Control of electron transfer by the electrochemical potential gradient in cytochrome-c oxidase reconstituted into phospholipid vesicles.

P Sarti,F Malatesta,G Antonini,B Vallone,M Brunori

doi:10.1016/s0021-9258(19)39396-2

P Sarti, F Malatesta + Show 3 more

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https://doi.org/10.1016/s0021-9258(19)39396-2

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Abstract

The kinetics of electron transfer between cytochrome-c oxidase and ruthenium hexamine has been characterized using the native enzyme or its cyanide complex either solubilized by detergent (soluble cytochrome oxidase) or reconstituted into artificial phospholipid vesicles (cytochrome oxidase-containing vesicles). Ru(NH3)2+6 (Ru(II] reduces oxidized cytochrome a, following (by-and-large) bimolecular kinetics; the second order rate constant using the cyanide complex of the enzyme is 1.5 x 10(6) M-1 s-1, for the enzyme in detergent, and slightly higher for COV. In the case of COV the kinetics are not affected by the addition of ionophores. Upon mixing fully reduced cytochrome oxidase with oxygen (in the presence of excess reductants), the oxidation leading to the pulsed enzyme is followed by a steady state phase and (eventually) by complete re-reduction. When the concentrations of dioxygen and oxidase are sufficiently low (micromolar range), the time course of oxidation can be resolved by stopped flow at room temperature, yielding an apparent bimolecular rate constant of 5 x 10(7) M-1 s-1. After exhaustion of oxygen and end of steady state, re-reduction of the pulsed enzyme by the excess Ru(II) is observed; the concentration dependence shows that the rate of re-reduction is limited at 3 s-1 in detergent; this limiting value is assigned to the intramolecular electron transfer process from cytochrome a-Cua to the binuclear center. Using the reconstituted enzyme, the internal electron transfer step is sensitive to ionophores, increasing from 2-3 to 7-8 s-1 upon addition of valinomycin and carbonyl cyanide m-chlorophenylhydrazone. This finding indicates for the first time an effect of the electrochemical potential across the membrane on the internal electron transfer rate; the results are compared with expectations based on the hypothesis formulated by Brunori et al. (Brunori, M., Sarti, P., Colosimo, A., Antonini, G., Malatesta, F., Jones, M.G., and Wilson, M.T. (1985) EMBO J. 4, 2365-2368), and their bioenergetic relevance is discussed with reference to the proton pumping activity of the enzyme.

Highlights

In spite of complexities due to the binding of Ru(I1) to the vesicles, the kinetics of reduction of the CN-inhibited enzyme in COV yields results consistent with a bimolecular process involving the reduction of cytochrome a by Ru(I1) with a second order rate constant of 2.5 x lo6 M-’ s-l
This process is not affected by ionophores, as shown by the identity of the time courses observed in the presence and absence of valinomycin and CCCP at micromolar concentrations
Avigliano for providing oxidase and Emilio D’Itri for skillful for critsamples technical

Summary

AND METHODS

Cytochrome oxidase from beef heart was prepared according to Yonetani [17] with minor modifications; the final pellet was dissolved using 0.1 M phosphate buffer, pH 7.4, containing 0.5% Tween 80. (b) The protein, fully reduced with Ru(I1) and ascorbate, was mixed with buffer containing variable amounts of oxygen and the same reductants (always in excess over oxygen). Under these conditions the enzyme is rapidly oxidized by oxygen and subsequently rereduced by the excess reductants, the number of turnovers elapsed depending on oxygen and oxidase concentrations. A typical experiment was carried out as follows: the protein (0.9-1.25 PM), degassed and nitrogen-equilibrated, was reduced in a gas-proof syringe with Ru(II), at different concentrations, in the presence of excess ascorbate (1 mM).

RESULTS

Summary

DISCUSSION