Cytochrome C Peroxidase Research Articles

Rational Design of a Functional Metalloenzyme: Introduction of a Site for Manganese Binding and Oxidation into a Heme Peroxidase

The design of a series of functionally active models for manganese peroxidase (MnP) is described. Artificial metal binding sites were created near the heme of cytochrome c peroxidase (CCP) such that one of the heme propionates could serve as a metal ligand. At least two of these designs, MP6.1 and MP6.8, bind Mn2+ with Kd congruent with 0.2 mM, react with H2O2 to form stable ferryl heme species, and catalyze the steady-state oxidation of Mn2+ at enhanced rates relative to WT CCP. The kinetic parameters for this activity vary considerably in the presence of various dicarboxylic acid chelators, suggesting that the similar features displayed by native MnP are largely intrinsic to the manganese oxidation reaction rather than due to a specific interaction between the chelator and enzyme. Analysis of pre-steady-state data shows that electron transfer from Mn2+ to both the Trp-191 radical and the ferryl heme center of compound ES is enhanced by the metal site mutations, with transfer to the ferryl center showing the greatest stimulation. These properties are perplexingly similar to those reported for an alternate model for this site (1), despite rather distinct features of the two designs. Finally, we have determined the crystal structure at 1.9 A of one of our designs, MP6.8, in the presence of MnSO4. A weakly occupied metal at the designed site appears to coordinate two of the proposed ligands, Asp-45 and the heme 7-propionate. Paramagnetic nuclear magnetic resonance spectra also suggest that Mn2+ is interacting with the heme 7-propionate in MP6.8. The structure provides a basis for understanding the similar results of Yeung et al. (1), and suggests improvements for future designs.

Energetics of Cation Radical Formation at the Proximal Active Site Tryptophan of Cytochrome c Peroxidase and Ascorbate Peroxidase

Despite very similar protein structures, ascorbate peroxidase (APX) and yeast cytochrome c peroxidase (CCP) stabilize different radical species during enzyme turnover. Both enzymes contain similar ...

Spectroscopic study of the compound ES and the oxoferryl compound II states of cytochrome c peroxidase: comparison with the compound II of horseradish peroxidase

Magnetic circular dichroism (MCD) spectroscopic data at −30°C are reported for the oxoferryl compound II state of cytochrome c peroxidase (CcP), which was generated by hydrogen peroxide oxidation of ferrous CcP, in comparison with compound ES (an oxoferryl heme with a protein based-radical) of CcP and compound II (an oxoferryl heme) of horseradish peroxidase (HRP-II). Detailed spectral analyses reveal that the protein-based free radical found in CcP-ES does not significantly perturb the electronic environment of the heme as measured by electronic absorption and MCD spectroscopy. Thus, the spectra of the compounds I and II (oxoferryl) states of CcP are similar. The compound I state of HRP (HRP-1), on the other hand, contains an oxoferryl heme coupled to a porphyrin π-cation radical. As a consequence, HRP-I and HRP-II are well known to have quite distinctive spectra. The similarity of the spectral properties of the two active species of CcP, and the contrasting dissimilarity between the spectral properties of the two active high valent species of HRP, clearly indicates the importance of the protein structure surrounding the heme group in determining the spectral properties of the active species.

Detection of a Tryptophan Radical as an Intermediate Species in the Reaction of Horseradish Peroxidase Mutant (Phe-221 → Trp) and Hydrogen Peroxide

The crucial reaction intermediate in the reaction of peroxidase with hydrogen peroxide (H2O2), compound I, contains a porphyrin pi-cation radical in horseradish peroxidase (HRP), which catalyzes oxidation of small organic and inorganic compounds, whereas cytochrome c peroxidase (CcP) has a radical center on the tryptophan residue (Trp-191) and oxidizes the redox partner, cytochrome c. To investigate the roles of the amino acid residue near the heme active center in discriminating the function of the peroxidases in these two enzymes, we prepared a CcP-like HRP mutant, F221W (Phe-221 --> Trp). Although the rapid spectral scanning and stopped-flow experiments confirmed that the F221W mutant reacts with H2O2 to form the porphyrin pi-cation radical at the same rate as for the wild-type enzyme, the characteristic spectral features of the porphyrin pi-cation radical disappeared rapidly, and were converted to the compound II-type spectrum. The EPR spectrum of the resultant species produced by reduction of the porphyrin pi-cation radical, however, was quite different from that of compound II in HRP, showing typical signals from a Trp radical as found for CcP. The sequential radical formation from the porphyrin ring to the Trp residue implies that the proximal Trp is a key residue in the process of the radical transfer from the porphyrin ring, which differentiates the function of peroxidases.

Protein conformer selection by ligand binding observed with crystallography.

A large-scale movement between "closed" and "open" conformations of a protein loop was observed directly with protein crystallography by trapping individual conformers through binding of an exogenous ligand and characterization with solution kinetics. The buried indole ring of Trp191 in cytochrome c peroxidase (CCP) was displaced by exogenous ligands, causing a conformational change of loop Pro190-Asn195 and exposing Trp191 to the protein surface. Kinetic measurements are consistent with a two-step binding mechanism in which the rate-limiting step is a transition of the protein to the open state, which then binds the ligand. This large-scale conformational change of a functionally important region of CCP is independent of ligand and indicates that about 4% of the wild-type protein is in the open form in solution at any given time.

Mediated electrochemistry of peroxidases—effects of variations in protein and mediator structures

The kinetics of a range of ferrocene derivatives with horseradish peroxidase (HRP), cytochrome c peroxidase (CCP) and 3 charge reversal mutants of cytochrome c peroxidase were measured using cyclic voltammetry. Substantial differences in rate constant (100 fold) were observed between HRP and CCP for the same mediator with smaller differences (4–5 fold) for different mediators with the same enzyme. The rate constant did not seem to be dependent on redox potential differences. Cluster analysis is proposed as a way of classifying mediator reactivity.

Structural analysis of compound I in hemoproteins: Study on Proteus mirabilis catalase

Ferryl catalysis has attracted considerable interest, because a diverse variety of enzymes use ferryl intermediates to perform difficult chemistry. The structure of the reactional intermediate compound I of Proteus mirabilis catalase (PMC) has been solved using time-resolved X-ray diffraction techniques and single crystal microspectrophotometry. Formation of compound I is characterized by significant changes in the absorbance spectrum, and the creation of an oxoferryl group on the distal side of the heme. This group is clearly visible in the X-ray electron density maps. An unidentified electron density, likely to be an anion because of the nature of its environment, appears during the reaction, in a site distant from the heme. The structure of compound I in PMC is compared with that of compound I in cytochrome c peroxidase (CCP).

Calculations of the structure and spectra of the putative transient peroxide intermediates of peroxidases

Peroxidases are oxidative metabolizing heme proteins that require hydrogen peroxide to be transformed to the catalytically active Compound I species from the ferric resting state. Although a peroxide complex with the heme iron has been proposed as a key intermediate in this reaction, this intermediate is too transient to have thus far been definitively characterized. While electronic spectra attributed to it have been observed, in the absence of any addition information, it is not possible from these reported spectra alone to identify the transient species that give rise to them. Results of previous molecular dynamic (MD) simulations of a peroxide complex with cytochrome C peroxidase (CCP) indicated that peroxide does indeed form a stable complex with the heme unit and binds in a nonsymmetric end-on mode in which the oxygen atoms systematically exchange places as ligands for the heme iron. To further assess this mode of binding, ab initio quantum chemical methods have been used to determine the optimized geometry and stability of a peroxide complex with a model heme peroxidase. The results obtained provide confirmation of the important characteristics of this transient intermediate found in the previous MD simulations. Specifically, two equivalent stable minima were identified with a binding energy of about 10 kcal mole −1 in which the peroxide binds in an end-on mode with alternative oxygen atoms as the Fe ligand and a small energy barrier between them. To further explore evidence for this species, the semiempirical INDO/ROHF/CI quantum chemical method has been used to calculate its electronic spectra and the results were compared with the observed spectra for the transient species of horse radish peroxidase and a mutant of it. Comparison of the calculated spectra of the neutral (HOOH) and anionic (OOH −) form of the peroxide intermediate with the two experimental data, indicated that both had been observed, the anionic form in the wild type and the neutral form in the mutant horse radish peroxidase. Thus, these calculations, by providing the missing correspondence between species and spectra, have allowed the identification of the transient intermediate species in the pathway from the resting state to Compound I of peroxidases as the long proposed peroxide intermediate.

Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase.

Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.

Electrostatic modulation of electron transfer in the active site of heme peroxidases

The heme enyzmes cytochrome c peroxidase (CCP) and pea cytosolic ascorbate peroxidase (APX) show a high level of sequence identity. The main difference near the active sites is the presence of a cation binding site in APX located about 1 nm from the Trp-179 side chain, which is hydrogen-bonded to Asp-208. It is possible that this difference in electrostatics provided by the protein environment is an essential determinant of the stabilization of the ion-pair or neutral form of the Trp...Asp couple in APX and CCP. Semiempirical molecular orbital calculations support the hypothesis that the position of the moving proton inside the couple influences the location of the free electron, leading to radical formation either on the heme or on the Trp side chain of these enzymes.

Mutation of Distal Residues of Horseradish Peroxidase: Influence on Substrate Binding and Cavity Properties

The manner in which the distal heme pocket residues of peroxidases control the reaction mechanism and ligand binding has been investigated further by analysis of the electronic absorption and resonance Raman (RR) spectra of distal site mutants of recombinant horseradish peroxidase (HRP-C*). The roles of the conserved distal histidine and arginine residues, particularly in the context of the catalytic mechanism originally proposed for cytochrome c peroxidase (CCP), have been evaluated by studying the His42 --> Leu, His42 --> Arg, Arg38 --> Gly, and Arg38 --> Leu variants of HRP-C*. Spectra of the ferric forms, their complexes with benzohydroxamic acid (BHA), and the ferrous forms have been recorded at neutral pH. In addition, the ferric forms have been studied at alkaline pH. The relative populations of the three heme spin states characteristic of HRP-C* and its mutants were found to vary markedly from mutant to mutant. This diversity of heme spin state populations among the various mutants has allowed a well-defined set of RR frequencies to be compiled for the three heme spin states. These frequencies support the analysis of wild-type HRP-C* in terms of two heme states, five- (5cHS#) and six-coordinate high-spin (6cHS#), which exhibit anomalous RR frequencies compared to those of model heme systems. The third heme spin state is identified as being six-coordinate high-spin, displaying typical RR frequencies (6cHS). The 6cHS# and the 6cHS heme states are characterized by H bonding between the iron-bound water molecule and the Arg38 residue or the His42 residue, respectively. The proportion of six-coordinate high-spin heme states is at a minimum in the Arg38Leu mutant, indicating that the occupancy of the distal water molecule site is reduced in this mutant. The His42Arg mutant is distinguished from the other mutants by the unexpected presence of an iron-bound hydroxyl group at neutral pH. The spectral changes induced upon complexation with BHA indicate that both the distal histidine and arginine are involved in BHA binding; however, the arginine residue appears to play a more critical role. Measurements at pH 12 suggest there is a concerted involvement of both distal residues in mediating the alkaline transition of HRP-C*. Arg38 appears to be essential for stabilization of the OH- ligand, while His42 acts as a H bond acceptor. A striking similarity between the roles of these residues in the reaction of H2O2 with the enzyme and the alkaline transition is noted. By comparison with the results from corresponding mutants of CCP, it appears that although the hydrogen-bonding network linking the distal and proximal sides of the heme is conserved the distal cavity in HRP-C differs significantly from that of CCP. However, some similarities in the local environment of the distal arginine are suggested.

Solution and Crystal Structures of the H175G Mutant of Cytochrome c Peroxidase: A Resonance Raman Study

Site-directed mutagenesis offers a powerful probe of heme proteins. Recent attention has focused on mutants whose proximal iron ligand is replaced by smaller, noncoordinating amino acids. These mutants form artificial cavities where the native proximal ligand resided and which are capable of binding exogenous ligands such as imidazoles. Such reconstituted systems permit detailed studies of the electronic and stereochemical influences of the proximal ligand on catalysis and activity.1-6 The paradigm of these mutants is H175G cytochrome c peroxidase (CCP). Its X-ray crystal structure revealed that the iron is close to two water molecules.1 In heme oxygenase, the H25A mutant has been proposed to contain a single water ligand on the heme iron.4 The iron in the H170A mutant of horseradish peroxidase is probably bound by a water molecule at pH 4 and an additional sixth ligand (possibly a distal histidine) at pH 5-7.6 The crystal structure of the imidazole adduct of H93G myoglobin has been reported,2 but structural and spectroscopic data for the H93G mutant in the absence of imidazole are not yet available. Interestingly, UV-vis spectra of wild-type metmyoglobin and ferric Coprinus cinereus peroxidase in acidic buffer7,8 bear resemblance to those of the proximal ligand-deficient heme proteins; the native proximal ligation may well be disrupted under such conditions. Among these proximal-ligand mutants, H175G CCP is unique in that its crystal structure indicates two water molecules axial to the heme. However, it is not certain whether one or both of these waters is strongly coordinated, what the water protonation states are, and whether the axial coordination of two water molecules can be maintained in solution. In this study, we have examined the crystal and solution structures of the H175G mutant by UVvis, resonance Raman (RR), and EPR spectroscopy. Ferric H175G CCP9 undergoes a transition from a “red form” near pH 6 to a “green form” near pH 7 (Figure 1). The absorbance at 408 nm (λmax of the red form) changes with pH to give an apparent pKa of ∼6.5 (inset, Figure 1). A fit to these data indicates that the transition involves a cooperative twoproton process. One proton is proposed to be from the ironbound water molecule, but the source of the second proton is unknown. The UV-vis and RR spectra of H175G at pH 10.0 are almost identical to those at pH 7.2 (not shown). FeIII(TMP)s [TMP ) 5,10,15,20-tetrakis(1-methylpyridinium)porphines] exhibit similar ligand ionization in aqueous solution where the five-coordinate iron is ligated by a water at pH 11.10,11 Resonance Raman spectra of heme proteins are dominated by porphyrin skeletal vibrations, i.e., totally symmetric ν2, ν3, and ν4 with Soret excitation, nonsymmetric modes ν10, ν11, and ν19 with Q-band excitation, and ν2 and ν10 with near-UV (∼350 nm) excitation.12-15 The frequencies of these modes are determined by the iron coordination and spin states. The RR spectra of H175G16 are shown in Figure 2. The well-defined ν3 and ν10 frequencies at 1494 and 1630 cm-1, respectively, of the red solution (pH 5.9) are clearly characteristic of a fivecoordinate high-spin (5cHS) ferric heme.15,17-20 The RR spectra of the green solution (pH 7.2) also indicate a dominant 5cHS

Photoinduced Electron Transfer between CytochromecPeroxidase (D37K) and Zn-Substituted Cytochromec: Probing the Two-Domain Binding and Reactivity of the Peroxidase

Cytochrome c peroxidase (CcP) binds cytochrome c (Cc) at two distinct surface binding domains, one having high affinity for Cc and the other having low affinity. The identity of the surface binding domains on CcP has been probed by studying photoinduced interprotein heme−heme electron transfer (ET) between Zn-substituted horse Cc, ZnCc, and a cloned cytochrome c peroxidase, CcP(D37K). Charge-reversal substitution of the negatively-charged residue Asp 37 of CcP by a positively-charged residue lysine greatly decreases the stoichiometric constant for 1:1 binding of Cc, from 8.5 × 105 M-1 for the wild-type CcP to 1.2 × 104 M-1 for CcP(D37K) (μ = 18 mM), thereby identifying residue 37 as part of the high-affinity binding domain of CcP(WT) (domain 1). This assignment is consistent with domain 1 as being that observed in a crystal of the Cc−CcP complex. The diminished ability of CcP(D37K) to bind Cc at domain 1 also suppresses binding of a second Cc molecule to form a ternary complex. However, the mutation sharp...

Charge reversal of a critical active-site residue of cytochrome-c peroxidase: characterization of the Arg48-->Glu variant.

A new variant of cytochrome-c peroxidase in which the positively charged Arg48 present in the distal heme-binding pocket has been replaced with a Glu residue has been prepared and characterized to explore, in part, the possibility that a negative charge close to the heme could contribute to stabilization of a porphyrin-centered pi-cation radical in the compound I derivative of the variant. Between pH 4 and 8, this variant forms three pH-linked spectroscopic species. The electronic absorption and 1H-NMR spectra of the predominant form at low pH (HS1) are indicative of a high-spin, pentacoordinate heme iron system. Near neutral pH, a second high-spin species (HS2) is dominant, in which the heme iron center is hexacoordinated, with a water molecule as the sixth axial ligand. At high pH, the third form (LS) exhibits the spectroscopic characteristics of a low-spin, hexacoordinate heme center with bishistidine axial ligation. The apparent pKa values for these transitions are 4.4 and 7.4, respectively, in phosphate buffers and 5.0 and 7.1, respectively, in phosphate/nitrate buffers. Replacement of Arg48 with Glu reduces the thermal stability of the enzyme and also decreases the Fe(III)/Fe(II) reduction potential of the enzyme by approximately 50 mV relative to that of the wild-type enzyme. The stability of compound I formed by the variant is decreased although the rate at which it forms is just one order of magnitude less than that of the wild-type enzyme, thus confirming previous results which indicate that the function of residue 48 in the wild-type peroxidase is more related to the stability of compound I than to its formation [Erman, J. E., Vitello, L. B., Miller, M. A. & Kraut, J. (1992) J. Am. Chem. Soc. 114, 6592-6593; Vitello, L. B., Erman, J. E., Miller, M. A., Wang, J. & Kraut, J. (1993) Biochemistry 32, 9807-9818]. Stopped-flow studies failed to detect even transient formation of a porphyrin-centered radical following addition of hydrogen peroxide to the Fe(III)-enzyme. The consequences of this drastic electrostatic modification of the active site on the steady-state kinetics of the variant are relatively minor.

The homologous tryptophan critical for cytochrome c peroxidase function is not essential for ascorbate peroxidase activity

The crystal structures of ascorbate peroxidase (APX) and cytochrome c peroxidase (CCP) show that the active site structures are nearly identical. Both enzymes contain a His-Asp-Trp catalytic triad in the proximal pocket. The proximal Asp residue hydrogen bonds with both the His proximal heme ligand and the indole ring nitrogen of the proximal Trp. The Trp is stacked parallel to and in contact with the proximal His ligand. This Trp is known to be the site of free radical formation in CCP compound I and also is essential for activity. However, APX forms a porphyrin radical and not a Trp-centered radical, even though the His-Asp-Trp triad structure is the same in both peroxidases. We found that conversion of the proximal Trp to Phe has no effect on APX enzyme activity and that the mutant crystal structure shows that changes in the structure are confined to the site of mutation. This indicates that the paths of electron transfer in CCP and APX are distinctly different. The Trp-to-Phe mutant does alter the stability of the APX compound I porphyrin radical, by a factor of two. Electrostatic calculations and modeling studies show that a potassium cation located about 8 A from the proximal Trp in APX, but absent in CCP, makes a significant contribution to the stability of a cation Trp radical. This underscores the importance of long-range electrostatic effects in enzyme catalyzed reactions.

Cytochrome c peroxidase complexed with cytochrome c has an unperturbed heme moiety.

Transient resonance Raman, Raman difference, circular dichroism (CD), and optical absorption studies have been carried out on the electrostatic complexes formed by yeast cytochrome c peroxidase (CCP) with horse cytochrome c (Cytc) in low ionic strength solutions. In all the complexes examined [e.g., CCP(II)/Cytc(II), CCP(III)/Cytc(II), CCP(III)/Cytc(III)], the local heme environments of both proteins are largely unperturbed upon complexation. Specifically, CCP preserves a completely pentacoordinate high-spin heme in both its ferric and ferrous forms in CCP/Cytc complexes and uncomplexed mixtures. We found no evidence corroborating the previously reported increase in the low-spin fraction of CCP heme upon complexation with Cytc [Hildebrandt et al. (1992) Biochemistry 31, 2384-2392]. Instead, our Raman data strongly suggest that the H-bonding networks in the distal and proximal pockets of CCP are well maintained in the complexes. On the other hand, CD spectra of CCP(III)/Cytc(III) complexes showed substantial variations (relative to the uncomplexed mixtures) in the far-UV region, reflecting some protein conformational rearrangements. In addition, the spectral data suggest that complexation with Cytc affects the previously observed pH-dependent flexibility of the heme structure of CCP and thus influences the photodynamics of the CCP active site.

Altering substrate specificity at the heme edge of cytochrome c peroxidase.

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.

Structure of a Model Transient Peroxide Intermediate of Peroxidases by ab Initio Methods

Peroxidases are oxidative metabolizing heme proteins that require hydrogen peroxide to be transformed to the catalytically active, compound I, species from the ferric resting state. Although a peroxide complex has been proposed as a key intermediate in this reaction, this intermediate is too transient to have thus far been definitively characterized. Results of previous molecular dynamics (MD) simulations of a peroxide complex with cytochrome C peroxidase (CCP) indicated that peroxide forms a stable complex and binds in a nonsymmetric, end-on mode in which the oxygen atoms systematically exchange places as ligands for the iron. These results provided support for a plausible pathway from the peroxide complex to compound I, involving the participation of nearby histidine and arginine residues. To further explore the reliability of this bonding description, we report here, for the first time, the use of ab initio methods to determine the optimized geometry and stability of a peroxide complex with a model heme peroxidase. Two stable minima were identified, with binding energies of 9.7 kcal/mol. In both of these complexes, the peroxide binds in an end-on mode but with alternative oxygen atoms as the Fe ligand. No minimum corresponding to a bridged structure could be found. The two end-on minima are connected by a low-energy ridge. These results provide confirmation of three mechanistically important characteristics found in the previous 300 K MD simulations of a peroxidase−HOOH complex: (i) formation of a stable peroxide complex, (ii) binding of peroxide in an asymmetric end-on mode, and (iii) dynamic interchange between the oxygen atoms that bind to the Fe, implying a small energy barrier between them.

Probing the cytochrome c peroxidase-cytochrome c electron transfer reaction using site specific cross-linking.

Engineered cysteine residues in yeast cytochrome c peroxidase (CCP) and yeast iso-1-cytochrome c have been used to generate site specifically cross-linked peroxidase-cytochrome c complexes for the purpose of probing interaction domains and the intramolecular electron transfer reaction. Complex 2 was designed earlier [Pappa, H.S., & Poulos, T.L. (1995) Biochemistry 34, 6573-6580] to mimic the known crystal structure of the peroxidase-cytochrome c noncovalent complex [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. Complex 3 was designed such that cytochrome c is tethered to a region of the peroxidase near Asp148 which has been suggested to be a second site of interaction between the peroxidase and cytochrome c. Using stopped flow methods, the rate at which the ferrocytochrome c covalently attached to the peroxidase transfers an electron to peroxidase compound I is estimated to be approximately 0.5-1 s-1 in complex 3 and approximately 800 s-1 in complex 2. In both complexes the Trp191 radical and not the Fe4+=O oxyferryl center of compound I is reduced. Conversion of Trp191 to Phe slows electron transfer about 10(3) in complex 2. Steady state kinetic measurements show that complex 3 behaves like the wild type enzyme when either horse heart or yeast ferrocytochrome c is used as an exogenous substrate, indicating that the region blocked in complex 3 is not a functionally important interaction site. In contrast, complex 2 is inactive toward horse heart ferrocytochrome c at all ionic strengths tested and yeast ferrocytochrome c at high ionic strengths. Only at low ionic strengths and low concentrations of yeast ferrocytochrome c does complex 2 give wild type enzyme activity. This observation indicates that in complex 2 the primary site of interaction of CCP with horse heart and yeast ferrocytochrome c at high ionic strengths is blocked. The relevance of these results to the pathway versus distance models of electron transfer and to the interaction domains between peroxidase and cytochrome c is discussed.

The affinity and specificity of Ca(2+)-binding sites of cytochrome-c peroxidase from Paracoccus denitrificans.

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.