Activation by reduction of the resting form of cytochrome c oxidase: Tests of different models and evidence for the involvement of Cu B

Abstract

(1) The reaction of the resting form of oxidised cytochrome c oxidase from ox heart with dithionite has been studied in the presence and absence of cyanide. In both cases, cytochrome a reduction in 0.1 M phosphate (pH 7) occurs at a rate of 8.2 · 10 4 M −1 · s −1. In the absence of cyanide, ferrocytochrome a 3 appears at a rate ( k obs) of 0.016 s −1. Ferricytochrome a 3 maintains its 418 nm Soret maximum until reduced. The rate of a 3 reduction is independent of dithionite concentration over a range 0.9 mM–131 mM. In the presence or cyanide, visible and EPR spectral changes indicate the formation of a ferric a 3/cyanide complex occurs at the same rate as a 3 reduction in the absence of cyanide. A g = 3.6 signal appears at the same time as the decay of a g = 6 signal. No EPR signals which could be attributed to copper in any significant amounts could be detected after dithionite addition, either in the presence or absence of cyanide. (2) Addition of dithionite to cytochrome oxidase at various times following induction of turnover with ascorbate/TMPD, results in a biphasic reduction of cytochrome a 3 with an increasing proportion of the fast phase of reduction occurring after longer turnover times. At the same time, the predominant steady state species of ferri-cytochrome a 3 shifts from high to low spin and the steady-state level of reduction of cytochrome a drops indicating a shift in population of the enzyme molecules to a species with fast turnover. In the final activated form, oxygen is not required for fast internal electron transfer to cytochrome a 3. In addition, oxygen does not induce further electron uptake in samples of resting cytochrome oxidase reduced under anaerobic conditions in the presence of cyanide. Both findings are contrary to predictions of certain O-loop types of mechanism for proton translocation. (3) A measurement of electron entry into the resting form of cytochrome oxidase in the presence of cyanide, using TMPD or cytochrome c under anaerobic conditions, shows that three electrons per oxidase enter below a redox potential of around +200 mV. An initial fast entry of two electrons is followed by a slow ( k obs ≈ 0.02 s) entry of a third electron. Above +200 mV, the number of electrons taken up in the initial fast phase drops as a redox center (presumably Cu A) titrates with an apparent mid-point potential of +240 mV. The slow phase of reduction remains at the more positive redox values. (4) The results are interpreted in terms of an initial fast reduction of cytochrome a (and Cu A at redox values more negative than +240 mV) followed by a slow reduction of Cu B. Cu B reduction is proposed to spin-uncouple cytochrome a 3 to form a cyanide sensitive center, and trigger a conformational change to an activated form of the enzyme with faster intramolecular electron transfer.