The steady state behaviour of cytochrome c oxidase in proteoliposomes

Abstract

Electron transfer to oxygen catalysed by cytochrome c oxidase is accompanied by spectral changes at the binuclear a 3CuB centre, both in the soluble enzyme and in membranous systems, indicating spin or ligand state transitions of an iron that remains ferric. The other haem group, cytochrome a, does not change its spectral characteristics significantly during the steady state, but remains partially reduced until anaerobiosis. Cytochrome a 3 is fully oxidized in each of its major steady state forms, and reduced upon anaerobiosis to a single ferrous species. Although cytochrome a is normally the immediate electron donor to the binuclear centre, its redox state does not alter under conditions in which the flux through the enzyme is changing significantly. A second electron transfer pathway to the binuclear centre may therefore exist, possibly one in which direct reduction of the binuclear a 3, CuB centre by CuA occurs. Both cytochrome a and CuA behave as simple electron transfer centres. The energy‐conserving chemistry takes place at the binuclear centre in concert with the four‐electron reduction of molecular oxygen.

Cytochrome c Oxidase: Biphasic Kinetics Result from Incomplete Reduction of Cytochrome a by Cytochrome c Bound to the High-Affinity Site

“…This is true not only when entry of electrons into cytochrome a is limited to the primary pathway, i.e., with a one to one complex of cytochrome c and cytochrome c oxidase (Figure 3), but also with higher concentrations of cytochrome c when electrons can enter by both pathways (Figure 5). These data are consistent with a number of studies on both detergent-solubilized and membrane-bound cytochrome c oxidase that have shown cytochromes c and a are partially oxidized during steady-state turnover (Kimelberg & Nicholls, 1969;Thomstrom et al, 1988; Morgan & Wikstrom, 1991;Nicholls, 1993).…”

“…None of these three models, however, gives a completely satisfactory explanation of the biphasic steady-state kinetic behavior of cytochrome c oxidase; e.g., Kd,2 is an order of magnitude greater than Km,2, which is inconsistent with the two binding site model (Garber & Margoliash, 1990). One possible explanation for the biphasic kinetics is the inability of electron transfer through a single, tightly bound cytochrome c to fully reduce cytochrome a. Cytochrome c, cytochrome a, and Cua are all known to be partially oxidized during steady-state turnover when the artificial electron donor Af.AUV'./V'-tetramethyl-p-phenylenediamine (TMPD)1 is used as the electron donor (Thomstrom et al, 1988; Morgan & Wikstrom, 1991;Alleyne et al, 1992;Nicholls, 1993). Incomplete reduction of these redox centers is probably due to the rate-limiting reduction of tightly bound cytochrome c by TMPD (Osheroff et al, 1983).…”

The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres

“…The K m for oxygen [9] is very small (1 μM or lower). Consequently at ambient air oxygen levels in vitro, though maybe not in vivo [10], the levels of the oxygen-reactive ferrous haem a 3 species are very low [11,12]. However, the measured partial rates appear inconsistent with the simple overall model of the sub-reactions described above.…”

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes

“…These approaches also have limitations. In the case of steady-state kinetics, a primary difficulty is incomplete reduction of cco redox centers by cyt c, due to the rate-limiting interaction between tightly bound cyt c molecules and the nonphysiological reductant normally used to initiate the reaction (Morgan and Wikstrom, 1991;Nicholls, 1993;Ortega-Lopez and Robinson, 1995). Because optical methods are usually based upon changes in the absorption spectrum of cco, which is dominated by heme a, such spectroscopic monitoring can only measure binding events related to this center (Malatesta et al, 1995).…”