Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)-tris-bipyridyl derivative of cytochrome c as electron donor

AbstractYeast iso-1-cytochrome c covalently modified at cysteine-102 with (4-bromomethyl-4′-methylbipyridine)[bis(bi-pyridine)]Ru2+ (Ru-102-Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash-induced photoreduction of Ru-102-Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipy-ridyl)3 (RuBpy) as the photoreductant [D.L. Zaslavsky et al. (1993) FEBS Lett. 336, 389–393]. At low ionic strength, when Ru-102-Cyt c forms a tight electrostatic complex with COX, flash-activation results in a polyphasic electrogenic response corresponding to transfer of a negative charge to the interior of the vesicles. The initial rapid phase is virtually identical to the 50 μs transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by CuA. CuA reduction by Ru-102-Cyt c turns out to be not electrogenic in agreement with the peripheral location of visible copper in the enzyme. A millisecond phase (τ ca. 4 ms) following the 50 μs initial part of the response and associated with vectorial translocation of protons linked to oxygen intermediate interconversion in the binuclear centre, can be resolved both with RuBpy and Ru-102-Cyt c as electron donors; however, this phase is small in the absence of added H2O2. In addition to these two transients, the flash-induced electrogenic response in the presence of Ru-102-Cyt c reveals a large slow phase of Δψ generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 μM ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru-102-Cyt c2+ generated by the flash in a solution