Control of proteoliposomal cytochrome c oxidase: the partial reactions

Abstract

The steady-state spectroscopic behaviour and the turnover of cytochrome c oxidase incorporated into proteoliposomes have been investigated as functions of membrane potential and pH gradient. The respiration rate is almost linearly dependent on [cytochrome c2+] at high flux, but while the cytochrome a redox state is always dependent on the [cytochrome c2+] steady state, it reaches a maximum reduction level less than 100% in each case. The maximal aerobic steady-state reduction level of cytochrome a is highest in the presence of valinomycin and lowest in the presence of nigericin. The proportion of [cytochrome c2+] required to achieve 50% of maximal reduction of cytochrome a varies with the added ionophores; the apparent redox potential of cytochrome a is most positive in the fully decontrolled system (plus valinomycin and nigericin). At low levels of cytochrome a reduction, the rate of respiration is no longer a linear function of [cytochrome c2+], but is dependent upon the redox state of both cytochromes a and c. That is, proteoliposomal oxidase does not follow Smith-Conrad kinetics at low cytochrome c reduction levels, especially in the controlled states. The control of cytochrome oxidase turnover by delta pH and by delta psi can be explained either by an allosteric model or by a model with reversed electron transfer between the binuclear centre and cytochrome a. Other evidence suggests that the reversed electron transfer model may be the correct one.

Conformational switching at cytochrome a during steady-state turnover of cytochrome c oxidase.

“…The duration and heme reduction level of the steady-state phase vary with experimental conditions (6,11,12). Under the conditions used here, the absorbance change at 444 nm during steady-state turnover was consistently 12-15% of the total absorbance change seen upon reaching anaerobicity.…”

“…For optimum efficiency, this conformational switch should be in allosteric communication with the oxygen binding site of the enzyme (5,6). These two conformational states [E1 and E2 in the nomenclature ofMalmstrom (5)] would be present in both the oxidized and reduced forms of the metal center which serves as the site of coupling; the equilibrium between E1 and E2 would be strongly dependent on electron occupancy at the coupling site.…”

Electron microscopy of cytochrome c oxidase-containing proteoliposomes: imaging analysis of protein orientation and monomer-dimer behaviour

“…vided substantial information concerning enzyme function and properties, both in this laboratory (Nicholls and Shaughnessy, 1985;Nicholls et al, 1988aNicholls et al, ,b, 1990Wrigglesworth et al, 1990;Nicholls, 1990) and elsewhere (Krab and Wikstr6m, 1978;Moroney et al, 1984;Papa et al, 1987;Brunori et al, 1987;Gregory and Ferguson-Miller, 1989;Wilson and Prochaska, 1990). Such COV have also been used to study lipid-protein interactions (Longmuir et al, 1977;Falk and Karlsson, 1979;Costello and Frey, 1982;Robinson, 1982;Rigell et al, 1985;Rietveld et al, 1987;Fajer et al, 1989;Powell et al, 1990) and the lipid requirements for reconstituted enzyme orientation, activity and respiratory control (Vik and Capaldi, 1977;Casey et al, 1982;Zhang et al, 1985).…”

Probe and protein orientations in proteoliposomes: electron microscopy and topobiochemistry

“…These are somewhat higher than those obtained previously by water soluble probes [6], but still smaller in millivolt terms than the AY measured in the absence of valinomycin. The major control exerted by such pH gradients, whether these are measured in bulk internal phase or in the surface region probed by HC, must therefore be kinetic rather than thermodynamic in nature, as emphasized previously [7].…”