A complex of cardiac cytochrome c1 and cytochrome c.

Y L Chiang,L S Kaminsky,T E King

doi:10.1016/s0021-9258(17)33922-4

Abstract

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.

Highlights

The concept of the respiratory chain as the basis of bioenergetics was conceived some 50 years ago by Keilin with his discovery of cytochrome [2] and further defined by Keilin and Hartree and other investigators in the field
Complex Formation with Cytochrome c, and Various Forms of Cytochrome c-Only a single band at the position of the void volume was obtained when equimolar quantities of beef heart cytochrome c1 and horse or beef heart cytochrome c were mixed in 10 mM phosphate buffer and passed through a Sephadex
S The molar ratios of the complexes formed from cytochrome c1 and cytochrome c or the modified forms of c described in this paper refer to cl:c in terms of heme

Summary

Introduction

The concept of the respiratory chain as the basis of bioenergetics was conceived some 50 years ago by Keilin with his discovery of cytochrome [2] and further defined by Keilin and Hartree (cf. Ref. 3) and other investigators in the field (cf. Ref. 4). It was only with the recent availability of a sufficient quantity of cytochrome c, [5, 6] that the opportunity arose to investigate the interaction and complex formation of these two lipid-free components of the chain We thought that such studies would be useful to bioenergetics, especially the mechanism of mitochondrial electron transfer. Cyanide reacts with ferricytochrome c and apparently displaces the axial ligand of methionine-80, while it is totally unreactive with cytochrome cI (l), probably as a consequence of the more deeply buried nature of its heme iron [9] Both cytochromes c, and c are reducible by ascorbate the difference in the rates of reduction is large [7] and sufficient to differentiate these two cytochromes in the complex. Cytochrome ct exhibits a much greater susceptibility than cytochrome c to photoreduction under anaerobic conditions in the absence of exogenous reducing agents [6]

Methods

Results

Conclusion