Titration and steady-state behaviour of the 830 nm chromophore in cytochrome c oxidase

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Abstract
Titration of cyanide-incubated cytochrome c oxidase (ox heart cytochrome aa3) with ferrocytochrome c or with NNN''N''-tetramethyl-p-phenylenediamine initially introduces 2 reducing equivalents/mol of cytochrome aa3. The first equivalent reduces the cytochrome a heme Fe; the second reducing equivalent is not associated with reduction of the 830 nm chromophores (EPR-detectable Cu) but is probably required for reduction of the EPR-undetectable Cu. Excess reductant introduces a third reducing equivalent into the cyanide complex of cytochrome aa3. During steady-state respiration in the presence of cytochrome c and ascorbate, the 830 nm chromophore is almost completely oxidized. It is reduced more slowly than cytochrome a on anaerobiosis. In the presence of formate or azide, some reduction at 830 nm can be seen in the steady state; in an oxygen-pulsed system, a decrease in steady-state reduction of cytochromes c and a is associated with an increased reduction of the 830 nm species. In the formate-inhibited system the reduction of a3 on anaerobiosis shows a lag phase, the duration of which corresponds to the time taken for the 830 nm species to be reduced. The EPR-undetectable Cu (CuU) is reduced early in the reaction sequence, whereas the detectable Cu (CuD) is reduced late. The latter species is probably responsible for reduction of these cytochrome a3 heme. The magnetic association between undetectable Cu and the a3 heme may not imply capability for electron transfer, which occurs more readily between cytochrome a3 and the 830 nm species.