active
20-hydroxyvitamin D and minor di- and trihydroxylated derivatives. In the absence of structural information, the molecular mechanisms that allow this enzyme to catalyze two different reactions while at the same time maintaining exquisite stereospecificity are unknown.
suggesting that they remain in the active site binding on the HEMECLR to iron(III) moiety active site until all three oxidative steps are completed (1, 3, 4). All enzymatic steps take place in the inner mitochondrial membrane of steroidogenic tissues and require a total of 3 mol of O₂, 3 mol of NADP⁺, and 3 mol of hydrogen hydride H^- with six electrons transferred separately to form reduced state intermediate 3 mol NADPH to adrenodoxin reductase, adrenodoxin (Adx), and CYP11A1. CYP11A1 is an important enzyme whose deficiency leads to lipoid congenital adrenal hyperplasia, a lethal disease if untreated. (22 H-O-C).
Oxygen molecule mono oxygenase CYT P450mechanism:
              singlet oxygen           water disociated                      water molecule
2C-H + -O-O-Fe³⁺-S^-Cys +2(H⁺ +   O-H^- )=> 2C-O-H     + 2H-O-H + Fe³⁺-S^-Cys
omega              iron            proton   hydroxyl     omega hydroxyl  water      iron cysteine

triplet oxygen                          singlet oxygen adsorbed on heme iron(III)
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
C-H + -O-O-Fe³⁺-S^-Cys =>C⁺ + H-O-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- )=> C-O-H     + H⁺
H⁺ + H-O-O-Fe²⁺-S^-Cys => H-O-H + -O-Fe³⁺-S^-Cys
C-H + -O-Fe³⁺-S^-Cys =>C⁺    + H-O-Fe²⁺-S^-Cys
C⁺     + (H⁺ +   O-H^- ) => C-O-H     + H⁺
H⁺ + H-O-Fe²⁺-S^-Cys => H-O-H + Fe³⁺-S^-Cys
OO     + Fe³⁺-S^-Cys => -O-O-Fe³⁺-S^-Cys
triplet                                        singlet oxygen adsorbed on heme iron(III)
Oxygenation of aliphatic 22C-H bond is thought to proceed by abstraction of a hydrogen hydride H^-by the iron(III) singlet oxygene or mono oxigen intermediate forms active iron(II)-bound hydroxyl radical
H-O-O-Fe²⁺-S^-Cys423 or H-O-Fe²⁺-S^-Cys423. Followed by recombination of the disociated water positive proton H⁺ with the transiently iron-bound hydroxyl radical in new water molekule recombinant formation H-O-H. Cholesterol hydroxilated product at carbon 22 H-O-C and similar hydroxilated product at carbon 20 H-O-C.

Third abstraction of a hydrogen hydride H^- from carbon 22 leads to cleavage of 22-20 carbon bond:
-(HO)C(H)-C(OH)-(CH₃)-=>-(HO)C⁺+H^-+ -C(OH)-(CH₃)-
C 22              C 20                     C 22  hydride H⁺+ O=C=(CH₃)-pregnenolone
isohexanal-CHO +H^-+ -O-O-Fe³⁺S^-Cys =>-CHO+ H-O-O-Fe²⁺S^-Cys
Singlet oxygene on iron(III) bound hydride and via C 20 -C(OH) deprotonation release water:
-pregnenolone=(CH₃)C=O+H⁺+H-O-O-Fe²⁺S^-Cys=>H-O-H + -O-Fe²⁺S^-Cys .
3N9YMarz3N9YAC(5-41-474-475-521+39)11A1Adx CholesterolOH,
HEM FeIII FES+ CLR HOH499,502,503,554 off Cys423 off thin off
3N9ZMarz3N9ZAC(5-41-474-475-521+39)11A1Adx CholesterolOH,
HEM FeIII FES+ HCD22 HOH496,499,516,531 off Cys423 off thin off
3NA1Marz3NA1AC(5-41-474-475-521+39)11A1Adx CholesterolOH,
HEM FeIII FES+ HCD20 HOH558,587,728,759 off Cys423 off thin off
3NA0Marz3NA0AC(5-41-474-475-521+39)11A1Adx CholesterolOH,
HEM FeIII FES+ DDC HOH12,24,562,591 off Cys462 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys423 in 46a1. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).

8-12,18-30,31-33,34-47,64-74,84-94,99-103,104-120,121-126,127-151,
159-177,188-206,207-209,212-220,220-253,261-269,273-290,290-306,306-324,327-332,
335-349,380-385,397-402,418-422,425-443 helixes
color chain A,C
H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23,H24,H25
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
28-37,60-65,71-80,92-94      FES  3N9YAC helixes

H1,H2,H3,H4 C
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
50-52,61-63,376-378,355-357 3MZS(6-41-475-520) beta sheet 1-4
B1,B2,B3,B4 4(B4)
     Active site of CYP11A1. (A) View of cholesterol binding within the sterol-binding pocket around the cholesterol 3beta-hydroxyl group. Residues F82, L84, and L460 contact the substrate from the flat side (alpha-face); S352, V353, T354 and Q356 approach the side with two methyl groups (beta-face); R81 and F458 side chains make contacts at its edges. CYP11A1 complexed with: (B) the reaction intermediate 22-HC; (C) substrate 20-HC; (D) reaction intermediate 20,22-DHC. Water molecules are represented as small red spheres; dashed lines indicate hydrogen bonds. Close proximity to the heme iron was suggested for 22-HC earlier based on NMR studies. Crystallographic observation of the hydroxyl-heme iron interaction was made with the hydroxylated product of P450cam.
      Upon 22-HC binding, the conformation of the Thr291 side chain is slightly changed so the interaction between the carbonyl oxygen of Gly287 and the hydroxyl group of Thr291 is loosened, which allows a new water molecule to bind in the I-helix groove. The carbonyl oxygen of Asp290 does not reorient to interact with Met294. A water molecule, which interacts with Thr291 and Gly287, is also hydrogen-bonded to the 22R-OH group. This network may contribute to the retention of the first stable intermediate, 22-HC, which displays very tight binding and remains bound in the active site for the next round of hydroxylation; i.e., until the reduction and following oxygen binding.
Thr291,Gly287,Asp290,Met294
A:CLR:H39,Y61,R81,F82,I84,W87,L101,F202,N210,S352,T354,Q356,Q377,I461,F458,L460
His39,Tyr61,Arg81,Phe82,Ile84,Trp87,Leu101,Phe202,Asn210,Ser352,Thr354,Gln356,Gln377,Phe458,Leu460,Ile461 off right button click on molecule>Select >Disply List> Create Molecular Surface>
B:22HC:H39,Y61,F82,I84,W87,F202,N210,G287,S352,T354,Q356,Q377,I461,F458
His39,Tyr61,Phe82,Ile84,Trp87,Phe202,Asn210,Gly287,Ser352,Thr354,Gln356,Gln377,Ile461,Phe458
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C:20HC:H39,Y61,F82,I84,W87,F202,N210,T291,S352,T354,Q356,Q377,I461,F458
His39,Tyr61,Phe82,Ile84,Trp87,Phe202,Asn210,Thr291,Ser352,Thr354,Gln356,Gln377,Ile461,Phe458
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D:20,22DHC:H39,Y61,F82,I84,W87,F202,N210,S352,T354,Q356,Q377,I461,F458+39
His78,Tyr100,Phe121,Ile123,Trp126,Phe241,Asn249,Ser391,Thr393,Gln395,Gln416,Phe497,Ile500
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       The cholesterol side-chain cleavage reaction requires efficient transfer of electrons from ferredoxin to the heme iron of CYP11A1 twice during each catalytic cycle: to the ferric substrate-bound form and to the ferrous dioxygen-bound form. Each electron-transfer reaction is realized through the formation of a donor-acceptor complex. To obtain the complex of substrate-bound CYP11A1 with Adx, which would normally be transient and hence structurally inaccessible, the fusion proteins comprising
N-terminal Adx and C-terminal CYP11A1 were constructed. By varying the length and composition of the linker we were able to purify and crystallize catalytically competent fusion proteins. Independent of the type of linker between proteins, which is not visible in the crystalline state, the Adx molecule is present in the structure and binds to the same site on the proximal surface of CYP11A1, although the regions distal from the interaction interface are not defined. We analyzed the cholesterol-bound CYP11A1-Adx structure (linker AAKKT), the highest resolution model reported here. Adx binds at the proximal surface of CYP11A1 via its F-helix (interaction domain) contacting the K-helix of CYP11A1 and with the loop surrounding the [2Fe-2S] cluster (core domain) interacting with the heme-binding loop, C- and L-helices of CYP11A1. The hydrogen bonding interactions predominate upon the Adx–CYP11A1 complex formation. The interface comprises two salt bridges, Lys339CYP11A1-Asp72Adxand Lys343CYP11A1-Asp76Adx, and is consistent with site-directed mutagenesis and chemical modification data (26–29). Residue Asp79 of Adx interaction domain, which affects Adx–CYP11A1 complex formation and activity (28–30), is not involved in direct contact but close (approximately 5 Å) to the “meander” region of CYP11A1, more specifically, to Lys406, the latter being implicated in redox partner binding. It is interesting to note that the effect of replacement of Asp79Adx (28–30) is more profound than the effect of replacement of either Lys404, Lys406, or Arg411 of CYP11A1 (31, 32). We hypothesize that during complex formation, Asp79Adx can initially interact with either of these residues in accordance with conformational changes of the flexible meander region, which would sterically complement a proximal surface for the redox partner.
Asp72,Asp76,Asp79,Lys339,Lys343,Lys404,Lys406,Arg411
     Direct hydrogen bonds between residues Lys109CYP11A1-Ala45Adx,, Trp418CYP11A1-Leu80Adx, and water-mediated hydrogen bonds, including residues Met120CYP11A1-Thr49Adx and Met120CYP11A1-Ala51Adx are displayed. Dashed lines indicate salt bridges and H-bonding interactions; water molecules are represented as small red spheres. (B) Predicted electron-transfer pathway from the [2Fe-2S] cluster to the heme iron proceeds from Fe1 of the [2Fe-2S] cluster via side- and main-chain atoms of Cys52Adx and Ala51Adx, through-space jump (3 Å), Gln422CYP11A1, Cys423CYP11A1 to the heme iron.
Lys109,Ala45,Trp418,Leu80,Met120,Thr49,Met120,Ala51,Cys52,Ala51,Gln422,Cys423
      The specific conserved motif C-GR/KR-E in mitochondrial P450s is important for interaction with Adx and enzymatic activity. The residues Arg426,Arg427,Glu430 of this motif form part of the interaction interface, but are not directly involved in the electrostatic interactions with Adx. Arg427CYP11A1 interacts with Glu430CYP11A1, the latter apparently facilitates Adx dissociation possibly by repulsing the negative charge of Glu73Adx. Notably, ordered water molecules are enclosed within the interaction interface surrounding these charged residues, and may facilitate electronic coupling between redox centers. Taking together, the basic patch on the surface of P450, formed by Arg465,Arg466 of the L-helix, can provide long range electrostatic steering for the recognition (guiding) of Adx, because a dipole moment of Adx does not contribute to electrostatic interactions.
Arg426,Arg427,Glu430,Arg427,Glu430,Glu73,Arg465,Arg466

        Inhibitory activity 3-heteroaromatic analogues 2a6
DNG . DNG

D2G DNG N-METHYL(5-(PYRIDIN-3-YL)FURAN-2-YL)METHANAMINE
     A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6 binding on the HEME to iron(III) moiety active site. The pyridyl moiety was positioned to accept a hydrogen bond from Asn297 with distance d=2.960 Å, and all three inhibitors exhibited orthogonal aromatic-aromatic interactions with protein side chains. For the 3-heteroromatic pyridines, N-methyl and N,N-dimethyl amino groups increased the apparent Ki and distorted helix I of the protein. Substitution of a phenyl ring for the pyridyl ring also increased the apparent Ki, which is likely to reflect the loss of the hydrogen bonding interaction with Asn297.
     A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6. Thiophene, substituted thiophene, furan, substituted furan, acetylene, imidazole, substituted imidazole, thiazole, pyrazole, substituted pyrazole, and aliphatic and isoxazol moieties were used to replace the
N-methylpyrrolidine ring of nicotine. A number of potent inhibitors were identified, and several exhibited high selectivity for CYP2A6 relative to -2E1, -3A4, -2B6, -2C9, -2C19, and -2D6. The majority of these inhibitors elicited type II difference spectra indicating the formation of a coordinate covalent bond to the heme iron.
DNG N-METHYL(5-(PYRIDIN-3-YL)FURAN-2-YL)METHANAMINE
2FDV2a6Marz,2a6(29-494 AA) cholesterol3sulfate, HEM FeIII+ DNG HOH2532,HOH2598,Asn297,Cys439 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys439 in 2a6. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
45-49,50-52,53-66,83-92,104-112,120-138,142-163,170-188,195-213,214-228,
232-257,266-278,287-320,320-335,342-348,349-364,394-400,412-417,441-459,467-471 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
68-73,76-81,390-393,372-373,100-101 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      Two distinct occupancy spaces for the ligand that depend on the orientation of F206 and F297 side chains. Most of the ligands observed in the crystal structures of CYP2B6 occupy space “A” where Phe206 moves in and Phe297 moves out. Ligands that occupy space “B” (4-CPI and amlodipine) were found in complexes showing the greatest movement of Phe206 (out) and Phe297 (in) within the active site. Now locates sabinene in the region near Ile300 in CYP2A6, where there is more space than in CYP2B6, which contains Phe297. Furthermore, a total of nine amino acid differences were found from 13 residues in the active site. These include Val104,Ile114,Ile209,Ser294,Phe297,Ala298,Leu363,Val367,Val477 in CYP2B6, which align to Phe107,Val117,Thr212,Asn297,Ile300,Gly301,Ile366,Leu370,Phe477 in CYP2A6. The distinct active site topology of CYP2A6 with larger hydrophobic side-chain substitutions led to a reduced active-site cavity volume of 289 ų compared with CYP2B6 (376 ų).
107,117,212,297,300,301,366,370,477
off 2a6
104,114,209,294,297,298,363,367,477
off 2b6

        Binding active site monoterpene sabinene and metoxsalen
SNE . SNE

SNE SABINENE (1S,5S)-4-METHYLIDENE-1-(PROPAN-2-YL) BICYCLO HEXANE
     Cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity binding on the HEME to iron(III) moiety active site.
     A series of 3-heteroaromatic analogues of nicotine were synthesized to delineate structural and mechanistic requirements for selectively inhibiting human cytochrome P450 (CYP) 2A6. Thiophene, substituted thiophene, furan, substituted furan, acetylene, imidazole, substituted imidazole, thiazole, pyrazole, substituted pyrazole, and aliphatic and isoxazol moieties were used to replace the
N-methylpyrrolidine ring of nicotine. A number of potent inhibitors were identified, and several exhibited high selectivity for CYP2A6 relative to -2E1, -3A4, -2B6, -2C9, -2C19, and -2D6. The majority of these inhibitors elicited type II difference spectra indicating the formation of a coordinate covalent bond to the heme iron.
     The cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. A much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with
(+)-alpha-pinene. The movement of the F206 side chain influences the volume of the binding pocket.
      Cytochrome P450 (P450) dependent monooxygenases are heme-containing enzymes that metabolize a vast array of drugs and endogenous chemicals. The 57 P450 enzymes found in humans are divided into 18 families and 44 subfamilies. Cytochromes that play a dominant role in the metabolism of drugs and xenobiotics, including environmental toxins, are members of the 1, 2, and 3 families, including CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4.
      Human CYP2B6 metabolizes approximately 3 to 12% of all available drugs and is inhibited by many clinically relevant drugs and small-molecule inhibitors. This enzyme, found in liver, lung, kidney, and brain, is highly polymorphic in nature with 51 known alleles.
SABINENE (1S,5S)-4-METHYLIDENE-1-(PROPAN-2-YL) BICYCLO[3.1.0]HEXANE
4RUI2a6Marz,2a6(29-494 AA) sabinene, HEM FeIII+ SNE Cys439 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys439 in 2a6. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
45-49,50-52,53-66,82-92,104-112,120-138,142-162,170-187,195-212,214-223,
223-228,232-257,266-278,287-320,320-335,342-347,349-364,394-400,412-417,441-460
helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
68-73,76-81,390-393,372-373,100-101 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      Two distinct occupancy spaces for the ligand that depend on the orientation of F206 and F297 side chains. Most of the ligands observed in the crystal structures of CYP2B6 occupy space “A” where Phe206 moves in and Phe297 moves out. Ligands that occupy space “B” (4-CPI and amlodipine) were found in complexes showing the greatest movement of Phe206 (out) and Phe297 (in) within the active site. Now locates sabinene in the region near Ile300 in CYP2A6, where there is more space than in CYP2B6, which contains Phe297. Furthermore, a total of nine amino acid differences were found from 13 residues in the active site. These include Val104,Ile114,Ile209,Ser294,Phe297,Ala298,Leu363,Val367,Val477 in CYP2B6, which align to Phe107,Val117,Phe118,Thr212,Asn297,Ile300,Gly301,Ile366,Leu370,Phe477,Phe480 in CYP2A6. The distinct active site topology of CYP2A6 with larger hydrophobic side-chain substitutions led to a reduced active-site cavity volume of 289 ų compared with CYP2B6 (376 ų).
107,117,118,212,297,300,301,366,370,477,480
off 2a6
107,117,118,212,297,300,301,366,370,477,480
off 2b6

      Human microsomal cytochrome P450 2A6 (CYP2A6) contributes extensively to nicotine detoxication but also activates tobacco-specific procarcinogens to mutagenic products. The CYP2A6 structure shows a compact, hydrophobic active site with one hydrogen bond donor, Asn297 distance 4.036 ų, that orients coumarin for regioselective oxidation. The inhibitor methoxsalen effectively fills the active site cavity without substantially perturbing the structure. The structure should aid the design of inhibitors to reduce smoking and tobacco-related cancers.
1Z10Marz,2a6,(29-494 AA) cholesterol3sulfate, HEM FeIII+ COU Cys439 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys439 in 2a6. Coordination of the heme iron by ionic bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
45-49,50-52,53-66,83-92,104-112,120-138,142-163,170-188,195-214,214-228, 232-257,266-278,287-302,303-320,320-335,342-348,349-364,394-400,412-417,441-459, 467-471 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
68-73,76-81,390-393,372-373,100-101 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      Two distinct occupancy spaces for the ligand that depend on the orientation of F206 and F297 side chains. Most of the ligands observed in the crystal structures of CYP2B6 occupy space ''A'' where Phe206 moves in and Phe297 moves out. Ligands that occupy space ''B'' (4-CPI and amlodipine) were found in complexes showing the greatest movement of Phe206 (out) and Phe297 (in) within the active site. Now locates sabinene in the region near Ile300 in CYP2A6, where there is more space than in CYP2B6, which contains Phe297. Furthermore, a total of nine amino acid differences were found from 13 residues in the active site. These include Val104,Ile114,Ile209,Ser294,Phe297,Ala298,Leu363,Val367,Val477 in CYP2B6, which align to
F107, V117, F118, T212, N297, I300, G301, I366, L370, and F480
Phe107,Val117,Phe118,Thr212,Asn297,Ile300,Gly301,Ile366,Leu370,Phe477,Phe480 in CYP2A6. The distinct active site topology of CYP2A6 with larger hydrophobic side-chain substitutions led to a reduced active-site cavity volume of 289 ų compared with CYP2B6 (376 ų).
104,114,209,294,297,298,363,367,477
off 2b6
107,117,118,212,297,300,301,366,370,480
off 2a6
2FDV2a6Marz,2a6(29-494)
HEM FeIII+ DNG HOH613,641,671,733 off Cys439 off thin off
50-63,79-89,91-96,104-109,117-135,141-160,167-185,192-210,211-225,229-254,
263-277,287-300,302-317,317-332,339-345,346-361,391-397,409-414,438-457,464-468
45-49,50-52,53-66,83-92,104-112,120-138,142-163,170-188,195-213,214-228,
232-257,266-278,287-320,320-335,342-348,349-364,394-400,412-417,441-459,467-471 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
65-69,74-78,387-390,369-370,97-98
68-73,76-81,390-393,372-373,100-101 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)

      Human microsomal cytochrome P450 2b6 (CYP2b6) (+)-alpha-Pinene TMH '. TMH

(+)-ALPHA-PINENE (+)-3,6,6-TRIMETHYLBICYCLO[3.1.1]HEPT-2-ENE C10 H16
is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding on HEME to iron(III) moiety.

5EM42b4Marz2b4(1-494 AA) ... empty active site
4I912b6Marz,(28-492 AA) alpha pinen, HEM FeIII+ TMH Cys436 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys439 in 2b6. Coordination of the heme iron by ionic donor acceptor bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
42-46,50-63,79-89,91-96,104-109,117-132,141-159,167-184,192-211,211-225,
229-255,263-275,284-317,317-332,339-345,346-361,391-397,409-414,431-435,438-456,
464-468 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
65-70,73-78,387-390,369-370,97-98 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      Rearrangements of two phenylalanine residues (F206 and F297) also contributed to a more compact active site in the (+)-alpha-pinene complex, with F206 moving in and F297 moved out. With 2B6, the hydrophobic nature of the monoterpene would provide the 'push' from solution, and the sum of the protein dynamics, the exclusion of water from the active site of the enzyme upon
(+)-alpha-pinene binding, and the hydrophobic nature of the active site could be responsible for the 'pull' phenomenon.
      Residues within a 5 Å radius of the ligand include I101, I114, F115, F206, S294, F297, A298, T302, L363, V367, and V477. These amino acids were previously identified in the active site of several complexes of 2B6 or 2B4. These include Ile101,Val104,Phe107,Phe115,Phe206,Ile114,Ile209,Ser294,Phe297,Ala298,Thr302,Leu363, Val367,Val477 in CYP2B6, which align to Phe107;Val117,Thr212,Asn297,Ile300,Gly301,Ile366,Leu370,Phe477 in CYP2A6. The distinct active site topology of CYP2A6 with larger hydrophobic side-chain substitutions led to a reduced active-site cavity volume of 289 ų compared with CYP2B6 (376 ų).
off
     These conformational changes could release water HOH661 from one or more small hydrophobic pockets where the water is confined and unable to make its full complement of hydrogen bonds. One such pocket seen in multiple X-ray crystal structures of 2B6 is that occupied by CYMAL-5 in the
(+)-alpha-pinene complex (4I91) and comprising residues Ala176,Cys180,Phe188,Phe195,Met198,Leu199,Phe202,Ile241,Tyr244,Ile245,Phe296,Thr300 within 5 Å of the CYMAL-5 molecule. Release of water from such a hydrophobic pocket could produce a significant enthalpic boost to binding.
off 4ZV82b6alphaPinenMarz,(30-491 AA) alpha pinen active site,

      Human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) CPZ '. CPZ

(+)-ALPHA-PINENE (+)-3,6,6-TRIMETHYLBICYCLO[3.1.1]HEPT-2-ENE C10 H16
Cytochromes P450 (P450s) belong to a superfamily of heme-containing monooxygenases and are the predominant enzyme responsible for phase I metabolism of clinically relevant drugs. Through the incorporation of a single oxygen atom, P450s generate products that are more water-soluble and are either readily excreted in the urine or more amenable substrates for phase II conjugation. Previous studies have demonstrated that many of these enzymes are highly flexible, allowing them to accommodate a wide range of substrates, including numerous steroids, pharmaceuticals, and environmental pollutants. P450 2B enzymes were among the first mammalian P450s to be purified and cloned and have served as a prototype for biochemical and biophysical experiments, as well as studies of substrate specificity and of interactions with the redox partners NADPH-cytochrome P450 reductase and cytochrome b5. There are 28 members of the subfamily, the best characterized being rat 2B1 and rabbit 2B4. The species, strain, and individual differences in P450 2B function with such substrates as steroids, polychlorinated biphenyls, and chloramphenicol and analogs have made these enzymes an excellent model system for structure-function analysis. However, little attention has been paid to the human 2B6 enzyme in the past because of a lack of selective substrates, inhibitors, and monoclonal antibodies for functional characterization. HEMECPZ to iron(III) moiety.

3IBD2b6Marz,(28-492 AA) alpha pinen, HEM FeIII+ CPZ Cys436 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys439 in 2b6. Coordination of the heme iron by ionic donor acceptor bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
42-46,50-63,79-89,91-96,104-109,117-132,141-159,167-184,192-211,211-225,
229-255,263-275,284-317,317-332,339-345,346-361,391-397,409-414,431-435,438-456,
464-468 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
65-70,73-78,387-390,369-370,97-98 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      Rearrangements of two phenylalanine residues (F206 and F297) also contributed to a more compact active site in the (+)-alpha-pinene complex, with F206 moving in and F297 moved out. With 2B6, the hydrophobic nature of the monoterpene would provide the 'push' from solution, and the sum of the protein dynamics, the exclusion of water from the active site of the enzyme upon
(+)-alpha-pinene binding, and the hydrophobic nature of the active site could be responsible for the 'pull' phenomenon.
      Residues within a 5 Å radius of the ligand include Leu363,Val367,Val477. These amino acids were previously identified in the active site of several complexes of 2B6. These include Ile101,Val104,Phe107,Phe115,Phe206,Ile114,Ile209,Ser294,Phe297,Ala298,Thr302,Leu363,Val367,Val477 in CYP2B6, which align to active site topology of CYP2A6 with larger hydrophobic side-chain substitutions led to a reduced active-site cavity volume of 289 ų for CYP2B4 compared with CYP2B6 (376 ų).
off
     These conformational changes could release water HOH497,HOH637,HOH645,HOH576,HOH549 from one or more small hydrophobic pockets where the water is confined and unable to make its full complement of hydrogen bonds. One such pocket seen in multiple X-ray crystal structures of 2B6 is that occupied by CYMAL-5 in the
(+)-alpha-pinene complex (4I91) and comprising residues Ala176,Cys180,Phe188,Phe195,Met198,Leu199,Phe202,Ile241,Tyr244,Ile245,Phe296,Thr300 within 5 Å of the CYMAL-5 molecule. Release of water from such a hydrophobic pocket could produce a significant enthalpic boost to binding.
off

      Human cytochrome P450 2B6 in complex with the inhibitor AMLODIPINE
C20 H25 CL N2 O5
O3-ETHYL O5-METHYL 2-(2-AZANYLETHOXYMETHYL)-4-(2-CHLOROPHENYL)-6-METHYL-1,4-DIHYDROPYRIDINE- 3,5-DICARBOXYLATE '. OAX

Human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with molecules of the calcium channel blocker amlodipine - inhibitor, and monoclonal antibodies for functional characterization. HEMEOAX to iron(III) moiety. The presence of two drug molecules suggests clear substrate access channels in each P450. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described (solvent) wtaer and oxygen channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B’, F, F’, G’ and part of helix G is substantially more open in the amlodipine complexes compared with the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F’ and B’ and the β4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states.  These conformational changes could release water HOH606,HOH624 from one or more small hydrophobic pockets where the water is confined and unable to make its full complement of hydrogen bonds.
3UA52b6Marz,27-28-491-492His AA amlodipine human,
HEM FeIII+ OAX HOH606,624 off Cys436 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys436 in 2b6. Coordination of the heme iron by ionic donor acceptor bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
50-63,79-89,91-96,104-109,117-135,141-160,167-185,192-210,211-225,229-254,
263-277,287-300,302-317,317-332,339-345,346-361,391-397,409-414,438-457,464-468 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
65-70,73-78,387-390,369-370,97-98 beta sheet 1-4
B1,B2,B3,B4 4
      The orientation of the F206 and F297 side chains, which were shown recently to rearrange in order to accommodate various ligands within the active site of human P450 2B6. Rearrangements of two phenylalanine residues (F206 and F297) also contributed to a more compact active site in the amlodipine complex, with F206 moving in and F297 moved out. With 2B6, the hydrophobic nature would provide the 'push' from solution, and the sum of the protein dynamics, the exclusion of water from the active site of the enzyme upon amlodipine binding, and the hydrophobic nature of the active site could be responsible for the 'pull' phenomenon.
      Residues within a 5 Å radius of the ligand include Leu363,Val367,Val477. These amino acids were previously identified in the active site of several complexes of 2B6. These include Ile101,Val104,Phe107,Phe115,Phe206,Ile114,Ile209,Ser294,Phe297,Ala298,Thr302,Leu363,Val367,Val477 in CYP2B6, which align to active site topology to active-site cavity volume of 289 ų for CYP2B4 compared with CYP2B6 (376 ų).
off except 2b4 residues T300,C475;R98,V104 in 2b6
     One such pocket seen in multiple X-ray crystal structures of 2B6 is that occupied by amlodipinee complex and comprising 31 residues
Arg49,Leu51,Leu70,Arg73,Arg98,Ile101,Ile114,Phe115,Phe206,Ile209,Ser210, Gln215,Glu218,Leu219,Phe297,Ala298,Gly299,Thr300,Glu301,Thr302,Ile363, Met365,Gly366,Val367,Pro368,Glu387,Phe389,Cys475,Gly476,Val477,Gly478
within 5 Å of the molecule. Release of water from such a hydrophobic pocket could produce a significant enthalpic boost to binding.
off B 3UA5.pdb (27-28-491-492His) human:

      Rabbit cytochrome P450 2B6 in complex with the inhibitor AMLODIPINE
C20 H25 CL N2 O5
O3-ETHYL O5-METHYL 2-(2-AZANYLETHOXYMETHYL)-4-(2-CHLOROPHENYL)-6-METHYL-1,4-DIHYDROPYRIDINE- 3,5-DICARBOXYLATE '. OAXAB
A and B conformation 180° for chlorbenzene ClC₆H₄-
         

Human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with molecules of the calcium channel blocker amlodipine - inhibitor, and monoclonal antibodies for functional characterization. HEMEOAXAB to iron(III) moiety. The presence of two drug molecules suggests clear substrate access channels in each P450. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described (solvent) wtaer and oxygen channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B’, F, F’, G’ and part of helix G is substantially more open in the amlodipine complexes compared with the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F’ and B’ and the β4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states.  These conformational changes could release water HOH611,HOH645,HOH704,HOH705 from one or more small hydrophobic pockets where the water is confined and unable to make its full complement of hydrogen bonds.
3TMZ2b4Marz,2b4,27-28-491-492His AA amlodipine rabbit,
HEM FeIII+ OAX HOH611,645,704,705 off Cys436 off thin off
The axial ligand at the fifth coordination site of the heme iron(III) is provided by the sulfide S^- of Cys436 in 2b6. Coordination of the heme iron by ionic donor acceptor bond Fe³⁺-S^- plays a central role in singlet oxygen bonding scission at the sixth coordination site on the heme iron(III).
42-46,50-63,79-89,92-96,104-109,112-116,117-133,141-160,167-184,192-210,
211-225,229-255,263-275,276-278,284-300,300-317,317-332,339-345,346-361,391-397,
409-414,431-435,438-456,464-468 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23,H24
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
65-69,74-78,387-390,369-370,97-98 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
      The orientation of the F206 and F297 side chains, which were shown recently to rearrange in order to accommodate various ligands within the active site of human P450 2B6. Rearrangements of two phenylalanine residues (F206 and F297) also contributed to a more compact active site in the amlodipine complex, with F206 moving in and F297 moved out. With 2B6, the hydrophobic nature would provide the 'push' from solution, and the sum of the protein dynamics, the exclusion of water from the active site of the enzyme upon amlodipine binding, and the hydrophobic nature of the active site could be responsible for the 'pull' phenomenon.
      Residues within a 5 Å radius of the ligand include Leu363,Val367,Val477. These amino acids were previously identified in the active site of several complexes of 2B6. These include Ile101,Val104,Phe107,Phe115,Phe206,Ile114,Ile209,Ser294,Phe297,Ala298,Thr302,Leu363,Val367,Val477 in CYP2B6, which align to active site topology to active-site cavity volume of 289 ų for CYP2B4 compared with CYP2B6 (376 ų).
off except 2b4 residues T300,C475;R98,V104 in 2b6
     One such pocket seen in multiple X-ray crystal structures of 2B6 is that occupied by amlodipinee complex and comprising 30 residues
Leu43,Leu51,Leu70,Arg73,Val75,Arg98,Ile101,Val104,Phe115,Phe206,Ile209, Ser210,Gln215,Glu218,Leu219,Phe297,Ala298,Gly299,Glu301,Thr302,Ile363, Phe365,Gly366,Val367,Pro368,Glu387,Phe389,Gly476,Val477,Gly478
within 5 Å of the molecule. Release of water from such a hydrophobic pocket could produce a significant enthalpic boost to binding.
off A 3TMZ.pdb rabit, 2b4

      Cytochrome P450 2B4 in complex with the inhibitor CPZ
5-(4-Chloro-phenyl)-1H-imidazole
     Cytochromes P450 1 are involved in steroidogenesis, fatty acid metabolism, synthesis of bile and retinoid acids, but it is their function in the elimination of xenobiotics that has received the most attention. Xenobiotic metabolizing P450s play the central role in detoxification of hydrophobic drugs, carcinogens, and toxins by decreasing the lipid solubility of these chemicals and, thus, promoting excretion. In contrast to the strict substrate selectivity of classical enzymes, xenobiotic-metabolizing P450s can each bind and oxidize a set of substrates with distinct sizes, shapes, and stereochemical features. Although the variety of substrates binding to a given P450 is often broad, the oxidation of each is usually remarkably regiospecific and stereospecific.
     The microsomal cytochrome P450 2B4 with the specific inhibitor 4-(4-chlorophenyl)imidazole (CPI) in the active site. The differences between the open and closed structures of 2B4 were primarily limited to the lid domain of helices F through G, helices B' and C, the N terminus of helix I, and the beta4 region. These large-scale conformational changes were generally due to the relocation of conserved structural elements toward each other with remarkably little remodeling at the secondary structure level. For example, the F' and G' helices were maintained with a sharp turn between them but are placed to form the exterior ceiling of the active site in the CPI complex. CPI was closely surrounded by residues from substrate recognition sites 1, 4, 5, and 6 to form a small, isolated hydrophobic cavity. The switch from open to closed conformation dramatically relocated helix C to a more proximal position. As a result, heme binding interactions were altered, and the putative NADPH-cytochrome P450 reductase binding site was reformed. This suggests a structural mechanism whereby ligand-induced conformational changes may coordinate catalytic activity. Comparison of the 2B4/CPI complex with the open 2B4 structure yields insights into the dynamics involved in substrate access, tight inhibitor binding, and coordination of substrate and redox partner binding.
1SUOMarz2b4(25-491)
HEM FeIII+ CPZ HOH1056,1107,1172 off Cys436 off thin off
and 2b6,5EM42b4Marz
The residues that comprise the active site in the CYP2B37 structure (magenta) within 5 Å from the
4-CPI (blue) coordinating heme are I101,V114,F115,F206,F297,A298,G299,T302,I363,V367.
off Alternate orientation and rearrangement of F206 and F297 side chains.
These include R98,I101,I104,I108,L114,F115 near the B' helix region, F206 and M209 (F helix), S294, F297, A298, T302 located on the I helix, and L362, A363, G366, A367, P368 surrounding helix K, and I477 and G478 of the beta4 loop. Residues 363 and 367 are alanine in the active site of CYP2B35, as opposed to L363 and V367 in human CYP2B6, and I363 and V367 in CYP2B4 and CYP2B37. These amino acid differences allow room for CYP2B35 to accommodate another 4-CPI molecule in the void region between the heme-ligating 4-CPI molecule and A363 and A367. The chloro group of this second 4-CPI molecule sits between the side chains of A367 and I114, facing the side chains of R98 and F115. The side chains of T302, L362, A363, and I477 surround the imidazole ring of the second 4-CPI molecule, and the imidazole nitrogen is within hydrogen bonding distance from the free nitrogen in the first 4-CPI molecule. Residues V104, M209, and I477 form a “roof” that closes the active site in the CYP2B35 complex. However, these residues in CYP2B37 are instead I104, I209, and F477, and are displaced considerably due to the second molecule of 4-CPI located at the active-site periphery and into the substrate access channel 2f.
98,101,104,108,114,115,206,209,294,297,298,302,362,363,366,367,368,477,478,
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In the CYP2B37 structure, nine residues are located within 5 Å from the single 4-CPI molecule in the active site coordinated to the heme-iron: I101,V114,F115,F297,A298,G299,T302,I363,V367. The residues closer to the second 4-CPI molecule observed in included E218 (F' helix), I365,P368,Y389,F477, which are predominantly within the 2f substrate access channel region. The side chain of F477 was disordered at the given resolution, and thus was not modeled in the structure. Residues within 5 Å from the third 4-CPI molecule were L43,M46,D47,F51 of the A' and A helices, and F212,Q215 of the F' helix, which are located near the surface at the substrate access region of the channel 2f. Furthermore, in the CYP2B37 complex, the orientation of the active-site 4-CPI molecule is the same as that previously observed in the CYP2B4 and CYP2B6 structures. However, the 4-CPI molecule coordinated to the heme iron in the CYP2B35 structure rotates toward the I helix by ∼90° to accommodate the neighboring 4-CPI molecule in the active site. In addition, the crucial residue side chains F206,F297 in the CYP2B37 structure are in similar orientation as that observed in the 4-CPI complexes of the CYP2B4 and CYP2B6 structures. As shown in, these residues in CYP2B35 now rotate by 90° around the axis to an alternate orientation, likely in response to the movement of the 4-CPI molecule ligated to the heme iron toward the I helix.
43,46,47,51,101,114,115,206,212,215,297,298,299,302,363,367,365,368,389,477,
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Residue side chains of F115,F206,F212,F297,Y389 that form stable -Cl- or -NH-pi interactions.
115,206,212,297,389
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Cytochrome P450 1A2 in human liver expressed
     Microsomal cytochrome P450 family 1 enzymes play prominent roles in xenobiotic detoxication and procarcinogen activation. P450 1A2 is the principal cytochrome P450 family 1 enzyme expressed in human liver and participates extensively in drug oxidations. This enzyme is also of great importance in the bioactivation of mutagens, including the N-hydroxylation of arylamines. P450-catalyzed reactions involve a wide range of substrates, and this versatility is reflected in a structural diversity evident in the active sites of available P450 structures. Enzymes of the cytochrome P450 (CYP)5 superfamily play a significant physiologic role in the detoxication of foreign compounds and the biosynthesis of endogenous compounds, including steroid hormones, bile acids, and cholesterol. The enzymes comprising P450 families 1, 2, and 3 contribute most extensively to the biotransformation of xenobiotics to more polar metabolites that are more readily excreted. In humans and most mammals, family 1 contains three well characterized P450 monooxygenases; 1A1, 1A2, and 1B1. These enzymes are generally distinguished from P450s in other families by their capacity to oxidize a variety of polynuclear aromatic hydrocarbons (PAHs). Some P450-catalyzed reactions can transform relatively inert compounds into genotoxic metabolites that can initiate mutagenesis and cancer. Human P450 1A2 is notable among family 1 enzymes for the capacity to N-oxidize arylamines, the major metabolic process in the bioactivation of arylamines to potent mutagenic or carcinogenic compounds. alpha-Naphthoflavone (ANF), a prototype flavonoid, is known to competitively inhibit P450s of family 1, albeit at different concentrations, and has been used to discriminate between P450 family 1 enzymes (3). Flavonoids have gained recent interest in view of their potential therapeutic and prophylactic effects on P450-mediated chemical carcinogenesis. CYP1A2 is the principal family 1 enzyme expressed in human liver, and CYP1A2 contributes significantly to the hepatic metabolism of drugs, as recently reviewed (5). Among liver P450 drug-metabolizing enzymes, P450 1A2 plays a predominant role in the metabolic clearance of caffeine and melatonin as well as of marketed drugs such as flutamide, lidocaine, olanzapine, tacrine, theophylline, triamterene, and zolmitriptan.
2HI4Marz,1a2(27-515)
HEM FeIII+ BHF HOH613,641,671,733 off Cys458 off thin off
53-58,60-73,90-99,101-105,111-116,130-147,159-182,187-206,207-209,213-221,
223-228,234-238,239-245,247-274,282-294,304-309,309-336,336-351,358-362,365-380,
410-417,428-433,441-446,453-457,460-479 helixes
color chain A,C
H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20,H21,H22,H23,H24,H25
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,                                            I,      J,K,L  
65-69,74-78,387-390,369-370,97-98
75-80,83-88,406-409,388-389 beta sheet 1-4
B1,B2,B3,B4 4
The overall size of the P450 1A2 cavity is 375 ų, which is about 50% larger than that of P450 2A6 (260 ų) but considerably smaller than that of P450 3A4 (1385 ų). Remarkable feature of the P450 1A2 structure is the disruption of helix F as it crosses the distal surface of the active site cavity Fig. 2. P450 family 2 structures 6-12 display an intact helix F for this region. Also, in the structure of P450 3A4 13, 14, a coiled structure connecting helices F and F' crosses above the active site cavity. In the P450 1A2 structure, the alpha-helical hydrogen-bonding pattern is lost at Val220,Lys221, causing one helical turn in the middle of helix F to unwind. Two water molecules HOH613,641,733 fill the space thus created, giving rise to water-bridged contacts between Val220 carbonyl oxygen and Thr223 Oγ, and Lys221 carbonyl oxygen and His224 amide nitrogen, respectively. The bending of helix F brings the C-terminal portion of the helix in toward the core of the protein, closing down the active site cavity. Substrates bind in the cavity located above the distal surface of the heme prosthetic group. In the P450 1A2 structure of the ANF complex, the active site is closed without evident solvent or substrate access channels. The volume of the cavity was estimated to be 375 ų, which is larger than that of P450 2A6 260 ų. the hydrophobic effect is likely to contribute to a favorable free energy difference for ANF binding. In addition, both orthogonal and parallel aromatic interactions between ANF and phenylalanine side chains 125,226 contribute to a tight binding affinity. The water molecule appears to be hydrogen-bonded to the carbonyl of ANF as well as to the carbonyl of Gly316 on helix I. F125,F226,V220,K221,T223,H224,G316
125,226,220,221,223,224,316
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The substrate binding cavity is uniformly narrow throughout its extent and is lined by residues on helix F and helix I that define a relatively planar substrate binding platform on either side (Fig. 5). Helix I bends as it crosses the heme prosthetic group. As a consequence, the helix I residues constituting one side of the substrate binding cavity adopt a relatively flat conformation of the peptide backbone, resulting in a near perfect coplanarity for the Ala-317 side chain, the Gly-316-Ala-317 peptide bond, and the Asp-320-Thr-321 peptide bond (Fig. 5). The occurrence of a water molecule within hydrogen-bonding distance from the backbone carbonyl group of Gly-318 on the backside of helix I helps to stabilize this deformation. On the other side of the cavity, the side chain of Phe-226 on helix F produces a parallel substrate binding surface. The distortion of helix F as it passes over the substrate binding cavity contributes to the narrow, extended cavity architecture. Not only is the C-terminal end of the helix positioned to produce a compact active site cavity, the observed bend in helix F also positions Phe-226 for the observed π-π stacking with ANF. The shape of the active site cavity is further stabilized by a strong hydrogen-bonding interaction between the side chain of Thr-223 on helix F with the side chain of Asp-320 on helix I, connecting both helices at the roof of the cavity. Both Thr-223 and Asp-320 participate in an extensive network of hydrogen-bonded water molecules and side chains, including Tyr-189, Val-220, Thr-498, and Lys-500. F125,F226,V220,K221,T223,H224,G316
125,226,220,221,223,224,316
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Helix I bends as it crosses the heme prosthetic group. As a consequence, the helix I residues constituting one side of the substrate binding cavity adopt a relatively flat conformation of the peptide backbone, resulting in a near perfect coplanarity for the Ala317 side chain, the Gly316,Ala317 peptide bond, and the Asp320,Thr321 peptide bond. The occurrence of a water molecule within hydrogen-bonding distance from the backbone carbonyl group of Gly318 on the backside of helix I helps to stabilize this deformation. On the other side of the cavity, the side chain of Phe226 on helix F produces a parallel substrate binding surface. The distortion of helix F as it passes over the substrate binding cavity contributes to the narrow, extended cavity architecture. Not only is the C-terminal end of the helix positioned to produce a compact active site cavity, the observed bend in helix F also positions Phe226 for the observed π-π stacking with ANF. The shape of the active site cavity is further stabilized by a strong hydrogen-bonding interaction between the side chain of Thr223 on helix F with the side chain of Asp320 on helix I, connecting both helices at the roof of the cavity. Both Thr223,Asp320 participate in an extensive network of hydrogen-bonded water molecules and side chains, including Tyr189,Val220,Thr498,Lys500. Taken together, these interactions produce a binding cavity that fits closely with planar compounds such as ANF and typical CYP1A2 substrates such as caffeine, melatonin, tacrine, olanzapine, arylamines, and alkoxyresorufins.
Y189,V220,T223,F226,Gly316,Ala317,G318,Asp320,Thr321,T498,K500
189,220,223,226,316,317,318,320,321,498,500
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The absence of solvent channels in the crystal structure of P450 1A2 in complex with ANF adds credence of idea in binding cavity to reduce the propensity of water to fill the cavity in a stable binding pattern. Amino acid substitutions for Phe226 and Asp320 have been shown to produce CYP1A2 mutant enzymes that display a predominantly low iron spin character. The substitutions F226I,F226Y,D320A have the potential to reduce intramolecular interactions between residues that constitute the compact, closed active site cavity, leading to open water channels that could stabilize the binding of water to the heme iron. In addition, these three mutants showed a reduced catalytic efficiency indicative of an effect on active site integrity. Mutations of Phe226,Asp320, which display a lowered catalytic activity in nearly all cases, the effect on the catalytic efficiency of single mutations in substrate recognition site regions largely depends on the substrate used in the activity assay. The increased Km values observed upon substitution of Phe226 can be rationalized based on the observed position in the active site cavity and point to its prominent role in binding a wide range of substrates. Although the D320A mutation generally decreases the catalytic efficiency of the enzyme, the catalytic efficiency for the O-demethylation of resorufin by the D320A mutant is similar to that of the wild-type CYP1A2 enzyme, although the D320A mutant exhibits a 2-fold lower Km for 7-ethoxyresorufin.

This is likely to reflect the effect of the mutation on substrate binding as well as the catalytic mechanism of the enzyme. The latter reflects the importance of Asp/Glu at this position for the protonation and cleavage of hydrated H^- singlet oxygen complex
(Asp/Glu)H⁺+H-O-O-Fe²⁺S^-Cys =>H-O-H + -O-Fe²⁺S^-Cys
during the P450 reaction cycle. It was seen that the D320A mutation impairs the formation of the iron-oxygen intermediate. This, together with the role of Asp320 in maintaining contacts that determine the substrate binding cavity architecture, leads to CYP1A2 mutants at this position that are relatively inefficient for most substrates.
226,320
off
The planar active site topology P450 1A2 structure is well adapted for the oxidation of relatively large aromatic compounds, is likely to be conserved among the family 1 enzymes. The 22 residues lining the active site cavity in the P450 1A2 structure.
F125,F226,V220,K221,T223,H224,G316
Y189,V220,T223,F226,Gly316,Ala317,G318,Asp320,Thr321,T498,K500
T118,T124,F125,T223,F226,D313,G316,A317,F319,D320,L382
118,124,125,189,220,221,223,224,226,313,316,317,318,319,320,321,382,498,500
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Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice.
     Human cytochrome P4503 2D6 mediates the principal route of metabolic clearance for >15% of the 200 most marketed drugs that are primarily cleared by P450-mediated hepatic metabolism. P450 2D6 oxidizes a variety of anti-psychotic, anti-depressant, and anti-arrhythmic drugs, which reflects the capacity of the enzyme to oxidize moderately sized, basic substrates. Genetic polymorphisms contribute to a wide variation of 2D6 expression and activity, leading to four phenotypes as follows: poor, intermediate, extensive, and ultra-fast drug metabolizers. Drugs such as thioridazine, debrisoquine, phenformin, and captopril, which exhibit comparatively narrow therapeutic windows, can be problematic when used by 2D6 poor metabolizers, which represent 10% of the Caucasian population. Prinomastat binding is pyridyl nitrogen bound to the heme iron(III). The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6. The pyridyl nitrogen is located 2.2 Å from the heme iron consistent with the observation that prinomastat shifts the
Soret maximum to 422 nm as typically seen for nitrogenous ligands that coordinate to the heme iron(III).

3TDA                      4WNU                 4WNV                     4WNT                      4WNW
2F9QMarzB2d6(29-494)3QM43d6Marz,
HEM FeIII+ PNO Cys443 off thin off
2d6(29-494)3TBG2d6Marz
HEM FeIII+ RTZ HOH15,504,552,615,623,628 off Cys443 off thin off
2d6(29-494)3TDAMarz
HEM FeIII+ PNO HOH9,513,519,557 off Cys443 off thin off
2d6(29-494)4WNUMarz
HEM FeIII+ QDN HOH736,737,743,753,790,822 off Cys443 off thin off
2d6(29-494)4WNVMarz
HEM FeIII+ QIN HOH717,729,739,747,758,795 off Cys443 off thin off
2d6(29-494)4WNTMarz
HEM FeIII+ AJN HOH714,715,718,720,728 off Cys443 off thin off
2d6(29-494)4WNWMarz
HEM FeIII+ RTZ+Na⁺ Cys443 off thin off
F' helix, residues 218–225; F residues 199–215, 2F9Q structure of P450 2D6, where a continuous helix encompasses residues 199–225, structural components that include helix A, the loop between the first two strands of sheet beta1 (residues 72–77), and helices F, G', G, H.
50-63,79-89,91-96,104-109,117-135,141-160,167-185,192-210,211-225,229-254,
263-277,287-300,302-317,317-332,339-345,346-361,391-397,409-414,438-457,464-468
45-49,50-52,53-66,83-92,104-112,120-138,142-163,170-188,195-213,214-228,
232-257,266-278,287-320,320-335,342-348,349-364,394-400,412-417,441-459,467-471 helixes

H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13,H14,H15,H16,H17,H18,H19,H20
      A  , B     B', C,  D  , E ,  F   F'  , G' ,   G,   H ,             I,      J,K,L  
65-69,74-78,387-390,369-370,97-98
68-73,76-81,390-393,372-373,100-101 beta sheet 1-5
B1,B2,B3,B4,B5 4(B5)
     One hydrogen bond is evident between Gln244 and one of the prinomastat sulfonyl oxygens. Another hydrogen bond is located between the amide hydrogen of the hydroxamic acid moiety and Ser304, and the third hydrogen bond occurs between the hydroxyl group of the hydroxamic acid moiety and Asp301. Glu216, the remaining contacts between the protein and prinomastat are nonpolar. Glu216,Gln244,Asp301,Ser304
216,244,301,304
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     Helix F' Ser217,Gly218 contribute to the flexibility of the polypeptide backbone in this turn because of the small size of the side chain and absence of side chain, respectively. The corresponding amino acids that form this turn are similar for other family 2 P450s. The position of helix F' in the prinomastat complex is similar to that seen for the structures 2A6 (PDB code 1Z10) and 2C8 (PDB code 2NNJ) as are the positions of helices F and G, the first turn in beta-sheet 1, and the N-terminal region before helix A. The shortened helix G is preceded by another short helix, which these authors designated G′. As this region is normally part of a longer G helix in other family 2 P450s, we have designated it as helix G'. This unusual feature is conserved for both the 2F9Q and 3QM4 structures. The prinomastat complex anti-parallel orientation of the helix F' and helix G' is seen. The loop between the two helices corresponds to the portion of the polypeptide chain that forms helix G' in structures of other family 2 enzymes, such as P450s 2A6 and 2C8. This loop and its side chains are defined in chain A but not in chain B of the 3QM4 structure.
Amino acid side chains (cyan carbons) contacting prinomastat (green carbons) are depicted along with the heme prosthetic group L108,F110,F120,L213,E216,Q244,F247,A300,D301,S304,A305,V307,S308,F483
108,110,120,213,216,244,247,300,301,304,305,307,308,483
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     The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp301,Glu216,Phe483,Phe120. The crystal structure helps to explain how Asp301,Glu216,Phe483 can act as substrate binding residues and suggests that the role of Phe120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme.
120,216,301,483
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The cumulative effects of these changes increase the volume of the active site cavity 3QM4 relative to that of the 2F9Q structure, 712 versus 582 ų uniformly above the surface of the heme. The differences are relatively small for residues on helix I with the exception of a distortion in the helix that accommodates the prinomastat pyridyl group bound to the heme iron. This distortion leads to significant changes in the positions of Ser304,Ala305,Val308,Thr309 with largest differences, ∼1.9 Å, seen for Ala305,Thr309, which contact the pyridyl group of prinomastat. In contrast, Ala300,Asp301 on helix I and Phe-120 on the adjacent helix B-C loop are not greatly affected. Nevertheless, larger differences of ∼1.5–1.9 Å are seen for Leu110,Phe112 on the helix B-C loop, which moves in to contact prinomastat. The largest differences are evident for residues on helix G, which exhibit >2-Å differences for Cα positions of Gln244,Phe247,Leu248, and in the case of Gln244, there is also a significant change in the side chain rotamer to accommodate hydrogen bonding to the sulfonyl oxygen of prinomastat.
Leu110,Phe112,Gln244,Phe247,Leu248,Ala300,Asp301,Ser304,Ala305,Val308,Thr309,
110,112,244,247,248,300,301,304,305,308,309
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Similarly, when the sector of the cavity formed by the intersection of beta-sheet 4 with helices F and I is examined, the largest changes in amino acid side-chain positions occur in the upper portion of the cavity with smaller changes observed for Val370 near the base of the cavity and the heme surface. Residues Ala209,Leu213,Glu216 on helix F are shifted to expand the cavity to accommodate prinomastat with the position of the carboxylate moiety of Glu216 displaced by more than 4 Å. In contrast, the side chain of Phe483 on the turn in the adjacent sheet beta-4 swings into the cavity by 4 Å to form favorable nonbonded interactions with prinomastat. The convergence of the amino acid side chains on helix F and sheet beta-4 with those on helix I close the open solvent channel evident in the 2F9Q structure. The largest differences for side-chain positions in the active site reflect the different locations of the helix F' segment of the polypeptide chain in the two structures. A turn following Glu216 in the prinomastat complex terminates helix F and orients the helix F' region so that its helical axis points toward the N-terminal side of the helix B-C loop. The polar surface of the helix F' region, which is on the exterior surface of the 2F9Q structure, is rotated so that Glu222,Arg221 are directed into the interior of the protein in the prinomastat complex, where a substrate access channel passes below helix F' and above the surface of the heme and the loop between helix K and beta-sheet 1. The outer surface of the entrance channel is formed by beta-sheet 1, helix A, and the loop between the proline-rich region at the N terminus of the model and helix A. This substrate access channel is not evident for the 2F9Q structure where the extended and combined helices F and F' pass through this region. Glu222 is likely to remain hydrated in the substrate access channel of the 3QM4 structure. The access channel is closed off from the active site by the close proximity of Glu216 on helix F with the guanidinium group of Arg221 on helix F', which face into the active site cavity. These amino acid side chains together with those of Val104,Phe120,Leu121 on the helix B-C loop and Phe483 on the turn in sheet beta-4 constrict the connection between the entrance channel and the prinomastat binding cavity, creating a separate solvent-accessible antechamber. Additionally, the interaction between Arg221,Glu216 provides a favorable electrostatic interaction between the oppositely charged side chains in the interior of the protein, but the overall electrostatic potential remains rather negative because of the presence of Asp301,Glu222. The constriction of the channel better accommodates the size and shape of prinomastat and increases van der Waal's contacts between prinomastat and the enzyme. Nevertheless, the overall flexibility, implied by comparison of the 3QM4 and 2F9Q structures, suggests that the constriction can open to form a passage to the outside for substrates and products. As shown by the comparisons in, these differences in the tertiary and secondary structure of the prinomastat complex lead to significant shifts in the positions of the contact residues relative to the positions seen in the 2F9Q structure.
Val104,Phe120,Leu121,Ala209,Leu213,Glu216,Arg221,Glu222,Asp301,Val370,Phe483
104,120,121,209,213,216,221,222,301,370,483
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The wild type (WT) residues L230,L231,V374
     P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes.The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu216, whereas the protonated nitrogen of quinidine is equidistant from Asp301,Glu216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds. In both (PDB3 code 3QM4 and 2F9Q) structures the protonated nitrogen of the thioridazine methyl piperidine ring forms a charge-stabilized hydrogen bond with Asp301 on helix I in the active site. The second thioridazine, which is bound in the entry channel, forms a charge-stabilized hydrogen bond with Glu222, which may serve as the initial site for binding cationic substrates in the entrance channel to facilitate their entry into the active site.
Glu216,Glu222,L230,L231,Asp301,V374
216,222,230,231,301,374
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2d6(29-494)3TBG2d6Marz
HEM FeIII+ RTZ Zn600 HOH15,504,506,614,615,628,567 off Cys443 off thin off
2d6(29-494)3TDAMarz
HEM FeIII+ PNO Zn600 HOH9,513,519,557 off Cys443 off thin off
2d6(29-494)4WNUMarz
HEM FeIII+ QDN HOH736,737,743,753,790,822 off Cys443 off thin off
2d6(29-494)4WNVMarz
HEM FeIII+ QIN HOH717,729,739,747,758,795 off Cys443 off thin off
2d6(29-494)4WNTMarz
HEM FeIII+ AJN HOH714,715,718,720,728 off Cys443 off thin off
2d6(29-494)4WNWMarz
HEM FeIII+ RTZ+Na⁺ Cys443 off thin off
     Structural features of the closed P450 2D6 thioridazine complex.
Leu110,Phe112,Phe120,Leu121,Ala209,Leu213,Glu216,Gln242,Phe247,Leu248,Ile297,Asp301,Val308,Thr309,Val370,Val374,Phe483,Leu484
110,112,120,121,209,213,216,242,247,248,297,301,308,309,370,374,483,484
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     The protonated lone pair of the RTZ piperidine nitrogen is directed toward the negatively charged oxygen of Asp301 at a distance of 3.0 Å. Glu216 is positioned more than 5.5 Å away from the piperidine nitrogen, and there are no intervening atoms that would shield the nitrogen from the negative charge of Glu216. A sodium ion was modeled into electron density based on B values and its close proximity to the side chain oxygens of Glu216. Additionally, the side-chain nitrogen of Gln242 is positioned for hydrogen bonding to an carboxyl oxygen of Glu216 as seen in the 3QM4 structure. Terminal nitrogens of the Arg221 side chain are positioned roughly equidistant from Glu216 and Glu222 (not shown), which resides in the entrance channel antechamber depicted. The remaining polar residues in the substrate binding site, Ser304,Thr309, are hydrogen-bonded to the backbone carbonyls of Ala300,Ala305, respectively. With the exception of residues in the vicinity of the N-methylpiperidine group, most of the active site residues did not exhibit significant Cα shifts or changes in side-chain torsion angles when compared with the 3QM4 structure of the co-crystallized prinomastat complex. The largest Cα movement was observed for Phe112 and adjacent residues on helix B-C loop, which moves out from the active site to accommodate the binding of the methylpiperidine ring near Asp-301. Changes in the dihedral angles for Phe120,Leu121 are also evident, which opens a channel between the active site cavity and an adjacent antechamber under helix F′ with a minimum radius of 1.7 Å. In general, the antechamber increases the potential range of motion for side chains that border both cavities to adapt to ligand binding. In this structure there is a channel between the antechamber and the exterior solvent with a minimum radius of 1.5 Å between Trp75,Thr76,Thr394 on beta-sheet 1 and Pro104 on the helix B-B′ loop. This channel is evident in other P450s and is designated as 2b in the nomenclature of Wade and co-workers. Trp75,Thr76,Thr394,Pro104,Phe112,Phe120,Leu121,Glu216,Arg221,Glu222,Ala300,Asp301,Ser304,Ala305,Thr309,Gln242
75,76,394,104,112,120,121,216,221,222,300,301,304,305,309,242
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     A nickel ion bound in the turn between helices G and H of chain B. The nickel ion is bound by the side chains of His258,Asp270,Glu273
258,270,273
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2d6(29-494)3TBG2d6Marz
HEM FeIII+ RTZ Zn600 HOH15,504,506,614,615,628,567 off Cys443 off thin off
2d6(29-494)3TDAMarz
HEM FeIII+ PNO Zn600 HOH9,513,519,557 off Cys443 off thin off
2d6(29-494)4WNUMarz
HEM FeIII+ QDN HOH736,737,743,753,790,822 off Cys443 off thin off
2d6(29-494)4WNVMarz
HEM FeIII+ QIN HOH717,729,739,747,758,795 off Cys443 off thin off
2d6(29-494)4WNTMarz
HEM FeIII+ AJN HOH714,715,718,720,728 off Cys443 off thin off
2d6(29-494)4WNWaMarz,4WNWbMarz
HEM FeIII+ RTZ Cys443 off thin off
     The RTZ molecule proximal to the heme forms an ionic hydrogen bond with Asp301. The 2B and 2F channels are constricted in chain D, but an alternative channel (ChS) has opened between helix I, helix F. and the turn in the C-terminal loop due in part to changes in the Phe483 and Phe51 rotamers and the different positioning of the pre helix A region. The thioridazine molecule bound in the entrance channel antechamber forms a charge-stabilized hydrogen bond with Glu222, suggesting that this acidic residue can stabilize initial substrate binding in the entrance channel. The passage from the active site to the antechamber remained constricted by residues Phe483,Phe120, Glu216. In the absence of thioridazine, Glu216 would likely adopt alternative orientations that would increase the size of the channel and allow the 2B and 2F channels to merge with each other.
L110,F112,F120,L213,E216,Glu222,Q244,F247,D301,S304,A305,V308,T309,F483
110,112,120,213,216,222,244,247,301,304,305,308,309,483
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