Photoinduced intracomplex electron transfer between cytochrome c oxidase and TUPS-modified cytochrome c.

A novel method for initiating intramolecular electron transfer in cytochrome c oxidase is reported. The method is based upon photoreduction of cytochrome c labeled with thiouredopyrene-3,6, 8-trisulfonate in complex with cytochrome oxidase. The thiouredopyrene-3,6,8-trisulfonate-labeled cytochrome c was prepared by incubating the thiol reactive form of the dye with yeast iso-1-cytochrome c, containing a single cysteine residue. Laser pulse excitation of a stoichiometrical complex between thiouredopyrene-3,6,8-trisulfonate-cytochrome c and bovine heart cytochrome oxidase at low ionic strength resulted in the reduction of cytochrome c by the excited form of thiouredopyrene-3,6, 8-trisulfonate and subsequent intramolecular electron transfer from the reduced cytochrome c to cytochrome oxidase. The maximum efficiency by a single laser pulse resulted in the reduction of approximately 17% of cytochrome a, and was achieved only at a 1 : 1 ratio of cytochrome c to cytochrome oxidase. At higher cytochrome c to cytochrome oxidase ratios the heme a reduction was strongly suppressed.

Use of thiouredopyrenetrisulfonate photochemistry for driving electron transfer reactions in aqueous solutions.

Photoexcitation of 1-thiouredopyrene-3,6,8-trisulfonic adducts (TUPS) of amino acids by the third harmonic frequency of a Nd:YAG laser (355 nm) generates the triplet state of the dye with high quantum efficiency. Relaxation of the triplet proceeds in anaerobiosis with a half decay time of 0.5 ms. The relaxation rate increases 100-fold in the presence of dioxygen. A radiative transition between the triplet and the ground state of the dye results in phosphorescent emission centered at 658 nm. The excited state of TUPS, being a strong reductant, can donate its electron to a variety of acceptors. Transient absorption spectroscopy was used to directly measure the photoinduced electron transfer from the excited dye to rhodamine B (RB) and cytochrome c. The reaction with RB was followed by monitoring the oxidation of the triplet state of TUPS at 487 nm (epsilon = 25 000 +/- 5 000 M-1 cm-1) or the reduction of RB at 553 nm. The second order rate constant for the reaction was found to be (2.5 +/- 0.2) x 10(9) M-1 s-1, a value compatible with that for diffusion controlled reactions. When directed to cytochrome c the photoinduced perturbation causes rapid reduction of the protein's heme group, seen as a monophasic increase of absorbance at 550 nm. The combination of appropriate redox properties with the capability of covalent protein modification makes the dye useful for initiation and analysis of electron transfer reactions in chemical and biological systems.

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate cytochrome c derivatives.

A novel method for initiation of intramolecular electron transfer reactions in cytochrome c is reported. The method is based on photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS) by the third harmonic frequency of a Nd:YAG laser (355 nm), the reaction that generates the low-potential triplet state of the dye with high quantum efficiency. TUPS derivatives of horse heart cytochrome c singly labeled at specific lysine residues were prepared and purified to homogeneity by ion-exchange high-pressure liquid chromatography. Eight derivatives were characterized by determination of the location of the modification, reduction potentials, and measurement of enzymatic activity with cytochrome oxidase. Transient absorption spectroscopy was used to directly measure the rate constants for the electron transfer reaction from the photoexcited triplet state of TUPS to the oxidized heme group and the back reaction from the ferrous heme to the oxidized dye. For all singly labeled derivatives, the rate constants for heme reduction were 1 or 2 orders of magnitude larger than for its reoxidation, consistent with the greater thermodynamic driving force for the oxidation reaction. Analysis of the variation of electron-transfer rates with the distance separating the dye and the heme reveals a value of coupling decay constant (beta) of 0.46 A-1. Rapid and effective photoreduction of cytochrome c makes it a useful tool for fast initiation of electron transfer in the reductive direction within complexes of cytochrome c with other redox proteins.