Photoinduced electron transfer in the cytochrome c/cytochrome c oxidase complex using thiouredopyrenetrisulfonate-labeled cytochrome c optical multichannel detection

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/ cytochrome c complex was investigated using a novel photoactivatable dye. L aser photolysis of thiouredopyrenetrisulfonate (TUPS), covalently linked to cysteine 102 on yeast iso-l-cytochrome c, generates a triplet state of the dye, which donates an electron to cytochrome c, followed by electron trans fer to cytochrome c oxidase. Time-resolved optical absorption difference sp ectra were collected at delay times from 100 ns to 200 ms between 325 and 6 50 nm. On the basis of singular value decomposition (SVD) and multiexponent ial fitting, three apparent lifetimes were resolved. A sequential kinetic m echanism is proposed from which the microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS donates an electron to cytochrome c with a forward rate constant of similar to2.0 x 10 (4) s(-1). A significant fraction of the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome c is followed b y faster electron transfer from cytochrome c to Cu-A, With the equilibrium favoring the reduced cytochrome c. Subsequently, CUA equilibrates with heme a with an apparent rate constant of similar to1 x 10(4) s(-1). On a millis econd time scale, the oxidized TUPS returns to the ground state and heme a becomes reoxidized. The extracted intermediate spectra are in excellent agr eement with model spectra of the postulated intermediates, supporting the p roposed mechanism.