Electrostatic Interaction between Redox Cofactors in Photosynthetic Reaction Centers

Jean Alric,Aude Cuni,Hideaki Maki,Kenji V.P Nagashima,André Verméglio,Fabrice Rappaport

doi:10.1074/jbc.m408888200

Abstract

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921-3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.

Highlights

Proteins exert a fine electrochemical tuning of the redox potential of the cofactors they bind in order to perform the various electron transfer reactions that are involved in biological processes
Redox Characteristics of the Pϩ/P Couple in the WT and Chimeric reaction centers (RC)—The primary electron donor P was titrated in membrane fragments purified from R. gelatinosus and B. viridis WT strains and the chimeric VC-F strain (Fig. 2, left panel) by measuring the flash-induced absorption changes at 605 nm, 50 ns after the actinic flash
Two main features arise from the combined study of the chimeric RC by either equilibrium redox titration or kinetic analysis

Summary

Electrostatic Interactions between Redox Centers

Such short distances may result in significant electrostatic interactions among the different cofactors. We further investigated this chimeric RC (hereafter named VC-F) and show that most of the various cofactors at the donor side of this RC show absorption spectra and relative orientations similar to those found in the respective native RCs, indicating that the backbone structure of the protein as well as the interactions between the hemes and the side chains are conserved (Fig. 1) These findings make the chimeric RC suited for the direct characterization of electrostatic interactions between cofactors by the mean of equilibrium redox titration. In this RC, two cofactors (the primary electron donor (P) and the closest heme c559) are expected to have redox potentials differing by less than 50 mV (see Fig. 1) Such a case allows the direct observation of a putative electrostatic interaction, because it should manifest itself by a deviation to a one-electron Nernst curve. These data prove that electrostatic interactions between cofactors in proteins modulate their redox potentials

EXPERIMENTAL PROCEDURES

RESULTS

EPcϩ EcPϩϭ ϭ

DISCUSSION