Peroxide-utilizing biocatalysts: structural and functional diversity of heme-containing enzymes

Heme-containing enzymes, such as peroxidases, catalase and peroxygenase P450 all utilize peroxides for their specific reactions. A variety of reactions catalyzed by such heme-containing enzymes involve a common, highly reactive intermediate, the so-called compound I (oxo-ferryl porphyrin pi-cation radical), which is generated via the reaction of peroxide with a ferric heme iron. However, the main reaction catalyzed by the heme-containing enzyme is determined by the accessibility of substrates to their active sites. Using the accumulated knowledge, we delineate a view, in which machineries of the heme-containing enzymes, especially the heme distal side structures, precisely regulate their functions in terms of sharing a common reactive intermediate. We also show the possibility that a hemoprotein of one functionality can be engineered to that with another functionality by modifying the heme distal side elements, on the basis of molecular-based mechanistic and structural data on these peroxide-utilizing enzymes.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

A biophysical characterization of the iron coordination environment in wheat germ peroxidase

 The heme protein wheat germ peroxidase (isoenzyme C₂) and its cyanide-inhibited form have been investigated by means of electronic, CD and paramagnetic NMR spectroscopy. The data indicate a protein environment of the active site distinct from that of horseradish peroxidase (HRP), with a larger solvent accessibility. The iron is pentacoordinated at neutral and low pH, whereas a hydroxyl anion may be bound at alkaline pH. The fifth axial ligand is a His residue with a partial anionic character, as found in other peroxidases. A spin equilibrium is observed at high enzyme concentrations. Received: 17 September 1996 / Accepted: 10 January 1997     

Salicylhydroxamic acid inhibits myeloperoxidase activity.

Salicylhydroxamic and benzohydroxamic acids were found to bind to the resting state of myeloperoxidase and inhibit ligand binding to the heme iron. An ionizable group on the enzyme with pKa = 4 affects salicylhydroxamic acid binding; binding occurs when this group is not protonated. The binding of the heme iron ligands (e.g. cyanide, nitrite, and chloride) is probably controlled by the same ionizable group. The equilibrium dissociation constant of the salicylhydroxamic acid-myeloperoxidase complex is about 2 x 10(-6) M, and the association rate constant is 7.4 x 10(6) M-1.s-1. Salicylhydroxamic acid serves as a donor to the higher oxidation state of myeloperoxidase and thereby inhibits guaiacol oxidation. Salicylhydroxamic acid was also found to bind to intestinal peroxidase and lactoperoxidase. Salicylhydroxamic acid binding to all three mammalian peroxidases was about 3 orders of magnitude stronger than benzohydroxamic acid binding. We conclude that the salicylhydroxamic and benzohydroxamic acids bind in the distal heme cavity of these peroxidases and interact with the heme ligand binding site.     

Roles of distal arginine in activity and stability of Coprinus cinereus peroxidase elucidated by kinetic and NMR analysis of the Arg51Gln, -Asn, -Leu, and -Lys mutants

In heme peroxidases, a distal His residue plays an essential role in the initial two electron oxidation of resting state enzyme to compound I by hydrogen peroxide. A distal Arg residue assists in this process. The contributions of the charge, H-bonding capacity, size, and mobility of this Arg residue to Coprinus cinereus peroxidase (CIP) reactivity and stability have been examined by substituting Arg51 with Gln (retains H-bond donor at N epsilon position), Asn (small size, H-bond donor and acceptor), Leu (similar to Asn, but hydrophobic), and Lys (charge and H-bond donor, but at N zeta position). UV-visible spectroscopy was used to monitor pH-linked heme changes, compound I formation and reduction, fluoride binding, and thermostability. (1)H NMR spectroscopy enabled heme pocket differences in both resting and cyanide-ligated states of the enzymes to be evaluated and compared with wild-type CIP. We found that the H-bonding capacity of distal Arg is key to fast compound I formation and ligand binding to heme, whereas charge is important for lowering the pK(a) of distal His and for the binding and stabilisation of anionic ligands at heme iron. The properties of the distal Arg residue in CIP, cytochrome c peroxidase (CCP) and horseradish peroxidase (HRP) differ significantly in their pH induced transitions and dynamics.     

Circular dichroism studies on cytochrome c peroxidase from baker's yeast (Saccharomyces cerevisiae).

Circular dichroism spectra of cytochrome c peroxidase from baker's yeast, those of the reduced enzyme, the carbonyl, cyanide and fluoride derivatives and the hydrogen peroxide compound, Compound I, have been recorded in the wavelength range 200 to 660 nm. All derivatives show negative Soret Cotton effects. The results suggest that the heme group is surrounded by tightly packed amino acid sidechains and that there is a histidine residue bound to the fifth coordination site of the heme iron. The native ferric enzyme is probably pentacoordinated. The circular dichroism spectra of the ligand compounds indicate that the ligands form a nonlinear bond to the heme iron as a result of steric hindrance in the vicinity of the heme. The spectrum of Compound I shows no perturbation of the porphyrin symmetry. The dichroic spectrum of the native enzyme in the far-ultraviolet wave-length region suggests that the secondary structure consists of roughly equal amounts of alpha-helical, beta-structure and unordered structure. After the removal of the heme group no great changes in the secondary structure can be observed.     

Escherichia coli flavohemoglobin is an efficient alkylhydroperoxide reductase

Escherichia coli flavohemoglobin (HMP) is shown to be capable of catalyzing the reduction of several alkylhydroperoxide substrates into their corresponding alcohols using NADH as an electron donor. In particular, HMP possesses a high catalytic activity and a low Km toward cumyl, linoleic acid, and tert-butyl hydroperoxides, whereas it is a less efficient hydrogen peroxide scavenger. An analysis of UV-visible spectra during the stationary state reveals that at variance with classical peroxidases, HMP turns over in the ferrous state. In particular, an iron oxygen adduct intermediate whose spectrum is similar to that reported for the oxo-ferryl derivative in peroxidases (Compound II), has been identified during the catalysis of hydrogen peroxide reduction. This finding suggests that hydroperoxide cleavage occurs upon direct binding of a peroxide oxygen atom to the ferrous heme iron. Competitive inhibition of the alkylhydroperoxide reductase activity by carbon monoxide has also been observed, thus confirming that heme iron is directly involved in the catalytic mechanism of hydroperoxide reduction. The alkylhydroperoxide reductase activity taken together with the unique lipid binding properties of HMP suggests that this protein is most likely involved in the repair of the lipid membrane oxidative damage generated during oxidative/nitrosative stress.     

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.     

X-ray absorption studies of intermediates in peroxidase activity

The structures of the enzyme-substrate compounds of peroxidases and catalase determined by X-ray absorption spectroscopy are presented. The valence state of the iron in Compounds I and II is determined from the edge to be higher than Fe+3. A short Fe-Ne (proximal histidine) distance is observed in all forms except Compound II, forcing the Fe-Np average distance to be long, a result which differentiates the peroxidases from the oxygen transport hemoproteins and plays a pivotal role in the mechanism. A correlation is shown between the ratio of peaks in the low k (ligand field indicator ratio) region, the Fe-Np (heme pyrrole nitrogen) average distance, and the magnetic susceptibility, which provides a sensitive indicator of spin state. The mechanism of H2O2 reduction is shown by analysis of the structural changes observed in the intermediates. Possible relationship of these compounds to that of the peroxidatic form of cytochrome oxidase is suggested by these results.     

Formation and decay of hydroperoxo-ferric heme complex in horseradish peroxidase studied by cryoradiolysis

Using radiolytic reduction of the oxy-ferrous horseradish peroxidase (HRP) at 77 K, we observed the formation and decay of the putative intermediate, the hydroperoxo-ferric heme complex, often called "Compound 0." This intermediate is common for several different enzyme systems as the precursor of the Compound I (ferryl-oxo pi-cation radical) intermediate. EPR and UV-visible absorption spectra show that protonation of the primary intermediate of radiolytic reduction, the peroxo-ferric complex, to form the hydroperoxo-ferric complex is completed only after annealing at temperatures 150-180 K. After further annealing at 195-205 K, this complex directly transforms to ferric HRP without any observable intervening species. The lack of Compound I formation is explained by inability of the enzyme to deliver the second proton to the distal oxygen atom of hydroperoxide ligand, shown to be necessary for dioxygen bond heterolysis on the "oxidase pathway," which is non-physiological for HRP. Alternatively, the physiological substrate H2O2 brings both protons to the active site of HRP, and Compound I is subsequently formed via rearrangement of the proton from the proximal to the distal oxygen atom of the bound peroxide.     

Resonance Raman study of ferric heme adducts of dehaloperoxidase from Amphitrite ornata

The study of axial ligation by anionic ligands to ferric heme iron by resonance Raman spectroscopy provides a basis for comparison of the intrinsic electron donor ability of the proximal histidine in horse heart myoglobin (HHMb), dehaloperoxidase (DHP), and horseradish peroxidase (HRP). DHP is a dimeric hemoglobin (Hb) originally isolated from the terebellid polychaete Amphitrite ornata. The monomers are structurally related to Mb and yet DHP has a peroxidase function. The core size marker modes, v2 and v3, were observed using Soret excitation, and DHP-X was compared to HHMb-X for the ligand series X = F, Cl, Br, SCN, OH, N3, and CN. Special attention was paid to the hydroxide adduct, which is also formed during the catalytic cycle of peroxidases. The Fe-OH stretching frequency was observed and confirmed by deuteration and is higher in DHP than in HHMb. The population of high-spin states of the heme iron in DHP was determined to be intermediate between HHMb and HRP. The data provide the first direct measurement of the effect of axial ligation on the heme iron in DHP. The Raman data support a modified charge relay in DHP, in which a strongly hydrogen-bonded backbone carbonyl (>C=O) polarizes the proximal histidine. The charge relay mechanism by backbone carbonyl >C=O-His-Fe is the analogue of the Asp-His-Fe of peroxidases and Glu-His-Fe of flavohemoglobins.     

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.     

The mechanism of Compound I formation revisited

The most recently proposed mechanisms for the formation of the Compound I intermediates of the peroxidases and catalases have been based on the crystallographic elucidation of the enzyme structures. It has been assumed that these mechanisms are compatible with an earlier proposal of the formation of a reversible enzyme-substrate intermediate called Compound 0, which was based on data that pre-dated the availability of the enzyme structures. However, it is argued here that this is not the case and some modifications of the existing mechanism are proposed which reconcile the structural, kinetic and energetic data for the reactions. This paper focuses attention on horseradish peroxidase isoenzyme C and particularly on the acid-base properties of the imidazole side chain of distal histidine 42. This imidazole group has an exceptionally low pK(a) value in the resting enzyme, which is higher in Compound I and higher still in Compound II. The pK(a) value must also be greatly increased following Compound 0 formation so that the imidazole can become an effective proton acceptor. An explanation is offered in a dielectric insertion (DI) model, in which the peroxide substrate, or fragments thereof, screens the influence of the positively charged heme iron on the pK(a) value of the imidazole group. It is proposed that Compound 0 is converted to a second intermediate, Compound 0*, by intramolecular proton transfer along a pre-existing hydrogen bond, a process which reduces the energy requirements of charge separation in the deprotonation of hydrogen peroxide.     

How nature tunes isoenzyme activity in the multifunctional catalytic globin dehaloperoxidase from Amphitrite ornata

The coelomic hemoglobin of Amphitrite ornata, termed dehaloperoxidase (DHP), is the first known multifunctional catalytic globin to possess biologically-relevant peroxidase and peroxygenase activities. Although the two isoenzymes of DHP, A and B, differ in sequence by only 5 amino acids out of 137 residues, DHP B consistently exhibits a greater activity than isoenzyme A. To delineate the contributions of each amino acid substitution to the activity of either isoenzyme, the substitutions of the five amino acids were systematically investigated, individually and in combination, using 22 mutants. Biochemical assays and mechanistic studies demonstrated that the mutants that only contained the I9L substitution showed increased i) k_cat values (peroxidase activity), ii) 5-Br-indole conversion and binding affinity (peroxygenase activity), and iii) rate of Compound ES formation (enzyme activation). Whereas the X-ray structures of the oxyferrous forms of DHP B (L9I) (1.96 Å), DHP A (I9L) (1.20 Å), and WT DHP B (1.81 Å) showed no significant differences, UV–visible spectroscopy (A_Soret/A₃₈₀ ratio) revealed that the I9L substitution increased the 5-coordinate high-spin heme population characterized by the “open” conformation (i.e., distal histidine swung out of the pocket), which likely favors substrate binding. The positioning of the distal histidine closer to the heme cofactor in the solution state also appears to facilitate activation of DHP via the Compound ES intermediate. Taken together, the studies undertaken here shed light on the structure-function relationship in dehaloperoxidase, but also help to establish the foundation for understanding how enzymatic activity can be tuned in isoenzymes of a multifunctional catalytic globin.     

Analysis of heme structural heterogeneity in Mycobacterium tuberculosis catalase-peroxidase (KatG)

Mycobacterium tuberculosis catalase-peroxidase (KatG) is a heme enzyme considered important for virulence, which is also responsible for activation of the anti-tuberculosis pro-drug isoniazid. Here, we present an analysis of heterogeneity in KatG heme structure using optical, resonance Raman, and EPR spectroscopy. Examination of ferric KatG under a variety of conditions, including enzyme in the presence of fluoride, chloride, or isoniazid, and at different stages during purification in different buffers allowed for assignment of spectral features to both five- and six-coordinate heme. Five-coordinate heme is suggested to be representative of "native" enzyme, since this species was predominant in the enzyme examined immediately after one chromatographic protocol. Quantum mechanically mixed spin heme is the most abundant form in such partially purified enzyme. Reduction and reoxidation of six-coordinate KatG or the addition of glycerol or isoniazid restored five-coordinate heme iron, consistent with displacement of a weakly bound distal water molecule. The rate of formation of KatG Compound I is not retarded by the presence of six-coordinate heme either in wild-type KatG or in a mutant (KatG[Y155S]) associated with isoniazid resistance, which contains abundant six-coordinate heme. These results reveal a number of similarities and differences between KatG and other Class I peroxidases.     

Molecular dynamics simulations of the resting and hydrogen peroxide-bound states of cytochrome c peroxidase.

The current hypothesis for the formation of the catalytically active compound I of peroxidases from the resting state and peroxide involves formation of a reversible "inner-sphere" complex in which the peroxide is bound to the heme iron. It is this precursor that is postulated to then form compound I. However, this crucial putative transient intermediate has not yet been definitively detected or characterized by experimental methods. We report here the use of energy minimization and molecular dynamics simulation together with the known X-ray structure of cytochrome c peroxidase to investigate the nature of this complex and comparisons of it with the resting state in which a water is bound as a ligand. Among the properties monitored in these simulations are the mode of binding of the peroxide to the heme iron, its interactions with neighboring amino acid residues, and the extent to which the binding of the peroxide perturbs both the local environment around the heme unit and more distant regions. The results of this study indicate that solvated, full protein dynamics is required to obtain reliable results for the known resting-state complex and hence for the uncharacterized peroxide complex. In this complex, the peroxide binds to the heme iron in a dynamically averaged end-on fashion, rather than a bridged structure, with approximately equal probability of each oxygen serving as the ligand to the iron. Binding of the peroxide as a ligand disrupts the H-bonded network of waters in the distal binding pocket which are present in the resting state, but there is no dramatic perturbation of the nearby amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nature of the ferryl heme in compounds I and II

Heme enzymes are ubiquitous in biology and catalyze a vast array of biological redox processes. The formation of high valent ferryl intermediates of the heme iron (known as Compounds I and Compound II) is implicated for a number of catalytic heme enzymes, but these species are formed only transiently and thus have proved somewhat elusive. In consequence, there has been conflicting evidence as to the nature of these ferryl intermediates in a number of different heme enzymes, in particular the precise nature of the bond between the heme iron and the bound oxygen atom. In this work, we present high resolution crystal structures of both Compound I and Compound II intermediates in two different heme peroxidase enzymes, cytochrome c peroxidase and ascorbate peroxidase, allowing direct and accurate comparison of the bonding interactions in the different intermediates. A consistent picture emerges across all structures, showing lengthening of the ferryl oxygen bond (and presumed protonation) on reduction of Compound I to Compound II. These data clarify long standing inconsistencies on the nature of the ferryl heme species in these intermediates.     

Molecular basis for the inability of an oxygen atom donor ligand to replace the natural sulfur donor heme axial ligand in cytochrome P450 catalysis. Spectroscopic characterization of the Cys436Ser CYP2B4 mutant

All cytochrome P450s (CYPs) contain a cysteinate heme iron proximal ligand that plays a crucial role in their mechanism of action. Conversion of the proximal Cys436 to Ser in NH₂-truncated microsomal CYP2B4 (ΔCYP2B4) transforms the enzyme into a two-electron NADPH oxidase producing H₂O₂ without monooxygenase activity [K.P. Vatsis, H.M. Peng, M.J. Coon, J. Inorg. Biochem. 91 (2002) 542–553]. To examine the effects of this ligation change on the heme iron spin-state and coordination structure of ΔC436S CYP2B4, the magnetic circular dichroism and electronic absorption spectra of several oxidation/ligation states of the variant have been measured and compared with those of structurally defined heme complexes. The spectra of the substrate-free ferric mutant are indicative of a high-spin five-coordinate structure ligated by anionic serinate. The spectroscopic properties of the dithionite-reduced (deoxyferrous) protein are those of a five-coordinate (high-spin) state, and it is concluded that the proximal ligand has been protonated to yield neutral serine (ROH-donor). Low-spin six-coordinate ferrous complexes of the mutant with neutral sixth ligands (NO, CO, and O₂) examined are also likely ligated by neutral serine, as would be expected for ferric complexes with anionic sixth ligands such as the hydroperoxo-ferric catalytic intermediate. Ligation of the heme iron by neutral serine vs. deprotonated cysteine is likely the result of the large difference in their acidity. Thus, without the necessary proximal ligand push of the cysteinate, although the ΔC436S mutant can accept two electrons and two protons, it is unable to heterolytically cleave the O–O bond of the hydroperoxo-ferric species to generate Compound I and hydroxylate the substrate.     

Investigations of the myoglobin cavity mutant H93G with unnatural imidazole proximal ligands as a modular peroxide O-O bond cleavage model system

A general inability to elucidate extensive variations in the electronic characteristics of proximal heme iron ligands in heme proteins has hampered efforts to obtain a clear understanding of the role of the proximal heme iron ligand in the activation of oxygen and peroxide. The disadvantage of the frequently applied site-directed mutagenesis technique is that it is limited by the range of natural ligands available within the genetic code. The myoglobin cavity mutant H93G [Barrick, D. (1994) Biochemistry 33, 6546-6554] has its proximal histidine ligand replaced with glycine, a mutation which leaves an open cavity capable of accommodating a variety of unnatural potential proximal ligands. We have carried out investigations of the effect of changing the electron donor characteristics of a variety of substituted imidazole proximal ligands on the rate of formation of myoglobin compound II and identified a correlation between the substituted imidazole N-3 pK(a) (which provides a measure of the electron donor ability of N-3) and the apparent rate of formation of compound II. A similar rate dependence correlation is not observed upon binding of azide. This finding indicates that O-O bond cleavage and not the preceding peroxide binding step is being influenced by the electron donor characteristics of the substituted imidazole ligands. The proximal ligand effects are clearly visible, but their overall magnitude is quite low (1.7-fold increase in the O-O bond cleavage rate per pK(a) unit). This appears to provide support for recent commentaries which concluded that the partial ionization of the proximal histidine ligand in typical heme peroxidases may not be enough of an influence to provide a mechanistically critical push effect [Poulos, T. L. (1996) JBIC, J. Biol. Inorg. Chem. 1, 356-359]. Further attempts were made to define the mechanism of the influence of N-3 pK(a) on O-O bond cleavage by using peracetic acid and cumene hydroperoxide as mechanistic probes. The observation of heme destruction in these reactions indicates that displacement of the proximal imidazole ligands by peracetic acid or cumene hydroperoxide has occurred. A combination mutation (H64D/H93G) was prepared with the objective of observing compound I of H64D/H93G with substituted imidazoles as proximal ligands upon reaction with H(2)O(2). This double mutant was found to simultaneously bind imidazole to both axial positions, an arrangement which prevents a reaction with H(2)O(2).