Yeast cytochrome c peroxidase (CcP) and horse metmyoglobin (Mb) bind HN3 with similar affinities at 25 °C. The pH-independent equilibrium association constants for formation of the CcP · HN3 and Mb · HN3 complexes are (1.05 ± 0.06) × 105 and (1.6 ± 0.8) × 105 M−1, respectively. However, the thermodynamic parameters for formation of the two complexes are quite different. The ΔH0 values for formation of CcP · HN3 and Mb · HN3 are −16.4 ± 0.7 and −9.0 ± 0.5 kcal/mol, respectively, and the ΔS0 values are −32 ± 2 and −16 ± 2 cal/deg mol, respectively. The proton associated with HN3 is retained in both protein complexes at low pH but dissociates with apparent pKA values of 5.5 ± 0.2 and ⩾8.2 for the Mb · HN3 and CcP · HN3 complexes, respectively. CcP and Mb differ significantly in their reactivity toward the azide anion, N3−. CcP binds N3− very weakly, if at all, and only an upper-limit of 18 ± 5 M−1 for the pH-independent equilibrium association constant for the CcP · N3− complex can be determined. Mb binds N3− with an association constant of (1.8 ± 0.1) × 104 M−1. The ratio of the equilibrium association constants for HN3 and N3− binding provides a discrimination factor between the neutral and charged forms of the ligand. The discrimination factor is greater than 5800 for CcP but only nine for Mb. Protonation of the distal histidines in the two proteins influences binding of HN3. Protonation of His-64 in Mb enhances HN3 binding due to a gating mechanism while protonation of His-52 in CcP decreases the affinity for HN3 due to loss of base-assisted association of the ligand to the heme iron.

The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25°C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M−1s−1 at pH 5 to a maximum value of 280 M−1s−1 at pH 9.5 and then decreasing to 10 M−1 s−1 at pH 11.5 in 0.1 M ionic strength buffers. The dissociation rate constant has a much smaller pH dependence, varying from 0.082 s−1 at low pH to 0.035 s−1 at high pH, with an apparent pKa of 6.5. The binding affinity of 4-nitroimidazole to horse metmyoglobin is about 2.5 orders of magnitude stronger than that for imidazole and this increased affinity is attributed to the much slower dissociation rate for 4-nitroimidazole compared to that of imidazole. Although the ionic strength dependence of the binding rate is small and secondary kinetic salt effects can account for the ionic strength dependence of the association rate constant, the pH dependence of the rate constants and microscopic reversibility arguments indicate that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than does the neutral form of the ligand. However, the spectrum of the complex is similar to model complexes involving neutral imidazole and not imidazolate. The latter observation suggests that the initial metmyoglobin/4-nitroimidazolate complex rapidly binds a proton and the neutral form of the bound ligand is stabilized, probably through hydrogen binding with the distal histidine.

The kinetics of formation and dissociation of the horse metmyoglobin/azide complex has been investigated between pH 3.5 and 11.5. The ionic strength dependence of the reaction has been determined at integral pH values between 5 and 10. Hydrazoic acid, HN3, binds to metmyoglobin with a rate constant of (3.8 +/- 1.0) x 10(5) M-1 s-1. Protonation of a group with an apparent pKa of 4.0 +/- 0.3 increases the rate of HN3 binding 6.5-fold to (2.5 +/- 0.8) x 10(6) M-1 s-1. The ionizable group is attributed to the distal histidine, His-64. The azide anion, N-3, binds to metmyoglobin with a rate constant of (4.7 +/- 0.3) x 10(3) M-1 s-1, about two orders of magnitude slower than HN3. Conversion of aquometmyoglobin to hydroxymetmyoglobin slows azide binding significantly. Binding of HN3 to hydroxymetmyoglobin cannot be detected, while N-3 binds to hydroxymetmyoglobin with a rate of 5.7 +/- 3.2 M-1 s-1, almost three orders of magnitude slower than N-3 binding to aquometmyoglobin. Protonation of the distal histidine facilitates HN3 dissociation from the complex. HN3 dissociates from the metmyoglobin/azide complex with a rate constant of 18 +/- 6 s-1, while the azide anion dissociates with a rate constant of 0.16 +/- 0.02 s-1, about 100 times slower. The apparent pKa for His-64 is essentially the same in metmyoglobin and the metmyoglobin/azide complex, 4.0 +/- 0.3 and 4.4 +/- 0.2, respectively. The ionic strength dependence of the observed association rate constant is influenced by both primary and secondary kinetic salt effects. The primary kinetic salt effect is anomalous, with the rate of N-3 binding decreasing with increasing ionic strength above the isoelectric point of metmyoglobin where the protein has a net negative charge. The ionic strength dependence of the dissociation rate constant can be described solely in terms of the ionic strength dependence of the acid dissociation constant for His-64 in the metmyoglobin/azide complex, a secondary kinetic salt effect.

The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength. The pH dependence of the reaction between CcP and peracetic acid has also been determined. The rate of the reactions are influenced by two heme-linked ionizations in the protein. The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated. The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively. These rates are about 60% slower than the reaction between hydrogen peroxide and CcP. A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme. The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids. These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions. However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion. The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.

Both cytochrome c peroxidase (CcP) and a mutant cytochrome c peroxidase in which the distal histidine has been replaced by leucine, CcP(H52L), are converted to hydroxy-ligated derivatives at alkaline pH. In CcP, the hydroxy-ligated derivative is subsequently converted to a bis-imidazole species prior to protein denaturation while the initial hydroxy-ligated CcP(H52L) is converted to a second, spectroscopically distinct hydroxy-ligated species prior to denaturation. The spectra of the alkaline forms of CcP and CcP(H52L) have been determined between 310 and 700 nm. The pH dependence of the rate of reaction between CcP(H52L) and hydrogen peroxide has been extended to pH 10. The hydroxy-ligated form of CcP(H52L) reacts with hydrogen peroxide 4 times more rapidly than the pentacoordinate, high-spin form of CcP(H52L) that exists at neutral pH. The rate of the reaction between p-nitroperoxybenzoic acid and CcP(H52L) has been measured between pH 4 and pH 8. Neutral p-nitroperoxybenzoic acid reacts with CcP(H52L) 10(5) times more slowly than with CcP while the negatively charged p-nitroperoxybenzoate reacts with CcP(H52L) 10(3) times more slowly than with CcP. These data indicate that the role of the distal histidine during the initial formation of the peroxy anion/heme iron complex is not simply base catalysis.

The kinetics of formation and dissociation of the horse metmyoglobin/fluoride complex has been investigated between pH 3.4 and 11. The ionic strength dependence of the reaction has been measured at integral pH values between pH 5 and 10. Hydrofluoric acid, HF, binds to metmyoglobin with a rate constant of (4.7 ± 0.7) × 104M−1s−1. An apparent ionization in metmyoglobin with a pKaof 4.4 ± 0.5 influences the rate of HF binding and is attributed to the distal histidine, His-64. Protonation of His-64 increases the HF binding rate by a factor of 2.6. The fluoride anion, F−, binds to metmyoglobin with a rate constant of (5.6 ± 1.4) × 10−2M−1s−1, about 106times slower than HF. Binding of either HF or F−to hydroxymetmyoglobin cannot be detected. Protonation of the distal histidine facilitates HF dissociation from the metmyoglobin/fluoride complex. HF dissociates with a rate constant of 1.9 ± 0.3 s−1. The fluoride anion dissociates 2000 times more slowly, with a rate constant of (8.7 ± 1.6) × 10−4s−1. The apparent pKafor His-64 ionization in the fluorometmyoglobin complex is 5.7 ± 0.1. The association and dissociation rate constants are relatively independent of ionic strength with secondary kinetic salt effects sufficient to account for the ionic strength variation of both, consistent with the idea that association and dissociation of neutral HF dominate the kinetics of fluoride binding to metmyoglobin.

The reaction between metmyoglobin and imidazole has been studied as a function of pH between pH 4.2 and 11.5 and as a function of ionic strength at integral pH values (5 to 10) between 0.001 and 1.0 M ionic strength. The reaction between metmyoglobin and 1-methylimidazole has also been investigated as a function of pH. Comparison of the pH dependence of the association rate constants for the two ligands indicates that the negatively charged imidazolate ion does not contribute to the observed rate of imidazole binding at pH < or = 11.5. At all pH values between pH 4.2 and pH 11.5 the initial complex formed involves the neutral form of bound imidazole. At pH 11.5, the neutral imidazole complex is converted slowly (t1/2 approximately 10 s) into an imidazolate complex. The kinetic data were analyzed according to two mechanisms, one involving the binding of neutral imidazole only and one involving the direct binding of both imidazole and the imidazolium ion to metmyoglobin. Although secondary kinetic salt effects account for the ionic strength dependence of the association rate constant, evidence which indicates that metmyoglobin reacts with imidazole and with the imidazolium ion with similar rates is provided. A self-consistent analysis indicates that the rate constants for imidazole and imidazolium ion binding to metmyoglobin are 350 and 230 M-1 s-1, respectively, at neutral pH and 0.1 M ionic strength. Imidazole can react directly with hydroxymetmyoglobin with a rate of 56 M-1 s-1 at 0.1 M ionic strength, about sixfold slower than binding to aquometmyoglobin. Protonation of a second heme-linked group, thought to be His-97, has little influence on the binding of imidazole but does decrease the rate of imidazolium binding by about eightfold to 29 M-1 s-1 at 0.1 M ionic strength.

The cytochrome c/cytochrome c peroxidase system has been extensively investigated as a model for long-range electron transfer in biology. Two models for the structure of the one-to-one cytochrome c/cytochrome c peroxidase complex in solution exist: one is based upon computer docking of the two proteins and the second is based upon the structure of the complex in the crystalline state. Titration calorimetry is used to investigate the interaction of horse ferricytochrome c with baker's yeast cytochrome c peroxidase and with six cytochrome c peroxidase mutants. Five of the six peroxidase mutants eliminate a negative charge in the cytochrome c binding site by replacing a side-chain carboxylate with an amide. The sixth mutation replaces a surface alanine residue with phenylalanine. The binding affinity between cytochrome c and the cytochrome c peroxidase mutants varies from no significant change in comparison to the wild-type enzyme to a 4-fold decrease in the equilibrium association constant. The pattern of decreasing cytochrome c binding affinity for the cytochrome c peroxidase mutants is consistent with the cytochrome c binding domain defined by X-ray crystallography [Pelletier, H., & Kraut, J. (1992) Science 258, 1748-1755]. For those mutants which have lower affinity for cytochrome c, the lower affinity is due to a decrease in the entropy change upon complex formation, consistent with the difference in hydration of carboxylate and amide groups.

The binding of horse ferricytochrome c to yeast cytochrome c peroxidase at pH 6.0 in 8.7 mM phosphate buffer (0.0100 M ionic strength) is characterized by a small, unfavorable enthalpy change (+1.91 +/- 0.16 kcal mol-1) and a large, positive entropy change (+37 +/- 1 eu). The free energy of binding depends strongly upon ionic strength, increasing from -9.01 to -4.51 kcal mol-1 between 0.0100 and 0.200 M ionic strength. The increase in free energy is due solely to the change in entropy over this ionic strength range, with the entropy change decreasing from 37 +/- 1 to 22 +/- 3 eu between 0.0100 and 0.200 M ionic strength. The observed enthalpy change remains constant over the same ionic strength range. At 0.0100 M ionic strength, complex formation is accompanied by the release of 0.54 +/- 0.11 proton, causing a variation in the observed enthalpy of reaction depending upon the buffer. After accounting for proton binding to the buffer, the corrected values for the enthalpy and entropy of binding are +2.84 +/- 0.26 kcal mol-1 and +21 +/- 3 eu, respectively. At 0.05 M ionic strength, the observed change in heat capacity, delta Cp, for the reaction between horse ferricytochrome c and cytochrome c peroxidase is essentially zero, 1.6 +/- 9.6 cal mol-1 K-1. The corrected delta Cp for binding is -28 +/- 10 cal mol-1 K-1 after accounting for proton binding to the buffer. No evidence for formation of a 2:1 horse ferricytochrome c/cytochrome c peroxidase complex was obtained in this study.

The reduction of cytochrome c peroxidase compound I by excess yeast iso-1 ferrocytochrome c is biphasic. Two pseudo-first-order rate constants can be measured by stopped-flow techniques. The fastest rate process is the reduction of cytochrome c peroxidase compound I to compound II, and the slower process is the reduction of II to the native enzyme. The yeast iso-1 ferrocytochrome c concentration dependence of the reduction of cytochrome c peroxidase compound I to compound II is consistent with a mechanism involving two binding sites for cytochrome c on cytochrome c peroxidase. Electron transfer from cytochrome c bound at the high-affinity binding site to the Fe(IV) site in cytochrome c peroxidase compound I is dependent upon ionic strength, increasing from 15 +/- 6 to 2000 +/- 100 s-1 over the ionic strength range 0.01-0.20 M. The reduction rate of the Fe(IV) site in the 2:1 yeast iso-1 ferrocytochrome c/cytochrome c peroxidase compound I complex is essentially independent of ionic strength with a value of 3800 +/- 300 s-1. The Fe(IV) site in cytochrome c peroxidase compound I is preferentially reduced by yeast ferrocytochrome c between 0.01 and 0.20 M ionic strength while the Trp-191 radical is preferentially reduced above 0.30 M ionic strength. The association rate constant for the binding of yeast iso-1 ferrocytochrome c to cytochrome c peroxidase compound I can be evaluated and varies from a remarkable 1 x 10(10) M-1 s-1 at 0.01 M ionic strength to 1.2 x 10(5) M-1 s-1 at 1.0 M ionic strength. Between 0.01 and 0.20 M ionic strength, the reduction of cytochrome c peroxidase compound II to the native enzyme is anomalous. The reaction is independent of the cytochrome c concentration and directly proportional to the initial cytochrome c peroxidase compound I concentration.

The crystallographic structures of two cytochrome c peroxidase (CcP) mutants, CcP(R48L) and CcP(R48K), have been determined. In addition, the electronic absorption spectrum and the hydrogen peroxide reactivity of these two mutants have been determined between pH 4 and 8. Both the crystallographic structure and the electronic absorption spectrum of CcP(R48L) are consistent with exclusive pentacoordination of the heme iron between pH 4 and 6.5. At higher pH, CcP(R48L) forms an alkaline bis-imidazole form of CcP with the distal histidine coordinated to the heme iron. The apparent pKA for this transition is 7.5 in CcP(R48L). The observed pseudo-first-order rate constant for the reaction between CcP(R48L) and hydrogen peroxide saturates at high peroxide concentrations. The data are consistent with a rate-limiting oxygen-oxygen bond scission at high peroxide concentrations. The observed rate of the bond scission step ranges between 1000 and 1950 s-1, an estimated 2 orders of magnitude slower than for wild-type enzyme. The data suggest that the protonated form of His-52 increases the bond scission step by a factor of 2. The properties of the CcP(R48K) mutant are significantly different from those of CcP(R48L). The crystal structure of CcP(R48K) shows Lys-48 occupying the putative peroxide binding site. The electronic absorption spectrum indicates that CcP(R48K) is predominantly pentacoordinate at neutral pH but with detectable amounts of hexacoordinate forms. Two ionizable groups affect the electronic absorption spectrum of CcP(R48K). An apparent ionization near pH 4 produces an enzyme with increased hexacoordination, while an apparent pKA of 6.9 generates the alkaline bis-imidazole form. The peroxide reaction saturates at high peroxide concentrations for CcP(R48K) and is attributed to a conformational-gating mechanism. The maximum rate for the reaction between CcP(R48K) and hydrogen peroxide is probably limited by the movement of either Lys-48 or His-52. This rate is 200 and 290 s-1 in nitrate-containing buffers and phosphate buffers, respectively. Evidence is provided that Arg-48 in wild-type enzyme is responsible for nitrate binding in the heme pocket and for stabilizing CcP Compound I.

The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate-containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pKA = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pKA = 5.4.(ABSTRACT TRUNCATED AT 250 WORDS)

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTActive-site mutations in cytochrome c peroxidase: a critical role for histidine-52 in the rate of formation of compound IJames E. Erman, Lidia B. Vitello, Mark A. Miller, and Joseph KrautCite this: J. Am. Chem. Soc. 1992, 114, 16, 6592–6593Publication Date (Print):July 1, 1992Publication History Published online1 May 2002Published inissue 1 July 1992https://pubs.acs.org/doi/10.1021/ja00042a068https://doi.org/10.1021/ja00042a068research-articleACS PublicationsRequest reuse permissionsArticle Views213Altmetric-Citations70LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pK(a) of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pK(a) of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.

Steady-State and transient-state kinetic studies of the cytochrome c peroxidase (CcP) catalyzed oxidation of ferrocytochrome c by hydrogen peroxide at pH 7.5 as a function on ionic strength have been carried out using both horse heart and yeast iso-1 cytochrome c. The ionic strength affects the apparent interaction between cytochrome c and the oxidized intermediates of CcP while the intracomplex electron transfer rates are essentially independent of ionic strength. The maximum enzyme turnover rates for the oxidation of yeast iso-1 and horse heart ferrocytochrome c are 780 ± 130 s−1 and 280 ± 40 s−1, respectively, independent of ionic strength at pH 7.5. The intramolecular electron transfer rates from bound ferrocytochrome c to the heme site in CcP compound I is about 1900 s−1 and 450 s−1 for yeast iso-1 and horse heart cytochrome c, respectively.

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 μM−1 · s−1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 μM−1 · s−1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin π-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions −2, −1, 53 and 152.

The effect of long-term storage on the electronic absorption spectrum and the kinetic properties of cytochrome c peroxidase has been investigated. No detectable differences were observed between freshly isolated enzyme and enzyme stored below -20 degrees C, in the crystalline state, for up to 41 months. The electronic absorption spectrum and the rate of the enzyme-hydrogen peroxide reaction are essentially independent of pH in 0.1 M potassium phosphate buffers for both fresh and stored enzyme. In buffers containing KNO3, the absorption spectrum and the kinetic properties of both fresh and stored enzyme vary with pH, consistent with the titration of an ionizable group with an apparent pKa of 5.5 +/- 0.1. The differences between phosphate- and nitrate-containing buffers are attributed to specific ion effects. In KNO3-containing buffers, the high-pH form of the enzyme reacts rapidly with hydrogen peroxide while the low-pH form is unreactive. Evidence is presented which indicates that both the low-pH and high-pH forms of the enzyme in KNO3-containing buffers are 5-coordinate, high-spin Fe(III) species.

Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme or the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl porphyrin pi-cation-radical species. At pH 6, 100 mM ionic strength, and 25 degrees C, the rate constant for formation of the oxyferryl pi-cation radical has a lower limit of 6 X 10(7) M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 +/- 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.

Cytochrome c (CC) is a small, peripheral membrane heme-protein which transfers electrons between the cytochrome b—c complex and the cytochrome oxidase complex in the mitochondrial respiratory chain. As a model for investigation of the mechanism of electron transfer reactions, the interaction of CC with cytochrome c peroxidase (CCP) has been widely studied. There have been two types of experimental approach: (i) electron transfer within a CC—CCP complex in solution, and (ii) the reduction and oxidation of both CC and CCP measured separately at an electrode in simple electrochemical systems. In this communication we report the first direct demonstration of an electron transfer between CC and CCP across a modified bilayer lipid membrane (BLM). Our calculated rate constant is five to six orders of magnitude lower than that reported for the complex. It has been demonstrated that the formation of a complex is not a necessary condition for electron transfer.