Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase

A covalent complex between purified rat liver microsomal NADPH-cytochrome P-450 reductase and horse cytochrome c was formed through cross-linking studies with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at low ionic strength. The purified cross-linked derivative shows that this product is a 1:1 complex containing one molecule each of the flavoprotein and cytochrome. The covalent complex had almost completely blocked the electron transfer from NADPH to exogenous cytochrome c or the rabbit liver microsomal cytochrome P-450 induced by phenobarbital, indicating that the cross-linked cytochrome c covers the electron-accepting site of the reductase. These results suggest that the covalently cross-linked derivative is a valid model of the noncovalent electron transfer complex. Although the exact number and site of the cross-linked location were not determinable, in cytochrome c the amide bond originates from Lys-13 and in reductase it might be at any one of six different side chain carboxyl groups in the two neighboring cluster acidic residues, Asp-207, -208, and -209, and Glu-213, Glu-214, and Asp-215. It is therefore proposed that the six clustered carboxyl groups on reductase are in an exposed location near the area where one heme edge comes close to the molecular surface.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.     

Proton NMR and electrophoretic studies of the covalent complex formed by cross-linking yeast cytochrome c peroxidase and horse cytochrome c with a water-soluble carbodiimide

The 1:1 covalently cross-linked complex between horse cytochrome c and yeast cytochrome c peroxidase (ccp) has been formed by a slight modification of the method of Waldmeyer and Bosshard [Waldmeyer, B., & Bosshard, H. R. (1985) J. Biol. Chem. 260, 5184-5190]. This earlier study has been extended to show that efficient cross-linking of the two proteins can occur in a variety of buffers over a broad ionic strength range. The substitution of ferrocytochrome c for ferricytochrome c in the cross-linking studies resulted in an increased yield of 1:1 complex (approximately 10-20%) under the conditions studied. An improved method for purifying the covalent complex in relatively large quantities is presented here as are the results of electrophoresis and proton NMR studies of the complex. Both electrophoresis and NMR studies indicate modification of some surface acidic amino acids in the covalent complex by the carbodiimide. The proton hyperfine-shifted resonances of cytochrome c are broadened in the covalent complex relative to free cytochrome c, and the resonances corresponding to the cytochrome c heme 3-CH3 and 8-CH3 groups are shifted closer together in the complex. Integration of NMR resonances confirms a 1:1 complex as the primary cross-linking reaction product. However, we also demonstrate that the covalent complex can be further coupled to ccp and to cytochrome c to form higher molecular weight aggregates.     

Kinetics of intracomplex electron transfer and of reduction of the components of covalent and noncovalent complexes of cytochrome c and cytochrome c peroxidase by free flavin semiquinones

The kinetics of reduction of free flavin semiquinones of the individual components of 1:1 covalent and electrostatic complexes of yeast ferric and ferryl cytochrome c peroxidase and ferric horse cytochrome c have been studied. Covalent cross-linking between the peroxidase and cytochrome c at low ionic strength results in a complex that has kinetic properties both similar to and different from those of the electrostatic complex. Whereas the cytochrome c heme exposure to exogenous reductants is similar in both complexes, the apparent electrostatic environment near the cytochrome c heme edge is markedly different. In the electrostatic complex, a net positive charge is present, whereas in the covalent complex, an essentially neutral electrostatic charge is found. Intracomplex electron transfer within the two complexes is also different. For the covalent complex, electron transfer from ferrous cytochrome c to the ferryl peroxidase has a rate constant of 1560 s-1, which is invariant with respect to changes in the ionic strength. The rate constant for intracomplex electron transfer within the electrostatic complex is highly ionic strength dependent. At mu = 8 mM a value of 750 s-1 has been obtained [Hazzard, J. T., Poulos, T. L., & Tollin, G. (1987) Biochemistry 26, 2836-2848], whereas at mu = 30 mM the value is 3300 s-1. This ionic strength dependency for the electrostatic complex has been interpreted in terms of the rearrangement of the two proteins comprising the complex to a more favorable orientation for electron transfer. In the case of the covalent complex, such reorientation is apparently impeded.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of amino acid replacements in yeast iso-1 cytochrome c on heme accessibility and intracomplex electron transfer in complexes with cytochrome c peroxidase

The kinetics of reduction of wild type and several site-specific mutants of yeast iso-1 cytochrome c (Arg-13----Ile, Gln-16----Ser, Gln-16----Lys, Lys-27----Gln, Lys-72----Asp), both free and in 1:1 complexes with yeast cytochrome c peroxidase, by free flavin semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H2O2-oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme within the electrostatically stabilized complex and the rate constants for intramolecular electron transfer at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Importantly, replacement by uncharged amino acids of Arg or Lys residues thought to be important in orientation and/or stabilization of the electron-transfer complex resulted in increased rates of electron transfer. In all cases, an increase in ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilized 1:1 complex is not optimized for electron transfer and that by neutralization of key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can orient themselves to allow the formation of a more efficient electron-transfer complex.     

Structure of an electron transfer complex. II. Chemical modification of carboxyl groups of cytochrome c peroxidase in presence and absence of cytochrome c

Cytochrome c peroxidase forms an electron transfer complex with cytochrome c. The complex is governed by ionic bonds between side chain amino groups of cytochrome c and carboxyl groups of peroxidase. To localize the binding site for cytochrome c on the peroxidase, we have used the method of differential chemical modification. By this method the chemical reactivity of carboxyl groups (toward carbodiimide/aminoethane sulfonate) was compared in free and in complexed peroxidase. When ferricytochrome c was bound to cytochrome c peroxidase, acidic residues 33, 34, 35, 37, 221, 224, and 1 to 3 carboxyls at the C terminus became less reactive by a factor of approximately 4, relative to the remaining 39 carboxylates of peroxidase. Of the less reactive residues those in the 30-40 region and the 221/224 pair are on opposite sides of the surface area which contains the heme propionates. We, therefore, propose that the binding site for cytochrome c on cytochrome c peroxidase spans the area where one heme edge comes close to the molecular surface. The results are in very good agreement with chemical cross-linking studies (Waldmeyer, B., and Bosshard, H.R. (1985) J. Biol. Chem. 260, 5184-5190); they also support a hypothetical model predicted on the basis of the known crystal structures of cytochrome c and peroxidase (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330).     

Metalloprotein complexes for the study of electron-transfer reactions. Characterization of diprotein complexes obtained by covalent cross-linking of cytochrome c and plastocyanin with a carbodiimide.

Cytochrome c (cyt) and zinc cytochrome c (Zncyt) are separately cross-linked to plastocyanin (pc) by the carbodiimide EDC according to a published method. The changes in the protein reduction potentials indicate the presence of approximately two amide cross-links. Chromatography of the diprotein complexes cyt/pc and Zncyt/pc on CM-52 resin yields multiple fractions, whose numbers depend on the eluent. UV-vis, EPR, CD, MCD, resonance Raman, and surface-enhanced resonance Raman spectra show that cross-linking does not significantly perturb the heme and blue copper active sites. Degrees of heme exposure show that plastocyanin covers most of the accessible heme edge in cytochrome c. Impossibility of cross-linking cytochrome c to a plastocyanin derivative whose acidic patch had been blocked by chemical modification shows that it is the acidic patch that abuts the heme edge in the covalent complex. The chromatographic fractions of the covalent diprotein complex are structurally similar to one another and to the electrostatic diprotein complex. Isoelectric points show that the fractions differ from one another in the number and distribution of N-acylurea groups, byproducts of the reaction with the carbodiimide. Cytochrome c and plastocyanin are also tethered to each other via lysine residues by N-hydroxysuccinimide diesters. Tethers, unlike direct amide bonds, allow mobility of the cross-linked molecules. Laser-flash-photolysis experiments show that, nonetheless, the intracomplex electron-transfer reaction cyt(II)/pc(II)----cyt(III)/pc(I) is undetectable in complexes of either type. Only the electrostatic diprotein complex, in which protein rearrangement from the docking configuration to the reactive configuration is unrestricted, undergoes this intracomplex reaction at a measurable rate.     

A hypothetical model of the cytochrome c peroxidase . cytochrome c electron transfer complex

A hypothetical three-dimensional model of the cytochrome c peroxidase . tuna cytochrome c complex is presented. The model is based on known x-ray structures and supported by chemical modification and kinetic data. Cytochrome c peroxidase contains a ring of aspartate residues with a spatial distribution on the molecular surface that is complementary to the distribution of highly conserved lysines surrounding the exposed edge of the cytochrome c heme crevice, namely lysines 13, 27, 72, 86, and 87. These lysines are known to play a functional role in the reaction with cytochrome c peroxidase, cytochrome oxidase, cytochrome c1, and cytochrome b5. A hypothetical model of the complex was constructed with the aid of a computer-graphics display system by visually optimizing hydrogen bonding interactions between complementary charged groups. The two hemes in the resulting model are parallel with an edge separation of 16.5 A. In addition, a system of inter- and intramolecular pi-pi and hydrogen bonding interactions forms a bridge between the hemes and suggests a mechanism of electron transfer.     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

Cytochrome b5, cytochrome c, and cytochrome P-450 interactions with NADPH-cytochrome P-450 reductase in phospholipid vesicles

Upon incubation of detergent-solubilized NADPH-cytochrome P-450 reductase and either cytochrome b5 or cytochrome c in the presence of a water-soluble carbodiimide, a 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), covalently cross-linked complex was formed. The cross-linked derivative was a heterodimer consisting of one molecule each of flavoprotein and cytochrome, and it was purified to 90% or more homogeneity. The binary covalent complex between the flavoprotein and cytochrome b5 was exclusively observed following incubation of all three proteins including NADPH-cytochrome P-450 reductase, cytochrome b5, and cytochrome c in L-alpha-dimyristoylphosphatidylcholine vesicles, and no heterotrimer could be identified. The isolated reductase-cytochrome b5 complex was incapable of covalent binding with cytochrome c in the presence of EDC. No clear band for covalent complex formation between PB-1 and reductase was seen with the present EDC cross-linking technique. More than 90% of the cross-linked cytochrome c in the purified derivative was rapidly reduced upon addition of an NADPH-generating system, whereas approximately 80% of the cross-linked cytochrome b5 was rapidly reduced. These results showed that in the greater part of the complexes, the flavin-mediated pathway for reduction of cytochrome c or cytochrome b5 by pyridine nucleotide was intact. When reconstituted into phospholipid vesicles, the purified amphipathic derivative could hardly reduce exogenously added cytochrome c, cytochrome b5, or PB-1, indicating that the cross-linked cytochrome shields the single-electron-transferring interface of the flavoprotein. These results suggest that the covalent cross-linked derivative is a valid model of the noncovalent functional electron-transfer complex.     

Binding of arylazidocytochrome c to yeast cytochrome c peroxidase

Cytochrome c derivatives modified with a photoactivatable arylazido group in selected lysine residues were irradiated in the presence of cytochrome c peroxidase (EC 1.11.1.5). A derivative modified at lysine 13 was able to cross-link to the enzyme and inhibit electron transfer activity. Complete inhibition of cytochrome c peroxidase activity was obtained when 1 mol of cytochrome c was covalently bound per mol of cytochrome c peroxidase. Chemical cleavage of the covalent complex has been used for a preliminary characterization of the site of cross-linking of cytochrome c to cytochrome c peroxidase. This linkage site was localized to the NH2 terminal part of cytochrome c peroxidase including residues 1-51.     

Sedimentation equilibrium studies on the interaction between cytochrome c and cytochrome c peroxidase

The interaction between cytochrome c and cytochrome c peroxidase was investigated using sedimentation equilibrium at pH 6,20 degrees C, in a number of buffer systems varying in ionic strength between 1 and 100 mM. Between 10 and 100 mM ionic strengths, the sedimentation of the individual proteins was essentially ideal, and sedimentation equilibrium experiments on mixtures of the two proteins were analyzed assuming ideal solution behavior. Analysis of the distribution of mixtures of cytochrome c and cytochrome c peroxidase in the ultracentrifuge cell based on a model involving the formation of a 1:1 cytochrome c-cytochrome c peroxidase complex gave values of the equilibrium dissociation constant ranging from 2.3 +/- 2.7 microM at 10 mM ionic strength to infinity (no detectable interaction) at 100 mM ionic strength. Attempts to determine the presence of complexes involving two cytochrome c molecules bound to cytochrome c peroxidase were inconclusive.     

A complex of cardiac cytochrome c1 and cytochrome c.

The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of cytochrome c on the methionine-80 side of the heme crevice.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

Definition of the interaction domain for cytochrome c on the cytochrome bc(1) complex. Steady-state and rapid kinetic analysis of electron transfer between cytochrome c and Rhodobacter sphaeroides cytochrome bc(1) surface mutants

The interaction domain for cytochrome c on the cytochrome bc(1) complex was studied using a series of Rhodobacter sphaeroides cytochrome bc(1) mutants in which acidic residues on the surface of cytochrome c(1) were substituted with neutral or basic residues. Intracomplex electron transfer was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 72 (Ru-72-Cc). Flash photolysis of a 1:1 complex between Ru-72-Cc and cytochrome bc(1) at low ionic strength resulted in electron transfer from photoreduced heme c to cytochrome c(1) with a rate constant of k(et) = 6 x 10(4) s(-1). Compared with the wild-type enzyme, the mutants substituted at Glu-74, Glu-101, Asp-102, Glu-104, Asp-109, Glu-162, Glu-163, and Glu-168 have significantly lower k(et) values as well as significantly higher equilibrium dissociation constants and steady-state K(m) values. Mutations at acidic residues 56, 79, 82, 83, 97, 98, 213, 214, 217, 220, and 223 have no significant effect on either rapid kinetics or steady-state kinetics. These studies indicate that acidic residues on opposite sides of the heme crevice of cytochrome c(1) are involved in binding positively charged cytochrome c. These acidic residues on the intramembrane surface of cytochrome c(1) direct the diffusion and binding of cytochrome c from the intramembrane space.     

Characterization of a covalently linked yeast cytochrome c-cytochrome c peroxidase complex: evidence for a single, catalytically active cytochrome c binding site on cytochrome c peroxidase

A covalent complex between recombinant yeast iso-1-cytochrome c and recombinant yeast cytochrome c peroxidase (rCcP), in which the crystallographically defined cytochrome c binding site [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755] is blocked, was synthesized via disulfide bond formation using specifically engineered cysteine residues in both yeast iso-1-cytochrome c and yeast cytochrome c peroxidase [Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580]. Previous studies on similar covalent complexes, those that block the Pelletier-Kraut crystallographic site, have demonstrated that samples of the covalent complexes have detectable activities that are significantly lower than those of wild-type yCcP, usually in the range of approximately 1-7% of that of the wild-type enzyme. Using gradient elution procedures in the purification of the engineered peroxidase, cytochrome c, and covalent complex, along with activity measurements during the purification steps, we demonstrate that the residual activity associated with the purified covalent complex is due to unreacted CcP that copurifies with the covalent complex. Within experimental error, the covalent complex that blocks the Pelletier-Kraut site has zero catalytic activity in the steady-state oxidation of exogenous yeast iso-1-ferrocytochrome c by hydrogen peroxide, demonstrating that only ferrocytochrome c bound at the Pelletier-Kraut site is oxidized during catalytic turnover.     

Binding of horse heart cytochrome c to yeast porphyrin cytochrome c peroxidase: a fluorescence quenching study on the ionic strength dependence of the interaction

The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.     

Formation of electrostatically-stabilized complex at low ionic strength inhibits interprotein electron transfer between yeast cytochrome c and cytochrome c peroxidase

Electron transfer from yeast ferrous cytochrome c to H2O2-oxidized yeast cytochrome c peroxidase has been studied using flash photoreduction methods. At low ionic strength (mu less than 10 mM), where a strong complex is formed between cytochrome c and peroxidase, electron transfer occurs rather slowly (k approximately 200s-1). However, at high ionic strength where the electrostatic complex is largely dissociated, the observed first-order rate constant for peroxidase reduction increases significantly reaching a concentration independent limit of k approximately 1500 s-1. Thus, at least in some cases, formation of an electrostatically-stabilized complex can actually impede electron transfer between proteins.     

A proton NMR study of the non-covalent complex of horse cytochrome c and yeast cytochrome-c peroxidase and its comparison with other interacting protein complexes

Cytochrome-c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) forms a noncovalent 1:1 complex with horse cytochrome c in low ionic strength solution that is detectable by proton NMR spectroscopy. When the entire proton hyperfine-shifted spectrum is considered only five hyperfine resonances exhibit unambiguously detectable shifts: the heme 8-CH3 and 3-CH3 resonances, single proton resonances near 19 ppm and -4 ppm and the methionine-80 methyl group. These shifts are very similar to those observed for the covalently crosslinked complex of cytochrome-c peroxidase and horse cytochrome c, but different from those reported for cytochrome c complexes with flavodoxin and cytochrome b5. By comparison with the shifts reported for lysine-13-modified cytochrome c we conclude that the results reported here support the Poulos-Kraut proposed structure for the molecular redox complex between cytochrome-c peroxidase and cytochrome c. These results indicate that the principal site of interaction with cytochrome-c peroxidase is the exposed heme edge of horse cytochrome c, in proximity to lysine-13 and the heme pyrrole II. The noncovalent cytochrome-c peroxidase-cytochrome c complex exists in the rapid-exchange time limit even at 500 mHz proton frequency. Our data provide an improved estimate of the minimum off-rate for exchanging cytochrome c as 1133 (+/- 120) s-1 at 23 degrees C.