SLOW (RESTING) FORMS OF MITOCHONDRIAL CYTOCHROME-C-OXIDASE CONSIST OF 2 KINETICALLY DISTINCT CONFORMATIONS OF THE BINUCLEAR CUB/A(3) CENTER - RELEVANCE TO THE MECHANISM OF PROTON TRANSLOCATION

We have purified slow ('resting') cytochrome oxidase from bovine heart, free of contamination with fast ('pulsed') enzyme. This form of the enzyme shows two kinetic phases of reduction of haem a3 by dithionite (k = 0.020 +/- 0.005 s-1 and k = 0.005 +/- 0.002 s-1). The presence of ligands that bind to the oxidized or reduced binuclear centre (formate or carbon monoxide respectively) has no effect on these rates. Varying the dithionite concentration also has no effect on either phase, although at low dithionite concentrations a lag phase is observed as the rate of haem a reduction is slower. The results are consistent with a model for reduction of the slow enzyme where the rate of electron transfer to the binuclear centre is the limiting step, rather than an equilibrium model where the haem a3 redox potential is low. Increasing the pH decreases the rate of the slower phase of dithionite reduction, but has no effect on the faster phase. EPR studies show that the slow phase (only) correlates with the disappearance of the g' = 12/g' = 2.95 signals, with the same pH dependence; again the presence of formate has no effect on these results. Deconvolution of the oxidized optical spectra shows that the enzyme reduced in the slow phase has a blue-shifted Soret band, relative to that reduced in the faster phase. Incubation of the oxidized enzyme at high pH causes a line-broadening of both the g' = 12 and g' = 2.95 EPR signals with no obvious effect on the amount of signal. The results are interpreted in a model where the presence of a carboxylate bridge between haem a3 and Cu(B) defines the slow enzyme. It is suggested that the two rates of dithionite reduction are the result of different ligation to Cu(B) - where water is the ligand the binuclear centre is Fe(IV)/Cu(I) (EPR-silent) and where hydroxide is the ligand the binuclear centre is Fe(III)/Cu(II) (g' = 121 g' = 2.95 EPR signals).