Photinduced Electron Transfer in the Cytochrome c/Cytochrome c Oxidase Complex Using Thiouredopyrenetrisulfonate-Labeled Cytochrome c. Optical Multichannel Detection

摘要

Intramolecular electron transfer in the electrostatic cytochrome c oxidase/cytochrome c complex Nas investigated using a novel photoactivatable dye. Laser photolysis of thiouredopyrenetrisulfonate TUPS), covalently linked to cysteine 102 on yeast iso-l-cytochrome c, generates a triplet state of the lye, which donates an electron to cytochrome c, followed by electron transfer to cytochrome c oxidase. rime-resolved optical absorption difference spectra were collected at delay times from 100 ns to 200 ms )etween 325 and 650 nm. On the basis of singular value decomposition (SVD) and multi exponential ltting, three apparent lifetimes were resolved. A sequential kinetic mechanism is proposed from which he microscopic rate constants and spectra of the intermediates were determined. The triplet state of TUPS lonates an electron to cytochrome c with a forward rate constant of 2.0 x 104 S-I. A significant fraction )f the triplet returns back to the ground state on a similar time scale. The reduction of cytochrome cis 'ollowed by faster electron transfer from cytochrome c to CUA, with the equilibrium favoring the reduced :ytochrome c. Subsequently, CUA equilibrates with heme a with an apparent rate constant of '""'"'1 x 104 :-1. On a millisecond time scale, the oxidized TUPS returns to the ground state and heme a becomes eoxidized. The extracted intermediate spectra are in excellent agreement with model spectra of the lostulated intermediates, supporting the proposed mechanism.