Horseradish peroxidase mutants that autocatalytically modify their prosthetic heme group: insights into mammalian peroxidase heme-protein covalent bonds

The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu(37), Phe(41), Gly(69), and Ser(73). The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H(2)O(2). As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.     

X-ray crystal structure of canine myeloperoxidase at 3 A resolution.

The three-dimensional structure of the enzyme myeloperoxidase has been determined by X-ray crystallography to 3 A resolution. Two heavy atom derivatives were used to phase an initial multiple isomorphous replacement map that was subsequently improved by solvent flattening and non-crystallographic symmetry averaging. Crystallographic refinement gave a final model with an R-factor of 0.257. The root-mean-square deviations from ideality for bond lengths and angles were 0.011 A and 3.8 degrees. Two, apparently identical, halves of the molecule are related by local dyad and covalently linked by a single disulfide bridge. Each half-molecule consists of two polypeptide chains of 108 and 466 amino acid residues, a heme prosthetic group, a bound calcium ion and at least three sites of asparagine-linked glycosylation. There are six additional intra-chain disulfide bonds, five in the large polypeptide and one in the small. A central core region that includes the heme binding site is composed of five alpha-helices. Regions of the larger polypeptide surrounding this core are organized into locally folded domains in which the secondary structure is predominantly alpha-helical with very little organized beta-sheet. A proximal ligand to the heme iron atom has been identified as histidine 336, which is in turn hydrogen-bonded to asparagine 421. On the distal side of the heme, histidine 95 and arginine 239 are likely to participate directly in the catalytic mechanism, in a manner analogous to the distal histidine and arginine of the non-homologous enzyme cytochrome c peroxidase. The site of the covalent linkage to the heme has been tentatively identified as glutamate 242, although the chemical nature of the link remains uncertain. The calcium binding site has been located in a loop comprising residues 168 to 174 together with aspartate 96. Myeloperoxidase is a member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and lactoperoxidase. The heme environment, defined by our model for myeloperoxidase, appears to be highly conserved in these four mammalian peroxidases. Furthermore, the conservation of all 12 cysteine residues involved in the six intra-chain disulfide bonds and the calcium binding loop suggests that the three-dimensional structures of members of this gene family are likely to be quite similar.     

Two alternative substrate paths for compound I formation and reduction in catalase-peroxidase KatG from Burkholderia pseudomallei

Five residues in the multifunctional catalase-peroxidase KatG of Burkholderia pesudomallei are essential for catalase, but not peroxidase, activity. Asp141 is the only one of these catalase-specific residues not related with the covalent adduct found in KatGs that when replaced with a nonacidic residue reduces catalase activity to 5% of native levels. Replacing the nearby catalytic residue Arg108 causes a reduction in catalase activity to 35% of native levels, whereas a variant with both Asp141 and Arg108 replaced exhibits near normal catalase activity (82% of native), suggesting a synergism in the roles of the two residues in support of catalase activity in the enzyme. Among the Asp141 variants, D141E is unique in retaining normal catalase activity but with modified kinetics, suggesting more favorable compound I formation and less favorable compound I reduction. The crystal structure of the D141E variant has been determined at 1.8-A resolution, revealing that the carboxylate of Glu141 is moved only slightly compared with Asp141, but retains its hydrogen bond interaction with the main chain nitrogen of Ile237. In contrast, the low temperature ferric Electron Paramagnetic Resonance spectra of the D141A, R108A, and R108A/D141A variants are consistent with modifications of the water matrix and/or the relative positioning of the distal residue side chains. Such changes explain the reduction in catalase activity in all but the double variant R108A/D141A. Two pathways of hydrogen bonded solvent lead from the entrance channel into the heme active site, one running between Asp141 and Arg108 and the second between Asp141 and the main chain atoms of residues 237-239. It is proposed that binding of substrate H(2)O(2) to Asp141 and Arg108 controls H(2)O(2) access to the heme active site, thereby modulating the catalase reaction.     

Unusual Cys-Tyr covalent bond in a large catalase

Catalase-1, one of four catalase activities of Neurospora crassa, is associated with non-growing cells and accumulates in asexual spores. It is a large, tetrameric, highly efficient, and durable enzyme that is active even at molar concentrations of hydrogen peroxide. Catalase-1 is oxidized at the heme by singlet oxygen without significant effects on enzyme activity. Here we present the crystal structure of catalase-1 at 1.75A resolution. Compared to structures of other catalases of the large class, the main differences were found at the carboxy-terminal domain. The heme group is rotated 180 degrees around the alpha-gamma-meso carbon axis with respect to clade 3 small catalases. There is no co-ordination bond of the ferric ion at the heme distal side in catalase-1. The catalase-1 structure exhibited partial oxidation of heme b to heme d. Singlet oxygen, produced catalytically or by photosensitization, may hydroxylate C5 and C6 of pyrrole ring III with a subsequent formation of a gamma-spirolactone in C6. The modification site in catalases depends on the way dioxygen exits the protein: mainly through the central channel or the main channel in large and small catalases, respectively. The catalase-1 structure revealed an unusual covalent bond between a cysteine sulphur atom and the essential tyrosine residue of the proximal side of the active site. A peptide with the predicted theoretical mass of the two bound tryptic peptides was detected by mass spectrometry. A mechanism for the Cys-Tyr covalent bond formation is proposed. The tyrosine bound to the cysteine residue would be less prone to donate electrons to compound I to form compound II, explaining catalase-1 resistance to substrate inhibition and inactivation. An apparent constriction of the main channel at Ser198 lead us to propose a gate that opens the narrow part of the channel when there is sufficient hydrogen peroxide in the small cavity before the gate. This mechanism would explain the increase in catalytic velocity as the hydrogen peroxide concentration rises.     

Catalase-peroxidase active site restructuring by a distant and "inactive" domain

Catalase-peroxidases are composed of two peroxidase-like domains. The N-terminal domain contains the heme-dependent, bifunctional active site. The C-terminal domain does not bind heme, has no catalytic activity, and is separated from the active site by >30 A. Nevertheless, without the C-terminal domain, the N-terminal domain exhibits neither catalase nor peroxidase activity due to the apparent coordination of the distal histidine to the heme iron. Here we report the ability of the separately expressed and isolated C-terminal domain (KatG(C)) to restructure the N-terminal domain (KatG(N)) to its bifunctional conformation. Addition of equimolar KatG(C) to KatG(N) decreased the hexacoordinate low-spin heme complex and increased the high-spin species (pentacoordinate and hexacoordinate). EPR spectra of the domain mixture showed a distribution between high-spin species nearly identical to that of wild-type KatG. The CD spectrum for the 1:1 physical mixture of the domains was identical to an arithmetic composite of individual spectra for KatG(N) and KatG(C). Both physical and arithmetic mixtures were nearly identical to the spectrum for wild-type KatG, suggesting that major shifts in secondary structure did not accompany active site reconfiguration. With the shift in heme environment, the parallel return of catalase and peroxidase activity was observed. Inclusion of bovine serum albumin instead of KatG(C) produced no activity, indicating that specific interdomain interactions were required to reestablish the bifunctional active site. Apparent constants for reactivation (k(react) approximately 4 x 10(-3) min(-1)) indicate that a slow process like movement of established structural elements may precede the restructuring of the heme environment and return of catalytic activity.     

Unprecedented access of phenolic substrates to the heme active site of a catalase: Substrate binding and peroxidase‐like reactivity of Bacillus pumilus catalase monitored by X‐ray crystallography and EPR spectroscopy

Heme‐containing catalases and catalase‐peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase‐peroxidase led us to investigate the enzyme for comparison with other catalase‐peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (k_cat = 339,000 s^?1). In addition, the enzyme supported a much slower (k_cat = 20 s^?1) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2‐chlorophenol were identified in crystal structures at 1.65–1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low‐spin conversion of the Fe^III high‐spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. Proteins 2015; 83:853–866. © 2015 Wiley Periodicals, Inc.     

The tuberculosis prodrug isoniazid bound to activating peroxidases

Isoniazid (INH, isonicotinic acid hydrazine) is one of only two therapeutic agents effective in treating tuberculosis. This prodrug is activated by the heme enzyme catalase peroxidase (KatG) endogenous to Mycobacterium tuberculosis but the mechanism of activation is poorly understood, in part because the binding interaction has not been properly established. The class I peroxidases ascorbate peroxidase (APX) and cytochrome c peroxidase (CcP) have active site structures very similar to KatG and are also capable of activating isoniazid. We report here the first crystal structures of complexes of isoniazid bound to APX and CcP. These are the first structures of isoniazid bound to any activating enzymes. The structures show that isoniazid binds close to the delta-heme edge in both APX and CcP, although the precise binding orientation varies slightly in the two cases. A second binding site for INH is found in APX at the gamma-heme edge close to the established ascorbate binding site, indicating that the gamma-heme edge can also support the binding of aromatic substrates. We also show that in an active site mutant of soybean APX (W41A) INH can bind directly to the heme iron to become an inhibitor and in a different mode when the distal histidine is replaced by alanine (H42A). These structures provide the first unambiguous evidence for the location of the isoniazid binding site in the class I peroxidases and provide rationalization of isoniazid resistance in naturally occurring KatG mutant strains of M. tuberculosis.     

Catalase-peroxidase KatG of Burkholderia pseudomallei at 1.7A resolution

The catalase-peroxidase encoded by katG of Burkholderia pseudomallei (BpKatG) is 65% identical with KatG of Mycobacterium tuberculosis, the enzyme responsible for the activation of isoniazid as an antibiotic. The structure of a complex of BpKatG with an unidentified ligand, has been solved and refined at 1.7A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are 15.3% and 18.6%, respectively. The crystallized enzyme is a dimer with one modified heme group and one metal ion, likely sodium, per subunit. The modification on the heme group involves the covalent addition of two or three atoms, likely a perhydroxy group, to the secondary carbon atom of the vinyl group on ring I. The added group can form hydrogen bonds with two water molecules that are also in contact with the active-site residues Trp111 and His112, suggesting that the modification may have a catalytic role. The heme modification is in close proximity to an unusual covalent adduct among the side-chains of Trp111, Tyr238 and Met264. In addition, Trp111 appears to be oxidized on C(delta1) of the indole ring. The main channel, providing access of substrate hydrogen peroxide to the heme, contains a region of unassigned electron density consistent with the binding of a pyridine nucleotide-like molecule. An interior cavity, containing the sodium ion and an additional region of unassigned density, is evident adjacent to the adduct and is accessible to the outside through a second funnel-shaped channel. A large cleft in the side of the subunit is evident and may be a potential substrate-binding site with a clear pathway for electron transfer to the active-site heme group through the adduct.     

Crystal structure of Nitrosomonas europaea cytochrome c peroxidase and the structural basis for ligand switching in bacterial di-heme peroxidases.

The crystal structure of the fully oxidized di-heme peroxidase from Nitrosomonas europaea has been solved to a resolution of 1.80 A and compared to the closely related enzyme from Pseudomonas aeruginosa. Both enzymes catalyze the peroxide-dependent oxidation of a protein electron donor such as cytochrome c. Electrons enter the enzyme through the high-potential heme followed by electron transfer to the low-potential heme, the site of peroxide activation. Both enzymes form homodimers, each of which folds into two distinct heme domains. Each heme is held in place by thioether bonds between the heme vinyl groups and Cys residues. The high-potential heme in both enzymes has Met and His as axial heme ligands. In the Pseudomonas enzyme, the low-potential heme has two His residues as axial heme ligands [Fulop et al. (1995) Structure 3, 1225-1233]. Since the site of reaction with peroxide is the low-potential heme, then one His ligand must first dissociate. In sharp contrast, the low-potential heme in the Nitrosomonas enzyme already is in the "activated" state with only one His ligand and an open distal axial ligation position available for reaction with peroxide. A comparison between the two enzymes illustrates the range of conformational changes required to activate the Pseudomonas enzyme. This change involves a large motion of a loop containing the dissociable His ligand from the heme pocket to the molecular surface where it forms part of the dimer interface. Since the Nitrosomonas enzyme is in the active state, the structure provides some insights on residues involved in peroxide activation. Most importantly, a Glu residue situated near the peroxide binding site could possibly serve as an acid-base catalytic group required for cleavage of the peroxide O--O bond.     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind     

Substitution of strictly conserved Y111 in catalase-peroxidases: Impact of remote interdomain contacts on active site structure and catalytic performance

Catalase-peroxidase function is strictly dependent on a gene-duplicated C-terminal domain. This domain no longer has a functioning active site, but from 25 to 30 Å away it is essential for preventing the coordination of an active site base (His106) to the heme. The mechanisms by which this distant structure supports active site function have not yet been elucidated. Tyr111 is a strictly conserved member of an interdomain H-bonding network that supports the loop connecting the N-terminal B (bearing His106) and C helices. Spectroscopic evaluation of the Tyr111Ala variant of KatG showed a substantial increase in hexa-coordinate low-spin heme, giving it the appearance of a transition between the wild type (primarily high-spin) and the N-terminal domain alone (pure low-spin). Concomitant with the spectral changes was decreased activity compared to the wild type enzyme, suggesting that Tyr111 does have a role in preventing His106 coordination. Substitution of Tyr111 diminishes catalase activity more substantially than peroxidase activity. Such an effect cannot be explained by His106 coordination alone, suggesting that these interdomain interactions may help tune the catalase-peroxidase active site for bifunctionality.     

Crystal structure of manganese catalase from Lactobacillus plantarum

BACKGROUND: Catalases are important antioxidant metalloenzymes that catalyze disproportionation of hydrogen peroxide, forming dioxygen and water. Two families of catalases are known, one having a heme cofactor, and the other, a structurally distinct family containing nonheme manganese. We have solved the structure of the mesophilic manganese catalase from Lactobacillus plantarum and its azide-inhibited complex. RESULTS: The crystal structure of the native enzyme has been solved at 1.8 A resolution by molecular replacement, and the azide complex of the native protein has been solved at 1.4 A resolution. The hexameric structure of the holoenzyme is stabilized by extensive intersubunit contacts, including a beta zipper and a structural calcium ion crosslinking neighboring subunits. Each subunit contains a dimanganese active site, accessed by a single substrate channel lined by charged residues. The manganese ions are linked by a mu1,3-bridging glutamate carboxylate and two mu-bridging solvent oxygens that electronically couple the metal centers. The active site region includes two residues (Arg147 and Glu178) that appear to be unique to the Lactobacillus plantarum catalase. CONCLUSIONS: A comparison of L. plantarum and T. thermophilus catalase structures reveals the existence of two distinct structural classes, differing in monomer design and the organization of their active sites, within the manganese catalase family. These differences have important implications for catalysis and may reflect distinct biological functions for the two enzymes, with the L. plantarum enzyme serving as a catalase, while the T. thermophilus enzyme may function as a catalase/peroxidase.     

Structure of an electron transfer complex. I. Covalent cross-linking of cytochrome c peroxidase and cytochrome c

Cytochrome c peroxidase and cytochrome c form a noncovalent electron transfer complex in the course of the peroxidase-catalyzed reduction of H2O2. The two hemoproteins were cross-linked in 40% yield to a covalent 1:1 complex with the aid of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The covalent complex was found to be a valid model of the noncovalent electron transfer complex for the following reasons. The covalent complex had only 5% residual peroxidase activity toward exogeneous ferrocytochrome c indicating that the cross-linked cytochrome c covers the electron-accepting site of cytochrome c peroxidase. The residual peroxidase activity was almost independent of ionic strength indicating that the electron-accepting site is much less accessible even when ionic bonds between the two cross-linked hemoproteins are severed. The rate of reduction of heme c by ascorbate is 15 times slower in the covalent complex than in free cytochrome c and is independent of ionic strength. Although the covalent complex may not have been entirely pure with respect to the number and location of the cross-links, two major cross-links could be localized to within a few residues. One is from Lys 13 of cytochrome c to an acidic residue in positions 32, 33, 34, 35, or 37 of cytochrome c peroxidase, the other from Lys 86 of cytochrome c to a carboxyl group in the same cluster of acidic residues. The result stresses the importance of a peculiar stretch of acidic residues of cytochrome c peroxidase and of Lys 13 and 86 of cytochrome c.     

Spatial organization of catalase proteins

The three-dimensional structure of the heme-containing fungal catalase fromPenicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2.0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct “X-ray” primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains aβ-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near theβ-barrel. A comparison of the structure of catalase fromPenicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteriaThermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2₁3, a = 133.4 Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme inThermus thermophilus catalase, the active site is between the four helices and contains two manganese ions.     

Substrate flow in catalases deduced from the crystal structures of active site variants of HPII from Escherichia coli.

The active site of heme catalases is buried deep inside a structurally highly conserved homotetramer. Channels leading to the active site have been identified as potential routes for substrate flow and product release, although evidence in support of this model is limited. To investigate further the role of protein structure and molecular channels in catalysis, the crystal structures of four active site variants of catalase HPII from Escherichia coli (His128Ala, His128Asn, Asn201Ala, and Asn201His) have been determined at approximately 2.0-A resolution. The solvent organization shows major rearrangements with respect to native HPII, not only in the vicinity of the replaced residues but also in the main molecular channel leading to the heme distal pocket. In the two inactive His128 variants, continuous chains of hydrogen bonded water molecules extend from the molecular surface to the heme distal pocket filling the main channel. The differences in continuity of solvent molecules between the native and variant structures illustrate how sensitive the solvent matrix is to subtle changes in structure. It is hypothesized that the slightly larger H(2)O(2) passing through the channel of the native enzyme will promote the formation of a continuous chain of solvent and peroxide. The structure of the His128Asn variant complexed with hydrogen peroxide has also been determined at 2.3-A resolution, revealing the existence of hydrogen peroxide binding sites both in the heme distal pocket and in the main channel. Unexpectedly, the largest changes in protein structure resulting from peroxide binding are clustered on the heme proximal side and mainly involve residues in only two subunits, leading to a departure from the 222-point group symmetry of the native enzyme. An active role for channels in the selective flow of substrates through the catalase molecule is proposed as an integral feature of the catalytic mechanism. The Asn201His variant of HPII was found to contain unoxidized heme b in combination with the proximal side His-Tyr bond suggesting that the mechanistic pathways of the two reactions can be uncoupled.     

Studies on phenotypic variation between Chinese hamster Kupffer cell lines : II. Correlation relationships and coordinate control of enzyme activities

This study examines somatic cell hybrids between parental cells of identical origin which exhibited quantitative differences in enzyme activities. Nine enzymes in cultured Chinese hamster Kupffer cell hybrids were studied. The parental Kupffer cell clones of identical genetic origin differed several-fold in the specific activities of catalase, arginase, microsomal heme oxygenase, peroxidase, β-glucuronidase, alcohol dehydrogenase, lactate dehydrogenase, isocitrate dehydrogenase and glucose-6-phosphate dehydrogenase. A selective system based on fusion of ouabain and vinblastine resistant clones of the parental cells was used to isolate hybrids. In hybrid clones, the specific activities of catalase, microsomal heme oxygenase, peroxidase and the dehydrogenases were expressed at the level characteristic of the parental clone with high activities of these enzymes. This result implied interaction between the parental genomes and suggested mechanisms regulating the quantitative levels of several enzyme activities. In contrast, the specific activities of arginase and β-glucuronidase in hybrid clones were intermediate between those possessed by the parental cells and indicated that for each parental genome in the hybrid there was autonomous regulation of the levels of these two enzyme activities.     

Vital roles of an interhelical insertion in catalase-peroxidase bifunctionality

The loop connecting the F and G helices of catalase-peroxidases contains a approximately 35 amino acid structure (the FG insertion) that is absent from monofunctional peroxidases. These two groups of enzymes share highly similar active sites, yet the monofunctional peroxidases lack appreciable catalase activity. Thus, the FG insertion may serve a role in catalase-peroxidase bifunctionality, despite its peripheral location relative to the active site. We produced a variant of Escherichia coli catalase-peroxidase (KatG) lacking its FG insertion (KatG(DeltaFG)). Absorption spectra indicated the heme environment of KatG(DeltaFG) was highly similar to wild-type KatG, but the variant retained only 0.2% catalase activity. In contrast, the deletion reduced peroxidase activity by only 50%. Kinetic parameters for the peroxidase and residual catalase activities of KatG(DeltaFG) as well as pH dependence studies suggested that the FG insertion supports hydrogen-bonded networks critical for reactions involving H2O2. The structure also appears to regulate access of electron donors to the active site.     

Structural design of the active site for covalent attachment of the heme to the protein matrix: studies on a thermostable cytochrome P450

The molecular basis of the post-translational modification involving covalent attachment of the heme with a glutamic acid observed in some enzymes of the CYP4 family of heme monooxygenases has been investigated using site-directed mutagenesis of CYP175A1 from Thermus thermophilus. Earlier studies of CYP4 as well as the G248E mutant of CYP101A1 showed covalent linkage of the heme to a conserved glutamic acid of helix I. We have introduced Glu/Asp at the Leu80 position in the β-turn of CYP175A1, on the basis of molecular modeling studies, to assess whether formation of such a covalent linkage is limited only to helix I or whether such modification may also take place with the residue that is spatially located at a position appropriate for activation by the heme peroxidase reaction. Tandem mass spectrometry analyses of the tryptic digest of the wild type and mutants of CYP175A1 were conducted to identify any heme-bound peptide. Tryptic digestion of the L80E mutant of CYP175A1 preincubated with H(2)O(2) showed formation of GLE(-heme)TDWGESWKEARK supporting covalent linkage of Glu80 with the heme in the mutant enzyme. No such heme-bound peptides were found if the sample was not preincubated in H(2)O(2), indicating no activation of the Glu by the heme peroxidase reaction, as proposed earlier. The wild type or L80D mutant of the enzyme did not give any heme-bound peptide. Thus, the results support the idea that covalent attachment of the heme to an amino acid in the protein matrix depends on the structural design of the active site.     

Structure of catalase HPII from Escherichia coli at 1.9 A resolution

Catalase HPII from Escherichia coli, a homotetramer of subunits with 753 residues, is the largest known catalase. The structure of native HPII has been refined at 1.9 A resolution using X-ray synchrotron data collected from crystals flash-cooled with liquid nitrogen. The crystallographic agreement factors R and R(free) are respectively 16.6% and 21.0%. The asymmetric unit of the crystal contains a whole molecule that shows accurate 222-point group symmetry. The structure of the central part of the HPII subunit gives a root mean square deviation of 1.5 A for 477 equivalencies with beef liver catalase. Most of the additional 276 residues of HPII are located in either an extended N-terminal arm or in a C-terminal domain organized with a flavodoxin-like topology. A small number of mostly hydrophilic interactions stabilize the relative orientation between the C-terminal domain and the core of the enzyme. The heme component of HPII is a cis-hydroxychlorin gamma-spirolactone in an orientation that is flipped 180 degrees with respect to the orientation of the heme found in beef liver catalase. The proximal ligand of the heme is Tyr415 which is joined by a covalent bond between its Cbeta atom and the Ndelta atom of His392. Over 2,700 well-defined solvent molecules have been identified filling a complex network of cavities and channels formed inside the molecule. Two channels lead close to the distal side heme pocket of each subunit suggesting separate inlet and exhaust functions. The longest channel, that begins in an adjacent subunit, is over 50 A in length, and the second channel is about 30 A in length. A third channel reaching the heme proximal side may provide access for the substrate needed to catalyze the heme modification and His-Tyr bond formation. HPII does not bind NADPH and the equivalent region to the NADPH binding pocket of bovine catalase, partially occluded in HPII by residues 585-590, corresponds to the entrance to the second channel. The heme distal pocket contains two solvent molecules, and the one closer to the iron atom appears to exhibit high mobility or low occupancy compatible with weak coordination.     

The active center of catalase

The refined structure of beef liver catalase (I. Fita, A. M. Silva, M. R. N. Murthy & M. G. Rossmann, unpublished results) is here examined with regard to possible catalytic mechanisms. The distal side of the deeply buried heme pocket is connected with the surface of the molecule by one (or possibly two) channel. The electron density representing the heme group, in each of the two crystallographically independent subunits, is consistent with degradation of the porphyrin rings. The heme group appears to be buckled, reflecting the high content of bile pigment in liver catalase. The spatial organization on the proximal side (where the fifth ligand of the iron is located) shows an elaborate network of interactions. The distal side contains the substrate pocket. The limited space in this region severely constrains possible substrate positions and orientations. The N delta atom of the essential His74 residue hydrogen bonds with O gamma of Ser113, which in turn hydrogen bonds to a water molecule associated with the propionic carbonylic group of pyrrole III. These interactions are also visible in the refined structure of Penicillium vitale catalase (B. K. Vainshtein, W. R. Melik-Adamyan, V. V. Barynin, A. A. Vagin, A. I. Grebenko, V. V. Borisov, K. S. Bartels, I. Fita, & M. G. Rossmann, unpublished results). Model building suggests a pathway for a catalase mechanism (compound I formation, as well as catalatic and peroxidatic reactions). There are some similarities in compound I formation of catalase and cytochrome c peroxidase.