Expression, purification, characterization, and NMR studies of highly deuterated recombinant cytochrome c peroxidase.

Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

Oxidation of bis(terpyridine)cobalt(II) chloride by cytochrome c peroxidase compounds I and II.

The bis(terpyridine)cobalt(II), Co(terpy)2(2+), reduction of cytochrome c peroxidase compound I, CcP-I, has been investigated using stopped-flow techniques as a function of ionic strength in pH 7.5 buffers at 25 degrees C. Co(terpy)2(2+) initially reduces the Trp191 radical site in CcP-I with an apparent second-order rate constant, k2, equal to 6.0+/-0.4x10(6) M(-1)s(-1) at 0.01 M ionic strength. A pseudo-first-order rate constant of 480 s(-1) was observed for the reduction of CcP-I by 79 microM Co(terpy)2(2+) at 0.01 M ionic strength. The one-electron reduction of CcP-I produces a second enzyme intermediate, CcP compound II (CcP-II), which contains an oxyferryl, Fe(IV), heme. Reduction of the Fe(IV) heme in CcP-II by Co(terpy)2(2+) shows saturation kinetics with a maximum observed rate constant, k3max, of 24+/-2 s(-1) at 0.01 M ionic strength. At low reductant concentrations, the apparent second-order rate constant for Co(terpy)2(2+) reduction of CcP-II, k3, is 1.2+/-0.5x10(6) M(-1) s-1. All three rate constants decrease with increasing ionic strength. At 0.10 M ionic strength, values of k2, k3, and k3max decrease to 6.0+/-0.8x10(5) M(-1) s(-1), 1.2+/-0.5x10(5) M(-1) s(-1), and 11+/-3 s(-1), respectively. Both the product, Co(terpy)2(3+), and ferricytochrome c inhibit the rate of Co(terpy)2(2+) reduction of CcP-I and CcP-II. Gel-filtration studies show that a minimum of two Co(terpy)2(3+) molecules bind to the native enzyme in low ionic strength buffers.

4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding.

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 degrees 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(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-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.

Yeast cytochrome c peroxidase expression in Escherichia coli and rapid isolation of various highly pure holoenzymes.

A more efficient 2-day isolation and purification method for recombinant yeast cytochrome c peroxidase produced in Escherichia coli is presented. Two types of recombinant "wild-type" CcP have been produced and characterized, the recombinant nuclear gene sequence and the 294-amino-acid original protein sequence. These two sequences constitute the majority of the recombinant "native" or wild-type CcP currently in production and from which all recombinant variants now derive. The enzymes have been subjected to extensive physical characterizations, including sequencing, UV-visible spectroscopy, HPLC, gel electrophoresis, kinetic measurements, NMR spectroscopy, and mass spectrometry. Less extensive characterization data are also presented for recombinant, perdeuterated CcP, an enzyme produced in >95% deuterated medium. All of these results indicate that the purified recombinant wild-type enzymes are functionally and spectroscopically identical to the native, yeast-isolated wild-type enzyme. This improved method uses standard chromatography to produce highly purified holoenzyme in a more efficient manner than previously achieved. Two methods for assembling the holoenzyme are described. In one, exogenous heme is added at lysis, while in the other heme biosynthesis is stimulated in E. coli. A primary reason for developing this method has been the need to minimize loss of precious, isotope-labeled enzyme and, so, this method has also been used to produce both the perdeuterated and the (15)N-labeled enzyme, as well as several variants.

Temperature, pH, and solvent isotope effects on cytochrome c peroxidase mutant N82A studied by proton NMR.

The mutant of baker's yeast cytochrome c peroxidase-CN with Ala82 in place of Asn82, [N82A]CcPCN, exhibits a complex solution behavior featuring dynamic interconversion among three enzyme forms that so far have only been detected by NMR spectroscopy. Proton NMR studies of [N82A]CcPCN reveal resonances from each of the three enzyme forms and show that the interconversion among forms is controlled by the pH, temperature, and isotope composition (H2O vs. D2O) of the buffer solution. No evidence for a key hydrogen bond between His52 and heme-coordinated cyanide is found in any of the enzyme forms, indicating that disruption of the extensive distal hydrogen bonding network is the source of this phenomenon.

Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix.

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.

Oxidation of the His-52 --> Leu mutant of cytochrome c peroxidase by p-nitroperoxybenzoic acid: role of the distal histidine in hydroperoxide activation.

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.

Metmyoglobin/azide: the effect of heme-linked ionizations on the rate of complex formation.

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.

Metmyoglobin/fluoride: effect of distal histidine protonation on the association and dissociation rate constants.

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) x 10(4) M-1 s-1. An apparent ionization in metmyoglobin with a pKa of 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) x 10(-2) M-1 s-1, about 10(6) times 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) x 10(-4) s-1. The apparent pKa for 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.

Imidazole binding to horse metmyoglobin: dependence upon pH and ionic strength.

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.

Cytochrome c/cytochrome c peroxidase complex: effect of binding-site mutations on the thermodynamics of complex formation.

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.

Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase.

Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-ferricytochrome c, while for CcP-complexed iso-1-ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-ferricytochrome c were elucidated. The results indicated that the iso-1-ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.

Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants.

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.

Regulation of interprotein electron transfer by Trp 191 of cytochrome c peroxidase.

Cytochrome c peroxidase (CcP) reacts with peroxide to form compound I, an intermediate that has an oxy-ferryl iron center and a stable indolyl radical at Trp 191. During the normal catalytic cycle, the oxy-ferryl heme and the Trp 191 radical are reduced by sequential electron transfers from ferrous cytochrome c (Cc). To investigate the role of protein structure in these electron transfer reactions, mutagenesis was used to replace Trp 191 with Phe. The Trp 191-->Phe enzyme [CcP(MI,F191)] reacts with peroxide to form an oxy-ferryl iron center and a transient porphyrin radical. The reaction of Cc from horse and yeast with peroxide-oxidized CcP(MI,F191) was characterized under transient and steady-state conditions. The rate of ET from Cc to the oxy-ferryl heme of CcP(MI,F191) was decreased by at least 10,000-fold relative to the CcP(MI) parent. This effect was observed at 20 and 100 mM ionic strength, with both yeast and horse cytochrome c as the substrate. Thus, Trp 191 is a critical component of all pathways that permit rapid reduction of the oxy-ferryl heme by Cc under these conditions. The reaction of the porphyrin radical with Cc was difficult to characterize, owing to the short half-life of this intermediate. The oxidation of Cc by this intermediate had a maximum rate constant of 32 s-1 at pH 6.0, 25 degrees C. Circumstantial evidence suggests that the porphyrin radical is not directly reduced by Cc, but is instead reduced via a protein-based radical intermediate. The steady-state activity of the mutant enzyme was 300-600-fold lower than the CcP(MI) parent, but kcat is 7-20 times greater than the rate constant for reduction of the oxy-ferryl heme under all conditions examined. Thus, the oxy-ferryl heme is not reduced to the ferric state under steady-state conditions. Transient changes in the absorption spectrum further indicate that steady-state oxidation of Cc2+ by CcP(MI,F191) occurs via reaction of peroxide with the oxy-ferryl enzyme.

Cytochrome c peroxidase-catalyzed oxidation of yeast iso-1 ferrocytochrome c by hydrogen peroxide. Ionic strength dependence of the steady-state parameters.

The cytochrome c peroxidase-catalyzed oxidation of yeast iso-1 ferrocytochrome c by hydrogen peroxide can be understood on the basis of a mechanism involving two cytochrome c-binding sites on cytochrome c peroxidase. Values of the equilibrium dissociation constants for both the high- and low-affinity binding sites determined from the steady-state kinetic measurements agree well with published values obtained by vastly different techniques, providing strong support for the two-binding site mechanism. Maximum enzyme turnover via oxidation of cytochrome c bound at the high-affinity site increases from 2 to 860 s-1 as the ionic strength is increased from 0.010 to 0.20 M. Oxidation of yeast iso-1 ferrocytochrome c is faster in the 2:1 complexes of cytochrome c peroxidase compounds I and II in comparison to the 1:1 complexes. The oxidation rates in the 2:1 complex are macroscopic rate constants equal to the sum of the oxidation rates via both the high- and low-affinity sites. The maximum enzyme turnover via the 2:1 complex increases from 1100 to 2700 s-1 over the ionic strength range 0.010-0.070 M.

Oxidation of yeast iso-1 ferrocytochrome c by yeast cytochrome c peroxidase compounds I and II. Dependence upon ionic strength.

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.

Calorimetric studies on the interaction of horse ferricytochrome c and yeast cytochrome c peroxidase.

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.

Studies of protein-protein association between yeast cytochrome c peroxidase and yeast iso-1 ferricytochrome c by hydrogen-deuterium exchange labeling and proton NMR spectroscopy.

Hydrogen-deuterium (H-D) exchange labeling and proton NMR have been applied to study the protein-protein association between cytochrome c peroxidase (CcP) and yeast iso-1 ferricytochrome c. Specifically, the exchange behavior of individual backbone amide protons of yeast iso-1 ferricytochrome c in both CcP-bound (i.e., complexed) and free (i.e., never in the complex) forms has been investigated and used in an attempt to map the binding site of CcP on yeast iso-1 ferricytochrome c when the noncovalent complex was formed in very low salt solution. The exchange rates of certain amino acid amide protons were significantly slowed down, by up to 40-fold, in the complex compared to the free form. The protected regions on iso-1 ferricytochrome c include parts of the 10's helix and the 70's helix surrounding the cytochrome c heme solvent-exposed edge (the so-called "front side" of iso-1 cytochrome c). These regions are very similar to the cytochrome c peroxidase binding interface on iso-1 ferricytochrome c that has been defined by X-ray crystallographic data. This further supports the direct involvement of the front side of iso-1 cytochrome c in binding with cytochrome c peroxidase. The results from our H-D exchange experiments also indicated that the amide proton exchange rates of Trp59, Asp60, and part of the 90's helix, all of which are located on the opposite side (the "back" side) of ferricytochrome c from the heme solvent-exposed edge, are also retarded upon complex formation.

The effect of the Asn82-->Asp mutation in yeast cytochrome c peroxidase studied by proton NMR spectroscopy.

Proton NMR studies of the mutant of baker's yeast cytochrome c peroxidase-cyanide with the Asn 82-->Asp mutation ([N82D]cytochrome c peroxidase-CN) are presented and compared to the wild-type enzyme. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is an important participant in the initial hydrogen peroxide decomposition step of cytochrome c peroxidase. In wild-type cytochrome c peroxidase-CN, His52 hydrogen bonds to the neighboring Asn82 peptide carbonyl group and to heme-coordinated cyanide. His52 thus manifests itself as an extensively hydrogen bonded histidinium moiety. The principal result from this study is the observation that three hyperfine-shifted resonances disappear from the spectrum of [N82D] cytochrome c peroxidase-CN compared to the wild-type enzyme. All three absent resonances in [N82D]cytochrome c peroxidase-CN belong to His52 and this leads to the conclusion that the result of the mutation has been elimination of the His52-Asn82 and His52-heme-coordinated cyanide hydrogen bonds.