Catalase Structure Tutorial Text

Catalase: H₂O₂: H₂O₂ Oxidoreductase

Elizabeth M. Boon '97, Aaron Downs '00, and David Marcey

I. Introduction

Catalase (EC 1.11.1.6), present in the peroxisomes of nearly all aerobic cells, serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without the production of free radicals. The mechanism of catalysis is not fully elucidated, but the overall reaction is as follows: 2 H₂O₂ --> 2 H₂0 + O₂ 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 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.

II. Structure of a Bovine Catalase Monomer

Primary structure.
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 <>. Alpha-helices account for 26% of its structure and beta-structure for 12%. Irregular structure includes a predominance of extended single stands and loops that play a major role in the assembly of the tetramer.

Tertiary structure.
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 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 type domain. It includes a beta-barrel, several helical segments of three to four turns each, and various loops <> . The beta-barrel <> 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 and is referred to as the wrapping domain. It lacks discernable secondary structure except for two helices <>, the largest of which (the essential helix) contains the heme phenolic ligand, Tyr357 <>.

The carboxy-terminal <> portion of the molecule contains residues 437 to 506 and is folded into a four-helical domain <> 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 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 ~23 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. Beta-strands from two pairs of adjacent wrapping domains form inter-subunit anti-parallel beta-sheets <>. There are numerous salt bridges at the interfaces between monomers, mostly involving arginine, asparagine, and glutamic acid partners <>. The tetrameric model shown shows a loss of 10633.2 Å² of solvent accessible surface area upon complex formation!

IV. The Heme Group and its Environment

The Channel to the 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 30 Å long and 15 Å wide <>. The channel is lined with hydrophilic residues at the entrance and with hydrophobic residues as the channel descends, constricting, toward the heme <>.

The Heme Cavity.
The heme groupis located between the internal wall of the beta-barrel and several helices <>. The heme pocket is hydrophobic with the exception of a few residues thought to be involved in binding the prosthetic group or in the catalysis of of peroxide dismutation (see below).

The Proximal and Distal Sides of the Heme.
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, His ²¹⁷, Pro ³³⁵, Arg³⁵³, Ala³⁵⁶, and Tyr³⁵⁷ <>. The 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 a the 5th heme iron (Fe) ligand, the other 4 being nitrogens of the heme protoporphyrin ring (see Introduction). Tyr³⁵⁷ is tightly juxtaposed to the Fe; the Fe-phenolic oxygen distance is 1.9 Å <>. 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. 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 <>.

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 and include Ser²⁰⁰, Arg²⁰², Asp²¹², Lys²³⁶, His³⁰⁴, Val³⁰¹, Trp³⁰², Tyr²¹⁴, His²³⁴ <>}.

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

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. The catalytic process is thought to occur in two stages: H₂O₂ + Fe(III)-E --> H₂O +O=Fe(IV)-E (1)
H₂O₂ + O=Fe(IV)-E --> H₂O + Fe(III)-E (2)

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 polarizes the O-O bond, which eventually breaks heterolytically as a peroxide oxygen is coordinated to the iron center. This coordination displaces water and forms Fe(IV)=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(IV)=O reacts with a second hydrogen peroxide to produce the original Fe(III)-E, another water, and a mole of molecular oxygen.

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 reaction intermediates. 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.

**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. 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 strongly-conserved mechanisms for peroxide detoxification, since mammalian and fungal catalases diverged from a common ancestor at least as early as the first eukaryotes.

Select Catalase WWW Sites

There is much information about catalase on the world wide web. Here are a few good starting points:

Mary Maj's research at Brock University.

Natalia Snarskaya's site at Moscow State University.

American Cancer Society Publication about catalase inhibition.

New binding site in catalase?

Test for presence of catalase in oxidant resistant bacteria.

Catalases.

SCOP: Catalase C-teminal Domain from Penicillium vitale.

References
Eventoff, William. (1976) Crystalline Bovine Liver Catalase. J. Mol. Biol. 103, 799-801.

Fita, et al. (1985) The active center of catalase. J. Mol. Biol. 185, 21-37.

Fita, et al. (1986) The refined structure of beef liver catalase. Acta Cryst. B42, 497-515.

Jouve, et al. (1991) Crystallization and crystal packing of Proteus mirabilis PR catalase. J. Mol. Biol. 221, 1075-77.

Mathur, et al. (1981) Structure of beef liver catalase. J. Mol. Biol. 152, 465-99.

Melik-Adamyan, et al. (1986) Comparison of beef liver and Penicillium vitale catalases. J. Mol. Biol. 188, 63-72.

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.

Vainshtein, et al. (1981) Three-dimensional structure of the enzyme catalase. Nature 293, 411-12.

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...

Catalase: H2O2: H2O2 Oxidoreductase