Preparation and properties of cardiac cytochrome c1

Abstract

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.

Structure and Function of CytochromebcComplexes

“…Kim & King (151,152) later developed improved preparations of one-and two-subunit cytochrome c 1 . Subunit 8 [the "hinge protein for interaction of cytochrome c 1 and cytochrome c"(51)] was shown to be required for high-affinity binding of cytochrome c (151)(152)(153).…”

Effect of the hinge protein on the heme iron site of cytochrome c1

“…Radiation damage is not a likely explanation, because no progressive changes in the spectra with time were observed, and the optical spectra appeared to be normal after X-ray irradiation. It is possible that the observed effect is due to the reaction of two-band cytochrome c, with X-ray photoproducts, but this seems to be unlikely, since the heme iron site in two-band cytochrome cx is less exposed than that in the one-band cytochrome Ci (Kim & King, 1987). One would expect that one-band cytochrome c, would be more easily altered than two-band cytochrome cx.…”

“…Recently we have reported the role of the hinge protein (Kim & King, 1981, 1983; Wakabayashi et al, 1982b; , which stabilizes the cytochrome Ci~c complex (Chiang et al, 1976; Kim & King, 1981), in electron-transfer reaction between cytochrome c, and cytochrome c (Kim et al, 1987a,b). The effect of the hinge protein on some properties of cytochrome c¡ has been also reported (Kim & King, 1987). In those reports, we proposed that the hinge protein may function as a regulator in the electron-transfer reaction between cytochromes c, and c by stabilizing cytochrome cl in a specific conformation.…”

“…from the cytochrome b-cx complex to cytochrome c in the respiratory chain of mitochondria. Although structural properties of cytochrome cu including the amino acid sequence (Wakabayashi et al, 1982a; King, 1983; Kim & King, 1987), have been extensively studied, the molecular mechanism of electron transfer between cytochrome c¡ and c is still not clearly understood because of a lack of detailed structural information.…”

“…In this paper we present the first X-ray spectroscopic evidence for conformational changes on the heme iron site of cytochrome ci when the hinge protein is bound to cytochrome Enzyme Preparations. One-band and two-band cytochromes Ci were prepared by the method previously reported (Kim & King, 1987). Cytochromes c, and c were oxidized with an excess amount of solid potassium ferricyanide and passed through a Sephadex G-25 column equilibrated with 50 mM phosphate buffer, pH 7.4.…”