SLOW (RESTING) FORMS OF MITOCHONDRIAL CYTOCHROME-C-OXIDASE CONSIST OF2 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 (format e or carbon monoxide respectively) has no effect on these rates. Varyi ng the dithionite concentration also has no effect on either phase, al though 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 tra nsfer to the binuclear centre is the limiting step, rather than an equ ilibrium model where the haem a3 redox potential is low. Increasing th e pH decreases the rate of the slower phase of dithionite reduction, b ut has no effect on the faster phase. EPR studies show that the slow p hase (only) correlates with the disappearance of the g' = 12/g' = 2.95 signals, with the same pH dependence; again the presence of formate h as no effect on these results. Deconvolution of the oxidized optical s pectra shows that the enzyme reduced in the slow phase has a blue-shif ted Soret band, relative to that reduced in the faster phase. Incubati on 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 am ount of signal. The results are interpreted in a model where the prese nce of a carboxylate bridge between haem a3 and Cu(B) defines the slow enzyme. It is suggested that the two rates of dithionite reduction ar e the result of different ligation to Cu(B) - where water is the ligan d the binuclear centre is Fe(IV)/Cu(I) (EPR-silent) and where hydroxid e is the ligand the binuclear centre is Fe(III)/Cu(II) (g' = 121 g' = 2.95 EPR signals).