Flavin‐photosensitized oxidation of reduced c‐type cytochromes

Abstract

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20 -100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome ~5 5 2 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin -cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.The mechanism of electron transfer between redox proteins has been the subject of extensive study in recent years (cf. [l -41 for reviews). The primary focus of these investigations has been on the reactions of soluble proteins containing a single redox center (e.g. c-type cytochromes, copper proteins, iron-sulfur proteins), although work has also been done with multicenter and membrane-associated systems. This emphasis is mainly a consequence of the substantial body of structural information which is available for the smaller and simpler redox proteins. With electron carriers of this type, electron transfer generally proceeds from a small substrate or from another protein into the prosthetic group of the redox protein, and then from the reduced protein to an acceptor, which again may be a small molecule such as dioxygen or another redox protein such as a cytochrome. Thus, a question arises as to the identity of the sites of reduction and oxidation: are they the same or different? This question has been studied most extensively with c-type cytochromes, and the general consensus appears to be that for these proteins the sites are probably the same (cf. [5, 61 f...

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