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The cytochrome c-cytochrome oxidase complex is formed when c reacts with cytochrome oxidase (Kuboyama et al. (1962) Biochem. Biophys. Res. Commun. 9, 534) and the cytochrome c1-cytochrome c complex is formed when c reacts with cytochrome c1 in the presence of the hinge protein (Kim, C.H. and King, T.E. (1981) Biochem. Biophys. Res. Commun. 101, 607). Both complexes are considered to be possible intermediates in electron transfer reaction between these cytochromes. Triply substituted modified cytochrome c by pyridoxal phosphate at lysine residues (Lys-79, 86 and one to be identified) abolishes both complex formations and electron transfer activity with succinate cytochrome c reductase or cytochrome oxidase.

A quantitative method has been developed to analyze the amino acid composition of protein subunits directly from the Coomassie Blue-stained band of polyacrylamide gel columns after electrophoresis. It is an improved method originally reported by Houston (Houston, L. L. (1971) Anal. Biochem. 44, 81--88). The results obtained can be thus used for the calculation of the molar ratios of subunit components of protein. The manipulation of the method and computation of the results are illustrated by a very complicated lipoprotein complex. The subunit molar ratios of the reconstitutively active cytochrome b-c1 complex were determined to be 2, 2, 2, 3, 2, 2, and 5 among the seven bands of the corresponding molecular weights of 53 000, 50 000, 37 000, 30 000, 28 000, 17 000, and 15 000, from gel electrophoretic columns. The amino acid composition of each subunit fraction determined directly from hydrolysis of gel was comparable with that obtained by actual isolation of each subunit.

The indispensability of phospholipid and ubiquinone (Q) in mitochondrial electron transfer was studied by depleting phospholipid and Q in succinate-cytochrome c reductase and then replenishing the depleted enzyme. More than 90% of phospholipid and Q was removed by repeated ammonium sulfate-cholate fractionation. The depleted succinate-cytochrome c reductase showed no enzymatic activity for succinate leads to c or QH2 leads to c and yet retained most of the succinate leads to Q activity. All enzymatic activity was restored upon the addition of Q and phospholipid. Restoration required the addition of Q prior to the addition of phospholipid. Reversing the addition sequence or addition of a mixture of phospholipid and Q resulted only in a small restoration of activities. The conditions for restoration are given in detail. Removal of phospholipid from succinate-cytochrome c reductase resulted in reduction of cytochrome c1 in the absence of exogenous electron donor. Replenishing the preparation with phospholipid brought about the reoxidation of cytochrome c1 in the absence of electron acceptor or oxygen.

A heme-not-containing protein was isolated from cytochrome c1 preparation by gel filtration after carboxymethylation and citraconylation. The amino acid sequence of this protein was determined by the analysis of tryptic and chymotryptic peptides as well as by solid-phase sequence analysis. It consisted of 78 amino acid residues and the molecular weight was calculated to be 9,175. This protein contained a high proportion of glutamic acid and glutamine (27% of the total residues) but no methionine, isoleucine, tyrosine, and tryptophan. The most notable feature was an acidic cluster of 8 consecutive glutamic acid residues near the amino(N)-terminus). The secondary structure was predicted to have a high proportion of helical content. The amino acid composition and N-terminal sequence of a protein independently prepared from bovine heart mitochondria, which is essential to the formation of the cytochrome c1-c complex, suggested that this colorless factor and the present heme-not-containing protein are identical. Evidence shows that another protein, called the non-heme protein, isolated from "two-band" cytochrome c1 preparation is also the same protein as that presented in this paper.

Cytochrome c1 forms an active complex with cytochrome c as previously reported (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36). It also forms a complex with cytochrome oxidase with heme ratio of 1:1. This cytochrome c1.oxidase complex has been purified by ammonium sulfate fractionation and is stable in media of high ionic strength (greater than 0.1 M) but dissociates as the pH deviates from neutral. The purified cytochrome c1 aggregates to an oligomer, presumably a pentamer. No agent has been found to depolymerize isolated c1 without denaturation. However, in the cytochrome c1.oxidase complex, these two cytochromes apparently were depolymerized to form smaller aggregates, if not monomeric units, as judged by sedimentation behavior. Cytochrome c1 also forms a ternary complex with cytochrome c and oxidase in the heme ratio of 1:1:1. This complex can be prepared by any of the following four methods: (i) c1 + c + oxidase: (ii) c1.c complex + oxidase; (iii) c1 + c.oxidase complex: or (iv) c + c1.oxidase complex. The mode of formation of these complexes is all from pure protein-protein interactions. Cytochrome c1 is also incorporated into phospholipid vesicles and these vesicles show about 200 molecules of phospholipid/cytochrome c1 in terms of heme. The spectrophotometric, circular dichroic, sedimentation behavior and enzymic properties of these complexes have been investigated.

1. Ferricytochrome c1 solution was reduced completely between pH 7 and 10 by illumination under anaerobic conditions. Photoreduction was not affected by the ionic strength of the medium. However, it did not take place at pH lower than 6 or higher than 10, or in the presence of p-hydroxymercuric benzoate. The ferricyanide-reoxidized photoreduced c1 was not further reduced upon illumination. The reductant was most probably a specific sulfhydryl group in the subunit containing the heme of the cytochrome since this subunit contained one less p-HMB-titratable group in the photoreduced sample than in the untreated preparation. 2. The photoreduced cytochrome c1 showed the same spectra as the native cytochrome, and was not reactive with carbon monoxide. The equilibrium constant of the reaction c12+ + c3+ equilibrium c13+ + c2+ for the photoreduced c1 was found to be slightly lower (Keq = 2.6) than that for the native c1 (Keq = 3.5). The antimycin A-sensitive electron acceptor activity of ferricyanide-reoxidized photoreduced c13+ catalyzed by succinate-cytochrome c reductase was about 80% of that of the native c1. 3. A somewhat simplified method for isolation of cytochrome c1 was developed. Anaerobic ammonium sulfate fractionation and calcium phosphate gel chromatography were still used in order to achieve the purity level of about 25 nmol of heme/mg of protein. The cytochrome c1 prepared by this procedure showed the same properties tested as that by the beta-mercaptoethanol method (Yu, C.A., Yu, L., and King, T.E. (1972) J. Biol. Chem. 247, 1012-1019).

A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.

A tryptic resistant heme peptide has been prepared and purified from cardiac cytochrome c1. This purified peptide is not further hydrolyzed by reactions of other proteolytic enzymes, such as pronase. The peptide contains 2 residues each of serine, cysteine and valine, and 1 residue each of alanine, methionine, tyrosine, histidine, arginine, proline, glutamic acid (glutamine) and aspartic acid. The intensity of the absorption spectrum of the peptide has been found to be dependent upon, but the positions of the absorption maxima do not vary with, concentration. The heme peptide does not show multiple splitting of absorption peaks at liquid N2 temperatures as does the intact cytochrome C1. However, cyanide rapidly reacts with the peptide and causes significant spectral changes. CD spectra of the peptide exhibit a typical profile of a non-structured heme peptide with positive CD bands in the Soret region and around 250 nm, and a broad negative extreme of 320-360 nm. The similarities and differences between the tryptic resistant heme peptides from cytochromes c1 and c have been compared.

A role of the hinge protein is studied in the electron transfer reaction between cytochromes c1 and c, using highly purified "one-band" cytochrome c1 and "two-band" cytochrome c1. The results show that the hinge protein (Hp), which is essential for a stable ionic strength-sensitive c1-Hp-c complex, seems to play a certain role in electron transfer between cytochromes c1 and c; Keq for electron transfer reaction between cytochromes c1 and c in the presence of the hinge protein is found to be about 40% higher than that in the absence of the hinge protein at low ionic strength, but no difference exists at high ionic strength. We propose a hypothesis that the hinge protein may function as regulator for the electron transfer reaction between cytochromes c1 and c, and this may be at least one of the roles of the hinge protein in mitochondria.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that

Highly purified cytochrome c1, which consists of only one heme peptide and does not form a stable c1-c complex (c1-H-c complex), was used in studies of electron transfer between cytochrome c1 and c. Results show that a stable and ionic-strength-sensitive c1-c complex (i.e., the c1-H-c complex) in the forms of the various oxidation states is not required, in contrast to the current belief of the participation of the complex in the electron transfer between cytochromes c1 and c. A minimum mechanism for electron transfer between these two cytochromes is suggested in accord with the experimental results.

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 complete primary structure of bovine heart cytochrome c1 was established by analyses of peptide fragments prepared by digestion using trypsin, staphylococcal protease, and chymotrypsin and by cyanogen bromide cleavage of cytochrome c1 and its derivatives. The total number of amino acid residues is 241, giving a molecular weight of 27,924 including the heme group. The NH2- and COOH-terminal residues are serine and lysine, respectively. One characteristic of the protein is that cytochrome c1 contains 43.6% hydrophobic residues and the polarity is estimated to be 41.1%. No clear homology was found between cytochrome c1 and other membranous proteins such as cytochrome b5 or the subunits of cytochrome oxidase for which sequences have been reported. Cytochrome c1 is predicted to have a high content of alpha-helix (46%). Partial sequence studies were also carried out on cytochrome c1 preparations obtained by different procedures and showed that there is no difference among the sequences of various preparations of cytochrome c1. The presence of a hydrophobic cluster near the COOH-terminal region indicates that the COOH-terminal region of cytochrome C1 associates with, or is buried in, the phospholipid bilayer of the mitochondrial membrane.

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].

Kinetics measurements of the electron transfer between ferricytochrome c and liposomal ferrocytochrome c1 (with and without the hinge protein) were performed. The observed rate constants(kobs) of electron transfer between liposomal ferrocytochrome c1 and ferricytochrome c at different ionic strengths were measured in cacodylate buffer, pH 7.4, at 2 C. The effect of ionic strength on the rate constant(kobs) of electron transfer between liposomal cytochrome c1 and cytochrome c is far greater than that in the solution kinetics (Kim, C.H., Balny, C. and King, T.E. (1987) J. Biol. Chem. 262, 8103-8108). The result demonstrates that the membrane bound cytochrome c1 creates a polyelectrolytic microenvironment which appears to be involved in the control of electron transfer and can be modulated by the ionic strength. The involvement of electrostatic potentials in the electron transfer between the membrane bound cytochrome c1 and cytochrome c is discussed in accord with the experimental results and a polyelectrolyte theory.

A method for the large-scale isolation of beef heart mitochondrial cytochrome c1 in high purity was developed. This method gave higher yield of "one-band" cytochrome c1 than previously reported [Kim, C. H., & King, T. E. (1981) Biochem. Biophys. Res. Commun. 102, 607-614]. In addition, the present method was effective in the preparation of "two-band" cytochrome c1 which was used to prepare the hinge protein according to the principle of sequential resolution [Kim, C. H., & King, T. E. (1983) J. Biol. Chem. 258, 13543-13551]. The isolation of one-band and two-band cytochrome c1 by this procedure could be completed within 3 or 4 days starting with succinate-cytochrome c reductase. One-band cytochrome c1 showed a molecular weight of 44,000 by sedimentation equilibrium and 29,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The disparities in these data from the actual value of 27,924 by amino acid sequence analysis, as previously reported [Wakabayashi, S., Matsubara, H., Kim, C. H., & King, T. E. (1982) J. Biol. Chem. 257, 9335-9344], are most probably due to the formation of detergent or detergent-phosphate complex. A comparison of some properties of one-band cytochrome c1 with those of two-band cytochrome c1 clearly showed significant differences between the two preparations. These results suggest the hypothesis that one of the possible roles of the hinge protein in the mitochondrial respiratory chain is to stabilize the conformation of cytochrome c1.

Investigations into the nature of the axial heme ligands, the strength of the heme crevice, the reactivity with cyanide, and the ascorbate reducibility of cytochrome c1 were performed to explore structure-function relationships of cytochrome c1. The existence of an absorbance band at 690 nm, which was quenched by raising the pH with a pK of 9.2 corresponding to a low spin-low transition, suggested that a methionine residue probably functioned as one of the axial heme iron ligands in this cytochrome. Spectral titrations of cytochrome c1 in the low pH range showed a markedly elevated pK for the low spin-high spin transition relative to cytochrome c. Denaturation studies with urea, the absence of any reaction with cyanide, and the evidence from other lines would appear to indicate that the heme group of cytochrome c1 was reduced by ascorbate at approximately 5% of the rate of reduction of cytochrome c but this rate dramatically increased with increasing pH concomitant with the disappearance of the 690 nm absorbance band. Circular dichroic spectra substantiated that elevated pH produced conformational changes localized to the heme crevice and probably also the regions containing aromatic residues. The enhanced rate of ascorbate reduction was perhaps a consequence of the increased accessibility of the heme iron to ascorbate. Major unfolding of the protein in 8 M urea, however, completely abolished the ascorbate reducibility of cytochrome c1. The buried nature of the heme group of cytochrome c1 would probably preclude transfer of an electron from cytochrome c1 to cytochrome c through a direct Fe-Fe or a heme-heme interaction. This poses an important question concerning the mechanism of this electron transfer between these two cytochromes not only in mitochondria but also in solution.