Environmental Control of Primary Photochemistry in a Mutant Bacterial Reaction Center Arlene L. M. Haffa, ‡,§,⊥,| Su Lin, ‡,§,⊥ Russell LoBrutto, §,# JoAnn C. Williams, ‡,§ Aileen K. W. Taguchi, ‡,§,⊥ James P. Allen, ‡,§ and Neal W. Woodbury* ,‡,§,⊥ Department of Chemistry and Biochemistry, Center for the Study of Early EVents in Photosynthesis, School of Life Sciences, and Center for BioOptical Nanotechnology/The Biodesign Institute, Arizona State UniVersity, Tempe, Arizona 85287-1604 ReceiVed: April 18, 2005; In Final Form: August 3, 2005 The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q y transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B A and B B ), or the bacteriopheophytins (H A and H B ) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B B . At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B B + H B - ) but no long-lived B B + H B - is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B B + H B - , including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B B + H B - stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent). Introduction The photosynthetic reaction center is responsible for the conversion of light energy into a transmembrane charge separation via a series of electron-transfer reactions between its redox-active cofactors. Rhodobacter (Rb.) sphaeroides is an anoxygenic purple non-sulfur bacterium whose reaction center is well characterized both functionally and structurally. 1 The Rb. sphaeroides reaction center consists of three protein subunits (L, M, and H) and ten cofactors. 2-4 The core of the reaction center is made up of the L and M subunits, which both contain five transmembrane helices arranged in a nearly C 2 symmetric fashion. The third protein subunit, H, lies predominantly on the cytoplasmic face of the intracytoplasmic membrane. Nine of the ten cofactors, a bacteriochlorophyll dimer (P composed of P A and P B ), two accessory bacteriochlorophylls B A and B B , two bacteriopheophytins H A and H B , two quinones, Q A and Q B , and a non-heme iron (Fe), are also related by the C 2 symmetry. P is near the periplasmic face of the membrane and is flanked by B A and B B on each side. H A and H B are farther into the membrane followed by Q A and Q B with the iron atom between the quinones near the cytoplasmic side of the membrane. A carotenoid molecule, the tenth cofactor, is asymmetrically placed near B B . Upon direct excitation of any of the lowest excited singlet states of the cofactors, rapid energy transfer to P results in the formation of P*, and then electron transfer occurs with a time constant of a few picoseconds forming P + H A - presumably via P + B A - . In about 200 ps, a subsequent electron transfer forms P + Q A - and then on longer time scales P + Q B - is formed. Under these excitation conditions, essentially no electron transfer from P to either B B or H B is observed despite the approximate C 2 symmetry relating the structure of the electron-transfer pathways on the two sides. 5-7 The quantum yield of A-side electron transfer in wild type under these conditions is nearly unity. 8 Since the elucidation of the structure two decades ago, much work has focused on understanding the ability of this pigment- protein complex to dictate the directionality of electron transfer. The lack of B-side charge separation in wild type reaction centers with P as the electron donor is predominantly the result of the energetic arrangement of charge-separated states in the system. Holten and co-workers have performed a number of studies of electron-transfer directionality using mutants in which the primary electron acceptor, H A , has been replaced with a bacteriochlorophyll making small changes in the ground-state absorbance band of H B (530 nm) easier to detect. The addition of mutations to modify the local environments of B A and B B has resulted in charge separation from P* forming the state P + H B - with a quantum yield as high as 30%. 9-12 Mutants have also been constructed that have modified the B-side such that * To whom correspondence should be addressed. E-mail: nwoodbury@ asu.edu. ‡ Department of Chemistry and Biochemistry. § Center for the Study of Early Events in Photosynthesis. ⊥ Center for BioOptical Nanotechnology/The Biodesign Institute. | Current address: Monterey Bay Aquarium Research Institute, Moss Landing, CA 95039-9644. # School of Life Sciences. 19923 J. Phys. Chem. B 2005, 109, 19923-19928 10.1021/jp052007e CCC: $30.25 © 2005 American Chemical Society Published on Web 10/05/2005