Rapid Kinetics of Membrane-Potential Generation by Cytochrome-C-Oxidase with the Photoactive Ru(ii)-Tris-Bipyridyl Derivative of Cytochrome-C as Electron-Donor

by Zaslavsky, Dmitry L.; Smirnova, I. A.; Siletsky, Sergey A.; Kaulen, A. D.; Millett, Francis Spencer; Konstantinov, Alexander A.

Yeast iso-1-cytochrome c covalently modified at cysteine-102 with (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]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)(bipyridyl)(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 mu s transient observed in the presence of RuBpy as the photoactive electron donor which originates from electrogenic reduction of heme a by Cu-A. Cu-A 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 (tau ca. 4 ms) following the 50 mu s 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 Delta Psi generation not observed with RuBpy. This phase is completely quenched upon inclusion of 100 mu M ferricyanide in the medium and originates from a second order reaction of COX with the excess Ru-102-Cyt c(2+) generated by the flash in a solution.

FEBS Letters
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1873-3468; 0014-5793