ROUTES OF ELECTRON-TRANSFER IN BEEF-HEART CYTOCHROME-C-OXIDASE - IS THERE A UNIQUE PATHWAY USED BY ALL REDUCTANTS

Cytochrome c oxidase oxidizes several hydrogen donors, including TMPD (N,N,N',N'-tetramethyl-p-phenyl-enediamine) and DMPT (2-amino-6,7-dimethyl-5,6,7,8-tetrahydropterine), in the absence of the physiological substrate cytochrome c. Maximal enzyme turnovers with TMPD and DMPT alone are rather less than with cytochrome c, but much greater than previously reported if extrapolated to high reductant levels and (or) to 100% reduction of cytochrome a in the steady state. The presence of cytochrome c is, therefore, not necessary for substantial intramolecular electron transfer to occur in the oxidase. A direct bimolecular reduction of cytochrome a by TMPD is sufficient to account for the turnover of the enzyme. Cu(A) may not be an essential component of the TMPD oxidase pathway. DMPT oxidation seems to occur more rapidly than the DMPT - cytochrome a reduction rate and may therefore imply mediation Of Cu(A). Both "resting" and "pulsed" oxidases contain rapid-turnover and slow-turnover species, as determined by aerobic steady-state reduction of cytochrome a by TMPD. Only the "rapid" fraction (almost-equal-to 70% of the total with resting and almost-equal-to 85% of the total with pulsed) is involved in turnover. We conclude that electron transfer to the a3Cu(B) binuclear centre can occur either from cytochrome a or Cu(A), depending upon the redox state of the binuclear centre. Under steady-state conditions, cytochrome a and Cu(A) may not always be in rapid equilibrium. Rapid enzyme turnover by either natural or artificial substrates may require reduction of both and two pathways of electron transfer to the a3Cu(B) centre.