Primary Electron Transfer in Membrane-Bound Reaction Centers with Mutations at the M210 Position L. M. P. Beekman,* ,† I. H. M. van Stokkum, † R. Monshouwer, † A. J. Rijnders, † P. McGlynn, ‡ R. W. Visschers, § M. R. Jones, ‡ and R. van Grondelle † Department of Physics and Astronomy and Department of Plant Physiology, Vrije UniVersiteit, De Boelelaan 1081, 1081 HV, Amsterdam, The Netherlands, and Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, UniVersity of Sheffield, Western Bank, Sheffield S10 2UH, United Kingdom ReceiVed: October 16, 1995; In Final Form: January 9, 1996 X The kinetics of primary electron transfer in membrane-bound Rhodobacter sphaeroides reaction centers (RCs) were measured on both wild-type (WT) and site-directed mutant RC’s bearing mutations at the tyrosine M210 position. The tyrosine was replaced by histidine (H), phenylalanine (F), leucine (L), or tryptophan (W). A mutant with histidine at both the M210 and symmetry-related L181 positions (YM210H/FL181H) was also examined. Rates of primary charge separation were determined by both single and multiple wavelength pump-probe techniques. The time constants for the decay of stimulated emission in the membrane-bound mutant RC’s were approximately 27 ps (F), 36 ps (L), 72 ps (W), 5.8 ps (H), and 4.2 ps (HH), compared with 4.6 ps in WT membrane-bound RC’s. For all RC’s, the decay of stimulated emission was found to be multiexponential, demonstrating that this phenomenon is not a consequence of the removal of the RC from the membrane. The source of the multiexponential decay of the primary donor excited state was examined, leading to the conclusion that a distribution in the driving force (ΔG) for electron transfer cannot be the sole parameter that determines the multiexponential character. Further measurements on membrane-bound mutant RC’s showed that chemical prereduction of the acceptor quinones resulted in a significant slowing of the rate of primary charge separation. This was most marked in those mutants in which the rate of charge separation had already been slowed down as a result of mutagenesis at the M210 position. The phenomenon is discussed in terms of the Coulombic interaction between Q A - and the other pigments involved in electron transfer and the influence of this interaction on the driving force for charge separation. Introduction The bacterial reaction center (RC) is an efficient optoelectric cell, which upon absorption of light-energy transfers an electron across the photosynthetic membrane before loss processes (e.g., fluorescence) become important. The crystal structure of the purple bacterial RC has been determined for two species, Rhodopseudomonas (Rps.) Viridis 1,2 and Rhodobacter (Rb.) sphaeroides. 3-5 The Rb. sphaeroides RC consists of three subunits, H, L, and M, of which L and M are related by a 2-fold axis of symmetry and bind the cofactors involved in electron transfer. These cofactors, four molecules of bacteriochlorophyll a (Bchl-a), two molecules of bacteriopheophytin a (Bphe-a), and two molecules of ubiquinone (Q), are arranged in two branches, but only the “L-branch” is active in electron transfer. 6 The primary electron donor (P) is a pair of Bchl molecules, which lie embedded in the RC protein close to the periplasmic face of the membrane. The formation of the first singlet excited state of the primary donor (P*), either by energy transfer from the antenna or by direct absorption by the RC pigments, triggers the transfer of an electron across the membrane dielectric. The kinetics of the primary electron transfer have been studied extensively with high time resolution using detergent-solubilized RC’s, and the lifetime of P* has been found to be approximately 3.5 ps at room temperature. 7-9 The mechanism of electron transfer from P* to the Bphe molecule (H L ) located halfway across the membrane is still a matter of debate. Two models have been proposed to describe this electron transfer process, which differ in the role played by the monomeric bacteriochlo- rophyll molecule (B L ), which in the crystal structure bridges the gap between P and H L . In the sequential model, the state P + B L - is formed as a distinct, but short-lived, intermediate between P* and P + H L - , 10-12 while in the superexchange model, P + B L - is a virtual state enhancing the electronic coupling between P* and P + H L - . 13,14 A model in which both the sequential and superexchange scheme contribute to the electron transfer proton has also been discussed. 14,15 In seeking to understand the striking asymmetry of electron transfer in the RC, much attention has been focused on the residue pair Tyr M210/Phe L181 (Tyr M210 in Rps. Viridis and Rb. capsulatus). 8,15,16 These conserved residues are in close contact with P and with the Bchl and Bphe on the active and inactive branches, respectively. As yet the precise role of Tyr M210 and the significance of the conserved Tyr/Phe arrange- ment are unclear; although the tyrosine has the capacity to form a hydrogen bond, there is no unequivocal evidence that it is H-bonded to P or the pigments on the active branch, with contrasting statements in the literature. 4,17 It has been proposed, on the basis of electrostatic calculations, that Tyr M210 acts to lower the energy of P + B L - relative to that of P + B M - , placing the energy of P + B L - in a region consistent with the operation of a sequential model for electron transfer. 18 Alternatively, it * Corresponding author. † Department of Physics and Astronomy, Vrije Universiteit. ‡ University of Sheffield. § Department of Plant Physiology, Vrije Universiteit. Current address: Department of Biochemistry, University of Pennsylvania, Philadelphia, PA 17104. X Abstract published in AdVance ACS Abstracts, April 1, 1996. 7256 J. Phys. Chem. 1996, 100, 7256-7268 0022-3654/96/20100-7256$12.00/0 © 1996 American Chemical Society