Catalase: H2O2: H2O2 dismutation

Elizabeth M. Boon '97, Aaron Downs '00, and David Marcey,
Aris Kaksis     Riga Stradin's University 2023

Contents:

  • Peptides and Backbones

    I. Introduction

    Catalase (EC 1.11.1.6), present in the peroxisomes of nearly all aerobic cells, serves Essential unsaturated fatty acid ω=6, ω=3 elongation to C20:4 with ethyl group
    -CH2-CH2- conversion to cis double bond H>C=C<H in peroxisomes
    is spontaneous.
          CATALASE in complex reaction sequence favors stabile unsaturated Essential
    fatty acid ω=6, ω=3 product efficiency 100%:
    Keq=108,43= catalyzing its decomposition into molecular life resources  products O2 + 2H2O + Q and avoiding the propagation in per-oxidation chain reactions of free radicals. The mechanism of catalysis is not fully elucidated, but the overall reaction is exothermic as follows:
    2H2O2+Fe3+=His74-H++HOO->Fe3+<-OOH+Asn147-H+=O2aqua+2H2O+O2 +Q+Fe3+.
      
    Protolysis activate    transition state active complex    oxygen+water+heat+CATALASE

    II. Structure of a Bovine Catalase Monomer

    Primary structure 1̊ .
         The beef liver catalase monomer (shown at left) consists of a 506 of 527 amino acid polypeptide chain plus one heme group and one NADH molecule.  Secondary structure.
    Only about 60% of catalase structure is composed of regular secondary structural motifs < >.
         13 Alpha-helices H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13
        account for 26% of its structure and beta-barrel structure 8 anti-parallel strands
         for 12%. Irregular structure includes a predominance of extended single strands and loops
    that play a major role in the assembly of the tetramer.

    Funtional catalase is a tetramer of four identical holo subunits. A model of a beef liver catalase
    tetramer is shown at left.

    IV. The Heme Group and its Environment

         The Funnel-Channel to the Iron in Heme Group.
    As noted above, the heme groups are deeply embedded in each subunit of a modeled tetramer. However, as can be seen in a monomer of beef liver catalase, each heme is exposed through a funnel-shaped channel
    Ser113,Glu118,Ser121,Arg126,Asp127,Gln167,Lys168,Lys176,Glu247,His254,Glu450,Asn461,His465,Val115,Ala116,Pro128,Phe153,Phe163,Ile164,Pro178,Val181,Leu198,Phe199
    25 Å long and 20-15 Å wide < >. The channel is lined with at the entrance
    14 hydrophilic residues < >
    Ser113,Glu118,Ser121,Arg126,Asp127,Gln167,Lys168,Lys176,Glu247,His255,Glu453,Glu454,Gln461,His466 and
    with 11 hydrophobic residues
    Val115,Ala116,Pro128, Phe152,Phe153,Phe163,Ile164,Pro178,Val181,Leu198,Phe199     as the channel descends, constricting, toward the heme.
         The Heme Cavity.
         The heme group is located between the internal wall of the beta-barrel and several helices
    H3=156-168 and H9=347-366     < >.
         The heme pocket is mainly 31 AA hydrophobic < >
    Val72,Val73,Ala75,Val115,Ala116,Pro128,Gly130,Val145,Gly146,Phe152,Phe153,Ile154,
    Ala157,Leu158,Leu159,Phe160,Pro161,Phe163,Ile164,Phe197,Leu198,Phe199,
    Leu298,Ala332,Phe333,Pro335,Met349,Leu350,Gly352,Phe355,Ala356,Pro358,Ala434
    Backbone thin off
    with the exception of a few 8, Arg353 and Tyr357residues
    thought to be involved in binding the heme prosthetic group or
    in the catalysis H+ desorbtion His74,Asn147 at peroxide dismutation on iron and
    binding heme propionic acid residues Arg71,Arg111,Glu329,Arg364 in pocket of CATALASE.
         Peroxide, upon entering the heme cavity, is severely sterically hindered and must interact with His74 and Asn147< >. It is in this position that the first stage of catalysis takes place. Transfer of a proton from one oxygen of the peroxide to the other, via His74, elongates and polarizes the O-O bond, which eventually breaks heterolytically to transferre proton through His74 and Asn147 for hydroxide ion making water molecule as a peroxide oxygen is coordinated to the iron center. This coordination displaces water and forms Fe(III)-O plus a heme radical. The radical quickly degrades in another one electron transfer to rid of the radical electron, leaving the heme ring unaltered. During the second stage, in a similar two electron transfer reaction, Fe(III)-O reacts with a second hydrogen peroxide to produce the original Fe(III)-E, another water, and a mole of molecular oxygen O2.

    Tertiary structure 3̊ monomer of beef liver catalase.
    Each monomer has four domains < >.
         The first domain < > is made up of the amino-terminal 75 residues. These form an arm with two alpha-helices H1,H2 and a large loop extending from the globular subunit < >.
         The second and largest domain contains the heme moiety < >. It is composed of residues 76 to 320 and may be classified as an alpha+beta-barrel 8 anti-parallel strands type domain . It includes a beta-barrel, fivel H3,H4,H5,H6,H7 helical segments of three to four turns each, and various loops < > . The beta-barrel 8 anti-parallel strands < > consists of two four stranded anti-parallel beta-sheets that twist to form a closed cylindrical surface.
         The third domain consists of residues 321-436 the largest of the essential helix H9 ASP347-GLY366 contains the heme phenolic ligand, Tyr357 < > His361 and is referred to as the wrapping domain . It lacks discernable secondary structure except for two helices H8 ASN323-GLU329,H9 ASP347-GLY366 < >, .
         The carboxy-terminal < > portion of the molecule contains residues 437 to 506 and is folded into a four-helical domain H10,H11,H12,H13 < > similar to the globin folds. Along with three alpha-helices from the heme-containing domain, these helices form one surface of the enzyme < >. 

    III. Quaternary 4̊ Structure: Assembly of the Catalase Tetramer

    Funtional catalase is a tetramer of four identical holo subunits. A model of a beef liver catalase tetramer is shown at left. Each monomer harbors a single heme and NADP. Whereas the NADPs lie on the surface, the heme moieties are embedded in the middle of each monomer, ~20 Å below the molecular surface, and using Disply options distance mesure by mous cklik on atoms the distance in angstrems between heme and calculate the distance Å from the center of the tetramer < >.

    The assembly of the multimeric complex is presumably more complicated than a simple combination of monomers, with changes in the folding pattern of each monomer occurring so as to optimize packing interactions < >.

    Most intersubunit contacts are confined to the amino-terminal arms and the wrapping domains < >. The most flexible parts of the protein are thus responsible for most of the quaternary structural interactions. The amino-terminal domain < > becomes almost completely buried between neighboring subunits in the tetramer. There are numerous salt bridges at the interfaces between monomers, mostly involving arginine, asparagine, and glutamic acid partners < >.
    Arg421A-Asp427D;Glu419A-Arg430D-Asp53C
    Arg421B-Asp427C;Glu419B-Arg430C-Asp53D
    Arg421C-Asp427B;Glu419C-Arg430B-Asp53A
    Arg421D-Asp427A;Glu419D-Arg430A-Asp53B
    Arg65A-Asp359B;Arg4A-Asp179B;Arg169A-Asp258B;Arg381A-Asp24C
    Arg65B-Asp359A;Arg4B-Asp179A;Arg169B-Asp258A;Arg381B-Asp24D
    Arg65C-Asp359D;Arg4C-Asp179D;Arg169C-Asp258D;Arg381C-Asp24A
    Arg65D-Asp359C;Arg4D-Asp179C;Arg169D-Asp258C;Arg381D-Asp24B
    The tetrameric model shows a loss of 10633.2 Å2 of solvent accessible surface area upon complex formation using 24 salt bridges between positive Arg charged residues and negative Asp,Glu charged residues! Plus 4
    between negative C-terminus charged residues and positive N-terminus charged residues.
    chain A -COO-...+HNH2- chain B
    chain B -COO-...+HNH2- chain A
    chain C -COO-...+HNH2- chain D
    chain D -COO-...+HNH2- chain C
    Beta-strands from two pairs of adjacent wrapping domains form inter-subunit
    2 sheets of 4 anti-parallel beta-strands < cpk colors >.
    Joined each with four hydrogen bonds between peptide bonds >C=O...HN<.
    beta

    The Proximal and Distal Sides of the Heme.
    monomer of beef liver catalase
    The proximal (facing the core of the tetramer) and distal (facing the surface) sides of the heme are quite different environments. The proximal side is crowded with residues Val145, Pro 335, His 217, Arg353, Ala356, and Tyr357 < >. The H9=347-366 essential helix of the wrapping domain (discussed above) provides three of these key residues, Arg353, Ala356, and Tyr357 < >.
    The phenolic sidechain of Tyr357 acts as the 5th heme iron (Fe) ligand, the other 4 being nitrogens of the heme protoporphyrin ring (see a heme< > prosthetic group). Tyr357 is tightly juxtaposed to the Fe; the Fe-phenolic oxygen distance < mesure distance is 1.835 Å
    and push button >. As a probable consequence, the phenolic oxygen is deprotonated due to the electron withdrawing power of Fe+++. Arg353 may also promote ionization of Tyr357 by lowering the pKa of the tyrosine phenol (the two sidechains are only 3.5 Å apart) < >.

    Tyr357 and Arg353 likely interact with other residues, as well.[31,218,157] Pro335, a nonpolar residue, is positioned to impede the movement of Tyr357, and interaction between Arg353 and His217 may play a role in the catalytic mechanism < > avoiding protonation of phenolic oxygen.

    In contrast to the heme's proximal side, its distal side (facing the channel) is much less confined. It contains many residues, some of which are contributed by the beta-barrel < >.

    {Note: a group of residues across the beta-barrel function to bind the NADP moiety include Ser200, Arg202, Asp212, Lys236, His304, Val301, Trp302, Tyr214, His234 < >}and funnel-heme moiety (see above).

    Phe160 < > is stacked parallel to one of the heme pyrrole rings and Val73 < > makes hydrophobic contact with a different pyrrole ring. His74< > is also parallel to the heme, with bond angles normally allowed for only glycine residues. This conformation is stabilized by interaction with Arg111 and Thr114 < > and probably relates directly to enzymatic activity. 

    V. Proposed Mechanism of Catalase

    monomer of beef liver catalase
    The chemistry of catalase catalysis has not been precisely solved yet, but the following, which is
    similar to the mechanism of cytochrome c peroxidase, has been proposed.
    On heme< > prosthetic group at the catalytic center.
    The catalytic process is thought to occur in two stages:
    H    OOH+His74+Fe3+O--Tyr357-E=H+-His7+HOO->Fe3+O--Tyr357-E
    H+-His7+HOO->Fe3+O--Tyr357-E=HO-+H+-His7+O-Fe4+O--Tyr357-E
    HO-+H+-His7+O-Fe4+O--Tyr357-E=H2O+His7+O-Fe4+O--Tyr357-E
    .=>H    OOH+Asn147+O-Fe4+O--Tyr357-E =H+-Asn147+HOO->+O-Fe4+O--Tyr357-E
    H+-Asn147+HOO->+O-Fe4+O--Tyr357-E=HO-+H+-Asn147+O-O-Fe3+O--Tyr357-E
    HO-+H+-Asn147+O-O-Fe3+O--Tyr357-E=H2O+Asn147+O-O-Fe3+O--Tyr357-E
    H2O+O-O-Fe3+O--Tyr357-E=>H2O+O=O+Fe3+O--Tyr357-E
    where Fe-E represents the iron center of the heme attached to the rest of the enzyme (E).
    Peroxide, upon entering the heme cavity, is severely sterically hindered and must interact
    with His74 and Asn147< >. It is in this position that the first stage of catalysis takes place.
    Transfer of a proton from one oxygen of the peroxide to the other, via His74, elongates and p
    olarizes the O-O bond, which eventually breaks heterolytically to transferre proton through
    His74     and Asn147 for hydroxide ion making water molecule as a peroxide oxygen is
    coordinated to the iron center. This coordination displaces water and
    forms Fe(III)-O plus a heme radical. The radical quickly degrades in another one electron
    transfer to rid of the radical electron, leaving the heme ring unaltered. During the second stage,
    in a similar two electron transfer reaction, Fe(III)-O reacts with a second hydrogen peroxide
    to produce the original Fe(III)-E, another water, and a mole of molecular oxygen O2.

       The heme reactivity is enhanced by the phenolate ligand of Tyr357 in the 5th iron ligand position < >, which may aid in the oxidation of Fe(III) to Fe(IV) and the removal of an electron from the heme ring. The efficiency of catalase may, in part, be due to the interaction of His74 and Asn147 with heterolytical proton transferes to heterolytical hydroxide part. This mechanism is supported by experimental evidence indicating modification of His74 with 3-amino-1,2,4-triazole inhibits the enzyme by hindering substrate binding.
    Catalyst (CAT) is involved in to reaction transition state active complex formation and released after reaction free, unchenged redy for next catalytic reaction.
    The protein exists as a dumbbell-shaped tetramer of four identical subunits (220,000 to 350,000 kD). Each monomer contains a heme< > prosthetic group at the catalytic center. Catalase monomers from certain species (e.g. cow) also contain one tightly bound NADP < > per subunit. This NADP may serve to protect the enzyme from per-oxidation by its H2O2 substrate.
       Catalase was one of the first enzymes to be purified to homogeneity, and has been the subject of intense study. The enzyme is among the most efficient known, with rates approaching 200,000 catalytic events/second/subunit (near the diffusion-controlled limit). Catalase structure from many different species has been studied by X-ray diffraction. Although it is clear that all catalases share a general structure, some differ in the number and identity of domains. In this display, beef liver catalase will be used as a model for catalase structure. It will then be compared to catalase structure from a fungus, Penicillium vitale.

    Biocatalyst enzyme-CATALASE according
    Cambridge University professor Alana Fersht shows great catalytic activity:

    VenzCatpoint[E]point[H2O2] =3.6point107point[E]point[H2O2]
         Usual catalase concentration is [E]=10-8M and Venz=0,36point[H2O2] s-1 .
    Activation energy catalase Ea=29 J/mol is smaller as catalase abscence Ea=79000 J/mol and
    active collision fraction is 0,988 that means 98,8% of total collisions are active
    restricted by geometric factor of catalase A=0.1311 but
    geometric factor A=0.1311 is beter as catalase absence A=0.01 and
    velocity constant value is 0.1296 M-1s-1 ,
    prodcing the life resources  products O2 +  2H2O  +  Q.
    Due to absence of catalyst and low geometric factor A=0.01 M-1s-1
    make the Arrhenius velocity constant expression negligible small:
    Arrenius0=
    = 0.01    point1.419point10-14=1.419point10-16 M-1s-1
    kpoint[H2O2] =1.191point10-8 M-1s-1
    Presence of CATALASE performs reaction rate constant 30 million times greater :
    kCATpoint[H2O2] =0.36point[H2O2] .

    If divide the velocity constant for CATALASE with enzyme absence
    can see 30 million times increase:
    kCAT-k=30point106 times greater velocity constant
    to producing the life resources  products O2 +  2H2O  +  Q
          Peroxisomes: peroxisomes Membrane-bound, micro body organelles
    that Essential unsaturated fatty acid ω=6, ω=3 elongation to C20:4 with ethyl group
    -CH2-CH2- conversion to cis double bond H>C=C<H in peroxisomes
    is spontaneous.
          CATALASE in complex reaction sequence favors stabile unsaturated
    fatty acid product efficiency 100%:
    Keq=108,43=
    because erasing peroxide H2O2 to zero [H2O2]=0 mol/liter and
    process velocity limits only dehydrogenase enzyme. It favors velocity
    of peroxide 2H-O-O-H conversion in to biological goods
    oxygen in water and heat
    O2aqua + 2H2O + Q thirty million times 30point106.

    VI. Comparison of Beef Liver and Penicillium vitale Catalase Structures

    670 residues of Penicillium vitale catalase (PVC) have been built into a 2 Å resolution
    electron density map and the backbone of this structure is compared to that of beef liver
    catalase (BLC) at left. The two proteins have many structural similarities, unsurprising given
    that they share the same catalytic function. Both catalases, as well as other catalases, bind
    heme groups in analogous binding pockets at similar positions. Both have a tyrosine as
    a proximal iron ligand, and a distal region containing a histidine and an asparagine necessary
    for activity (see above). However, there are differences in the two structures. PVC has
    an additional flavodoxin-like domain at its carboxy terminus < >. BLC contains
    a bound NADP < > molecule plus an extra 13 residues at the amino-terminus < >
    that are absent in PVC. heme< > prosthetic group The NADP molecule in BLC is bound
    in the region occupied by the extra flavodoxin-like domain in PVC. The presence of
    the flavodoxin-like domain in PVC may indicate the binding of a nucleotide.

    The three dimensional structure of proteins is often more conserved than their amino acid sequences. Comparison of three dimensional structures can reveal common origins and functions of evolutionarily distant proteins and can provide information on functionally important, conserved structural features. The above comparison shows that neither the flavodoxin-like domain of PVC nor the NADP of BLC are absolutely required for catalase function, but that the presence of catalase-bound nucleotides is important, presumably to protect the enzyme from oxidative damage. The structural similarities point to bly-conserved mechanisms for peroxide detoxification, since mammalian and fungal catalases diverged from a common ancestor at least as early as the first eukaryotes. 

    VII. Structure of a Human erytrocyte Catalase Monomer

    Primary structure 1̊ . monomer is shown at left.
         The beef liver catalase monomer (shown at left) consists of a 506 amino acid
    polypeptide chain plus one heme group and one NADH molecule.  Secondary structure.
    Only about 60% of catalase structure is composed of regular secondary structural
    motifs < >.
         13 Alpha-helices H1,H2,H3,H4,H5,H6,H7,H8,H9,H10,H11,H12,H13
        account for 26% of its structure and beta-barrel structure 8 anti-parallel strands
         for 12%. Irregular structure includes a predominance of extended single strands and
    loops that play a major role in the assembly of the tetramer.

    Funtional catalase is
    a tetramer of four identical holo subunits. A model of a beef liver catalase
    tetramer is shown at left.

    VIII. The Heme Group and its Environment

         The Funnel-Channel to the Iron in Heme Group.
    As noted above, the heme groups are deeply embedded in each subunit of a modeled
    tetramer. However, as can be seen in a monomer , each heme is exposed through
    a funnel-shaped channel
    Ser114,Glu119,Ser122,Arg127,Asp128,Gln168,Lys169,Lys177,Glu248,His255,
    Glu256,Glu453,Glu454,Asn462,His466,Val116,Ala117,Pro129,Phe153,Phe154,
    Phe164,Ile165,Pro179,Val182,Leu199,Phe200
    25 Å long and 20-15 Å wide < >. The channel is lined with at the entrance
    14 hydrophilic residues < >
    Ser114,Glu119,Ser122,Arg127,Asp128,Gln168,Lys169,Lys177,Glu248,Glu256,
    Glu453,Glu454,Gln461,His466
    and with 11 hydrophobic residues
    Val116,Ala117,Pro129,Phe153,Phe154,Phe164,Ile165,Pro179,Val182,Leu199,Phe200
    as the channel descends, constricting, toward the heme.
         The Heme Cavity.
         The heme group is located between the internal wall of the beta-barrel and several
    helices H3=156-168 and H9=347-366 < >.
         The heme pocket is mainly 31 AA hydrophobic < >
    Val73,Val74,Ala76,Val116,Ala117,Pro129,Gly131,Val146,Gly147,Phe153,Phe154,
    Ile155,Ala158,Leu159,Leu160,Phe161,Pro162,Phe164,Ile165,Phe198,Leu199,
    Phe200,Leu299,Ala333,Phe334,Pro336,Met350,Leu351,Gly353,Phe356,Ala357,
    Pro359,Ala435
    with the exception of a few 8, Arg354 and Tyr358residues
    thought to be involved in binding the heme prosthetic group or
    in the catalysis H+ desorbtion His75,Asn148 at peroxide dismutation on iron and
    binding heme propionic acid residues Arg72,Arg112,Glu330,Arg365 in pocket of
    CATALASE.
         Peroxide, upon entering the heme cavity, is severely sterically hindered and must interact
    with His75 and Asn148< >. It is in this position that the first stage of catalysis takes place.
    Transfer of a proton from one oxygen of the peroxide to the other, via His75, elongates and
    polarizes the O-O bond, which eventually breaks heterolytically to transferre proton through
    His75     and Asn148 for hydroxide ion making water molecule as a peroxide oxygen is
    coordinated to the iron center. This coordination displaces water and forms
    Fe    (III)-O plus a heme radical. The radical quickly degrades in another one electron transfer
    to rid of the radical electron, leaving the heme ring unaltered. During the second stage,
    in a similar two electron transfer reaction, Fe(III)-O reacts with a second hydrogen peroxide
    to produce the original Fe(III)-E, another water, and a mole of molecular oxygen O2.

    Tertiary structure 3̊ monomer .
    Each monomer has four domains < >.
         The first domain < > is made up of the amino-terminal 75 residues. These form an arm with two alpha-helices H1,H2 and a large loop extending from the globular subunit < >.
         The second and largest domain contains the heme moiety < >. It is composed of residues 76 to 320 and may be classified as an alpha+beta-barrel 8 anti-parallel strands type domain . It includes a beta-barrel, fivel H3,H4,H5,H6,H7 helical segments of three to four turns each, and various loops < > . The beta-barrel 8 anti-parallel strands < > consists of two four stranded anti-parallel beta-sheets that twist to form a closed cylindrical surface.
         The third domain consists of residues 321-436 the largest of the essential helix H9 ASP347-GLY366 contains the heme phenolic ligand, Tyr358 < > and is referred to as the wrapping domain . It lacks discernable secondary structure except for two helices H8 ASN323-GLU329,H9 ASP347-GLY366 < >, .
         The carboxy-terminal < > portion of the molecule contains residues 437 to 506 and is folded into a four-helical domain H10,H11,H12,H13 < > similar to the human globin folds. Along with three alpha-helices from the heme-containing domain, these helices form one surface of the enzyme < >. 

    References
    1. . J Inorg Biochem. 2017 ;167:60-67. 1QQW
    2. Journal of Molecular Biology Volume 296, Issue 1, 2000, Pages 295–309 8CAT,1DGB
    3. Acta Crystallogr D Biol Crystallogr. 2001 ;57(Pt 1):1-7.
    4. Proteins: Structure, Function, and Bioinformatics Volume 50, Issue 2, pages 261-271, 2003
    5. FEBS Lett. 1992;312(2-3):127-31.
    6. Acta Crystallogr D Biol Crystallogr. 2002;58(Pt 12):1972-82.
    7. Journal of Molecular Biology Volume 249, Issue 5, 1995, Pages 933–954
    8. Acta Crystallogr D Biol Crystallogr. 2003;59(Pt 12):2163-8.
    9.Proteins. 2003;50(2):261-71.
    10. Biochemistry. 2001 ;40(45):13734-43.
    11. Biochemistry. 2004 ;43(11):3089-103.
    12. Acta Crystallogr D Biol Crystallogr. 2004;60(Pt 8):1374-80.
    13. J Am Chem Soc. 2007 ;129(14):4193-205.
    14. Biochemistry. 2007 ;46(1):11-22.
    15. Acta Crystallogr D Biol Crystallogr. 2007;63(Pt 2):135-48.
    16. Biochim Biophys Acta. 2009;1790(8):741-53.
    17. Biochemistry. 2011;50(21):4491-503.
    18. Eventoff, William. (1976) Crystalline Bovine Liver Catalase. J. Mol. Biol. 103, 799-801.
    19. Fita, et al. (1985) The active center of catalase. J. Mol. Biol. 185, 21-37.
    20. Fita, et al. (1986) The refined structure of beef liver catalase. Acta Cryst. B42, 497-515.
    21. Jouve, et al. (1991) Crystallization and crystal packing of Proteus mirabilis PR catalase. J. Mol. Biol. 221, 1075-77.
    22. Mathur, et al. (1981) Structure of beef liver catalase. J. Mol. Biol. 152, 465-99.
    23. Melik-Adamyan, et al. (1986) Comparison of beef liver and Penicillium vitale catalases. J. Mol. Biol. 188, 63-72.
    24. Reid, et al. (1981) Structure and heme environment of beef liver catalase at 2.5 A resolution. Proc. Natl. Acad. Sci. USA 78, 4767-71.
    25. Vainshtein, et al. (1981) Three-dimensional structure of the enzyme catalase. Nature 293, 411-12.
    26. Vainshtein, et al. (1986) Three-dimensional structure of catalase from Penicillium vitale at 2.0 A resolution. J. Mol. Biol. 188, 49-61.    Back to the Index...