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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.

The reaction between cytochrome c (Cc) and Rhodobacter sphaeroides cytochrome c oxidase (CcO) was studied using a cytochrome c derivative labeled with ruthenium trisbipyridine at lysine 55 (Ru-55-Cc). Flash photolysis of a 1:1 complex between Ru-55-Cc and CcO at low ionic strength results in electron transfer from photoreduced heme c to Cu(A) with an intracomplex rate constant of k(a) = 4 x 10(4) s(-1), followed by electron transfer from Cu(A) to heme a with a rate constant of k(b) = 9 x 10(4) s(-1). The effects of CcO surface mutations on the kinetics follow the order D214N > E157Q > E148Q > D195N > D151N/E152Q approximately D188N/E189Q approximately wild type, indicating that the acidic residues Asp(214), Glu(157), Glu(148), and Asp(195) on subunit II interact electrostatically with the lysines surrounding the heme crevice of Cc. Mutating the highly conserved tryptophan residue, Trp(143), to Phe or Ala decreased the intracomplex electron transfer rate constant k(a) by 450- and 1200-fold, respectively, without affecting the dissociation constant K(D). It therefore appears that the indole ring of Trp(143) mediates electron transfer from the heme group of Cc to Cu(A). These results are consistent with steady-state kinetic results (Zhen, Y., Hoganson, C. W., Babcock, G. T., and Ferguson-Miller, S. (1999) J. Biol. Chem. 274, 38032-38041) and a computational docking analysis (Roberts, V. A., and Pique, M. E. (1999) J. Biol. Chem. 274, 38051-38060).

Beef heart cytochrome c oxidase was labeled at a single sulfhydryl group by treatment with 5 mM N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS) at pH 8.0 for 4 h. Sodium dodecyl sulfate gel electrophoresis revealed that the enzyme was exclusively labeled at subunit III, presumably at Cys-115. The high affinity phase of the electron transfer reaction with horse cytochrome c was not affected by acetylamidoethyl-1-aminonaphthalene-5-sulfonate (AEDANS) labeling. Addition of horse cytochrome c to dimeric AEDANS-cytochrome c oxidase resulted in a 55% decrease in the AEDANS fluorescence due to the formation of a 1:1 complex between the two proteins. Forster energy transfer calculations indicated that the distance from the AEDANS label on subunit III to the heme group of cytochrome c was in the range 26-40 A. In contrast to the results with the dimeric enzyme, the fluorescence of monomeric AEDANS-cytochrome c oxidase was not quenched at all by binding horse heart cytochrome c, indicating that the AEDANS label on subunit III was at least 54 A from the heme group of cytochrome c. These results support a model in which the lysines surrounding the heme crevice of cytochrome c interact with carboxylates on subunit II of one monomer of the cytochrome c oxidase dimer and the back of the molecule is close to subunit III on the other monomer. In order to identify the cysteine residues that ligand copper A, a new procedure was developed to specifically remove copper A from cytochrome c oxidase by incubation with 2-mercaptoethanol followed by gel chromatography. Treatment of the copper A-depleted cytochrome c oxidase preparation with 1,5-I-AEDANS resulted in labeling sulfhydryl groups on subunit II as well as on subunit III. No additional subunits were labeled. This result indicates that the copper A binding site is located at cysteines 196 and/or 200 of subunit II and that removal of copper A exposes these residues for labeling by 1,5-I-AEDANS. Alternative copper A depletion methods involving incubation with bathocuproine sulfonate (Weintraub, S.T., and Wharton, D.C. (1981) J. Biol. Chem. 256, 1669-1676) or p-(hydroxymercuri)benzoate (Li, P.M., Gelles, J., Chan, S.I., Sullivan, R.J., and Scott, R.A. (1987) Biochemistry 26, 2091-2095) were also investigated. Treatment of these preparations with 1,5-I-AEDANS resulted in labeling cysteine residues on subunits II and III. However, additional sulfhydryl residues on other subunits were also labeled, preventing a definitive assignment of the location of copper A using these depletion procedures.

Modification of the three carboxyl groups on adrenodoxin using a water-soluble carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) caused a weakening of the binding of this iron-sulfur protein to both its electron donor protein, adrenodoxin reductase, and its electron acceptor protein, cytochrome P-450scc. Based upon the proximity of the modified groups, the site on adrenodoxin for interaction with the other two proteins is likely to be either identical or highly overlapping, and formation of a ternary complex among the proteins is precluded. Upon incubation of adrenodoxin and either adrenodoxin reductase or cytochrome P-450 plus the carbodiimide (1:1), covalently cross-linked species were formed. When all three proteins were incubated with the cross-linker, only the binary complexes were formed, and no higher order (e.g. 1:1:1 or 1:2:1) complexes were seen. These studies indicate that adrenodoxin forms exclusive binary complexes with its electron transfer partner proteins, and thus provide a physical explanation for the proposed role of adrenodoxin as a mobile electron shuttle between NADPH-adrenodoxin reductase and cytochrome P-450scc.

The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 “hinge” peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.

Modification of carboxyl groups on putidaredoxin with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) resulted in loss of putidaredoxin reductase activity. The modification did not affect the visible absorption spectrum of putidaredoxin, indicating that the iron-sulfur center was not perturbed. In order to identify the carboxyl groups labeled by EDC, native and EDC-treated putidaredoxin were digested with a combination of trypsin and Staphylococcus aureus protease, and the resulting peptides were separated by high pressure liquid chromatography. The most heavily modified carboxyl groups were found to be those at residues 58, 65, 67, 72, and 77. These carboxyl groups are located in the same general region of the protein as those on adrenodoxin that have been shown to be involved in binding to both adrenodoxin reductase and cytochrome P-450scc. Chemical modification was also used to compare the role of lysine, arginine, and histidine residues on putidaredoxin and adrenodoxin. Modification of lysine and arginine residues had no effect on the reductase activity of either protein. The reductase activity of adrenodoxin was unaffected by labeling with 1 eq of diethyl pyrocarbonate/histidine residue, but labeling with a second equivalent completely abolished both activity and the iron-sulfur center spectrum. In contrast, modification of the 2 histidines in putidaredoxin with 1 eq each resulted in nearly complete loss of reductase activity. There was no significant activity for adrenodoxin in the putidaredoxin reductase assay or for putidaredoxin in the adrenodoxin reductase assay, demonstrating that, in spite of the structural similarity between the two proteins, they are not interchangeable functionally.

The single free cysteine at residue 95 of bovine adrenodoxin was labeled with the fluorescent reagent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonate (1,5-I-AEDANS). The modification had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc, suggesting that the AEDANS group at Cys-95 was not located at the binding site for these molecules. Addition of adrenodoxin reductase, cytochrome P-450scc, or cytochrome c to AEDANS-adrenodoxin was found to quench the fluorescence of the AEDANS in a manner consistent with the formation of 1:1 binary complexes. Förster energy transfer calculations indicated that the AEDANS label on adrenodoxin was 42 A from the heme group in cytochrome c, 36 A from the FAD group in adrenodoxin reductase, and 58 A from the heme group in cytochrome P-450scc in the respective binary complexes. These studies suggest that the FAD group in adrenodoxin reductase is located close to the binding domain for adrenodoxin but that the heme group in cytochrome P-450scc is deeply buried at least 26 A from the binding domain for adrenodoxin. Modification of all the lysines on adrenodoxin with maleic anhydride had no effect on the interaction with either adrenodoxin reductase or cytochrome P-450scc, suggesting that the lysines are not located at the binding site for either protein. Modification of all the arginine residues with p-hydroxyphenylglyoxal also had no effect on the interaction with adrenodoxin reductase or cytochrome P-450scc. These studies are consistent with the proposal that the binding sites on adrenodoxin for adrenodoxin reductase and cytochrome P-450scc overlap, and that adrenodoxin functions as a mobile electron carrier.

Yeast iso‐1‐cytochrome ccovalently modified at cysteine‐102 with (4‐bromomethyl‐4′‐methylbipyridine)[bis(bi‐pyridine)]Ru2+(Ru‐102‐Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome coxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash‐induced photoreduction of Ru‐102‐Cyt cheme has been measured and compared to photovoltaic responses observed with Ru(II)(bipy‐ridyl)3(RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389–393]. At low ionic strength, when Ru‐102‐Cyt cforms a tight electrostatic complex with COX, flash‐activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 μs transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme aby CuA. CuAreduction by Ru‐102‐Cyt cturns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (τ ca. 4 ms) following the 50 μs initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru‐102‐Cyt cas electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash‐induced electrogenic response in the presence of Ru‐102‐Cyt creveals a large slow phase of Δψ generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 μM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru‐102‐Cyt c2+generated by the flash in a solution.

Treatment of cytochrome P-450sccwith fluorescein isothiocyanate (FITC) resulted in covalent labeling with 1.0 ± 0.1 eq of FITC. Reverse-phase high performance liquid chromatography of tryptic and chymotryptic digests of the labeled protein revealed that a single FITC-labeled peptide accounted for 75% of the label. This peptide was found to be specifically labeled at lysine 338 by amino acid sequencing. The modification of lysine 338 with FITC resulted in 85 ± 15% inhibition of adrenodoxin binding to cytochrome P-450scc. In a complementary experiment it was found that if a complex between adrenodoxin and native cytochrome P-450scc was formed in the presence of cholesterol and then treated with FITC, there was almost no labeling of lysine 338. The modification of lysine 338 by FITC was not inhibited by 22(R)-hydroxycholesterol, the first intermediate in the side chain cleavage reaction which binds to the active site 300 times more tightly than cholesterol itself. These experiments suggest that lysine 338 is located at the binding site for adrenodoxin and electrostatically interacts with one of the carboxylate groups on adrenodoxin that has been implicated in binding. The fluorescence emission of the FITC label on cytochrome P-450scc was only 14% as large as that of an equivalent concentration of FITC-labeled bovine serum albumin, suggesting that it was quenched by Forster energy transfer to the heme group.

Modification of bovine adrenodoxin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) dramatically inhibited the reaction with adrenodoxin reductase (EC 1.18.1.2). The modification did not cause any change in the visible spectrum of adrenodoxin, indicating that the iron-sulfur center was not perturbed. Furthermore, the anomalous fluorescence of Tyr 82 was not changed in either intensity or wavelength. The inhibition was accompanied by the covalent incorporation of 14C-labeled EDC into adrenodoxin. The sites modified by EDC were determined by hydrolyzing adrenodoxin with either trypsin or Staphylococcus aureus protease and separating the resulting peptides by reverse phase high pressure liquid chromatography. The major carboxyl groups modified were found to be at Glu 74, Asp 79, and Asp 86, which are located in a sequence containing a high negative charge density. We propose that the conversion of negatively charged carboxylate groups at these residues to bulky, positively charged EDC-carboxyl groups inhibits the reaction with the reductase. EDC was also found to cross-link adrenodoxin to cytochrome c in yields up to 90%. The cross-links were found to involve the formation of amide linkages between carboxyl groups on adrenodoxin and the lysine amino groups surrounding the heme crevice of cytochrome c.

The interaction of the Rhodospirillum rubrum cytochrome bc1 complex with R. rubrum cytochrome c2 and horse cytochrome c was studied using specific lysine modification and ionic strength dependence methods. In order to define the reaction domain on cytochrome c2, several fractions consisting of mixtures of singly labeled carboxydintrophenyl-cytochrome c2 derivatives were employed. Fraction A consisted of a mixture of derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2, while fractions C1, C2, C3, and C4 were mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The rate of the reaction of fraction A was found to be nearly the same as that of native cytochrome c2. However, the rate constants of fractions C1-C4 were found to be more than 20-fold smaller than that of native cytochrome c2. These results indicate that lysine residues surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site on the cytochrome bc1 complex. Since the same domain is involved in the reaction with the photosynthetic reaction center, cytochrome c2 must undergo some type of rotational or translational diffusion during electron transport in R. rubrum. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, and 87 surrounding the heme crevice was found to significantly lower the rate of the reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse cytochrome c also involves the heme crevice domain.

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.

Flavocytochrome c552 from Chromatium vinosum catalyzes the oxidation of sulfide to sulfur using a soluble c-type cytochrome as an electron acceptor. Mitochondrial cytochrome c forms a stable complex with flavocytochrome c552 and may function as an alternative electron acceptor in vitro. The recognition site for flavocytochrome c552 on equine cytochrome c has been deduced by differential chemical modification of cytochrome c in the presence and absence of flavocytochrome c552 and by kinetic analysis of the sulfide:cytochrome c oxidoreductase activity of m-trifluoromethylphenylcarbamoyl-lysine derivatives of cytochrome c. As with mitochondrial redox partners, interaction occurs around the exposed heme edge at the “front face” of cytochrome c. However, the domain recognized by flavocytochrome c552 seems to extend to the right of the heme edge, whereas the site of interaction with mitochondrial cytochrome c oxidase and reductase is more to the left. Km but not Vmax of the electron transfer reaction with mitochondrial cytochrome c increases with increasing ionic strength. The correlation of chemical modification and ionic strength dependence data indicates that the electrostatic interaction between the two hemoproteins involves fewer ionic bonds than that with other redox partners of cytochrome c.

In order to define the interaction domain on Rhodospirillum rubrum cytochrome c2 for the photosynthetic reaction center, positively charged lysine amino groups on cytochrome c2 were modified to form negatively charged carboxydinitrophenyl lysines. The reaction mixture was separated into six different fractions by ion exchange chromatography on carboxymethylcellulose and sulfopropyl-Sepharose. Peptide mapping studies indicated that fraction A consisted of a mixture of singly labeled derivatives modified at lysines 58, 81, and 109 on the back of cytochrome c2. Fractions C1, C2, C3, and C4 were found to be mixtures of singly labeled derivatives modified at lysines 9, 13, 75, 86, and 88 on the front of cytochrome c2 surrounding the heme crevice. The photooxidation of the carboxydinitrophenyl-cytochrome c2 derivatives by reaction centers purified from R. rubrum was measured following excitation with a laser pulse. The second-order rate constant of fraction A modified at backside lysines was found to be 2.3 × 10(7) M-1 s-1, nearly the same as that of native cytochrome c2, 2.6 × 10(7) M-1 s-1. However, the rate constants of fractions C1-C4 were found to be 6 to 12-fold smaller than that of native cytochrome c2. These results indicate that lysines surrounding the heme crevice of cytochrome c2 are involved in electrostatic interactions with carboxylate groups at the binding site of the reaction center. The reaction rates of horse heart cytochrome c derivatives modified at single lysine amino groups with trifluoroacetyl or trifluoromethylphenylcarbamoyl were also measured. Modification of lysines 8, 13, 25, 27, 72, 79, or 87 surrounding the heme crevice was found to significantly lower the rate of reaction, while modification of lysines in other regions had no effect. This indicates that the reaction of horse heart cytochrome c with the reaction center also involves the heme crevice domain.