Kinetics of electron transfer between cardiac cytochromes c1 and c.

Abstract

Highly purified cytochrome cl, which consists of only one heme peptide and does not form a stable cl-c complex (cl-H-c complex), was used in studies of electron transfer between cytochromes cl and c. Results show that a stable and ionic-strength-sensitive cl-c complex (i.e., the cl-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 cl and c. A minimum mechanism for electron transfer between these two cytochromes is suggested in accord with the experimental results.The molecular mechanism for electron transfer between cytochrome c1 and cytochrome c remains to be clarified. The kinetics of this reaction were first studied (1) using purified preparations of cl (2) (vol/vol) ethylene glycol, the final concentration of the buffer being 20 mM. The protonic activities (paH) of this buffer were 7.4, 7.5, and 7.6 at 20, 0, and -20°C, respectively (cf. refs. 10 and 11). Fast Kinetics and Rapid-Scanning Measurements. Stoppedflow and rapid-scan measurements were performed using a special stopped-flow mixing device (12), designed and built by INSERM (Montpellier), adapted to a Union Giken (model RA 415 and 401) fast-response spectrophotometer (13,14). The temperature of syringes and mixing chamber of the stopped-flow were maintained at ±0.1°C with a circulating Haake bath model f3-Q. Rapid-scan absorption spectra were measured with a multichannel photodiode (maximum speed, 95 x 103 nm sec-1). The spectra were stored in a memory unit and then analyzed with a digital computer system. The kinetic responses were found to be first order, and the observed rate constants (kobs) were determined from a nonlinear least-squares analysis using a computer fitting program. Single values of rate constants reported are the average of at least five experimental determinations. Kinetic constants have been estimated with an error not exceeding ±20%.Absorption spectra were obtained at 20°C or 2°C using either a Cary 219 or an Aminco DW-2 spectrophotometer equipped with a thermostatted cells holder (15). RESULTS AND DISCUSSIONReactions of c2+ with c3+ were measured during stoppedflow rapid mixing of the two cytochromes in 20 mM sodium cacodylate buffer (pH 7.4) at 20C. A typical tracing of kinetics at 416 nm is shown in Fig. 1A. Fig. 1B (Fig. 2, spectrum A). Another difference spectrum was seen when both cytochromes in the oxidized form were mixed and compared with these two cytoAbbreviation: cl-H-c complex; cytochrome cl-hinge protein-cytochrome c complex. 2026The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Preparation and properties of cardiac cytochrome c1

“…The midpoint potential (+227 mV) for one-band cx is practically the same as that of two-band cx (+225 mV) (Chiang, 1976), and this suggests that the hinge protein does not affect the redox potential (£m) of cytochrome c{. This observation is in agreement with the fact that no direct participation of the hinge protein is observed in the electrontransfer reaction between cytochromes cl and c (Kim et al, 1984) in rapid kinetic studies. Moreover, the enzymatic activity of present cytochrome Ci preparations shows that either one-band or two-band cytochrome c, is active to serve as an electron donor as well as acceptor.…”

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

“…Since the discovery of the hinge protein and its obligatory role for the formation of cytochrome cx-c complex (Kim & King, 1981, 1983Wakabayashi et al, 1982b;, the function of the hinge protein in the mitochondria has remained to be investigated. Studies of structural and func- tional properties of cytochrome q in the presence and absence of the hinge protein (Kim & King, 1987;Kim et al, 1984Kim et al, , 1987a showed that the hinge protein may play a role for stabilizing the structure of cytochrome q. It appears that when the hinge protein is bound to the cytochrome q, the structure of cytochrome q is stabilized and somewhat protected from photoreduction, autoxidation, and other reactions (Kim & King, 1987).…”