Exploring the Energy Landscape for Q A - to Q B Electron Transfer in Bacterial Photosynthetic Reaction Centers: Effect of Substrate Position and Tail Length on the Conformational Gating Step † Qiang Xu, ‡ Laura Baciou, § Pierre Sebban, § and M. R. Gunner* ,‡ Department of Physics, City College of New York, 138th Street and ConVent AVenue, New York, New York 10031, and Centre de Ge ´ ne ´ tique Mole ´ culaire, CNRS, Gif/YVette, France ReceiVed January 22, 2002; ReVised Manuscript ReceiVed May 31, 2002 ABSTRACT: The ability to initiate reactions with a flash of light and to monitor reactions over a wide temperature range allows detailed analysis of reaction mechanisms in photosynthetic reaction centers (RCs) of purple bacteria. In this protein, the electron transfer from the reduced primary quinone (Q A - ) to the secondary quinone (Q B ) is rate-limited by conformational changes rather than electron tunneling. Q B movement from a distal to a proximal site has been proposed to be the rate-limiting change. The importance of quinone motion was examined by shortening the Q B tail from 50 to 5 carbons. No change in rate was found from 100 to 300 K. The temperature dependence of the rate was also measured in three L209 proline mutants. Under conditions where Q B is in the distal site in wild-type RCs, it is trapped in the proximal site in the Tyr L209 mutant [Kuglstatter, A., et al. (2001) Biochemistry 40, 4253-4260]. The electron transfer slows at low temperature for all three mutants as it does in wild-type protein, indicating that conformational changes still limit the reaction rate. Thus, Q B movement is unlikely to be the sole, rate-limiting conformational gating step. The temperature dependence of the reaction in the L209 mutants differs somewhat from wild-type RCs. Entropy-enthalpy compensation reduces the difference in rates and free energy changes at room temperature. Reactions in proteins generally require structural changes as the reactant passes the transition state to go on to product. It is difficult to follow these changes for a number of reasons. The motions can be too small to be seen in any but the highest resolution structures (1, 2), for nonphotoactive proteins it is difficult to synchronize reactions (3), and it is hard to trap proteins in identifiable substates for analysis (4). The photosynthetic reaction centers (RCs) 1 from purple bacteria have proved to be a useful model system, allowing synchronized single-turnover reactions that can be analyzed to study factors that control the electron- and proton-transfer processes in proteins (5-7). The light reaction of bacterial photosynthesis takes place in RCs embedded in the cell membrane. The absorption of a photon by the electron donor, a bacteriochrolophyll dimer (P), triggers a series of electron transfers between bound cofactors, creating a separation of charge across the protein. P is oxidized (P + ) and first the primary quinone (Q A ) and then the secondary quinone (Q B ) reduced. The ≈100 μs electron transfer from Q A - to Q B is rate-limited by a conformational change in isolated RCs from Rhodobacter sphaeroides with native ubiquinone as Q A and Q B (8, 9). As a result of the needed conformational changes, the electron transfer from Q A - to Q B shows significant activation enthalpy. If RCs are frozen in the dark, in the ground state, the reaction slows so it cannot be seen as the temperature is lowered. However, RCs frozen under illumination in the product P + Q B - state return to the ground state trapped in a different conformation. Now electron transfer from Q A - to Q B occurs even below 40 K with high yield (10, 11). Analysis of the temperature dependence of the reaction has begun to reveal a number of substates and the thermodynamic differ- ences and barriers between these states (11-13). In dark-adapted RCs at physiological pH (6-8) keeping the room temperature, little proton uptake accompanies electron transfer from Q A - to Q B . Previous measurements (13) have shown the reaction free energy change (ΔG° AB ) is -90 meV with favorable ∆H° AB (-230 meV) and unfavorable T∆S° AB (-140 meV) [pH 8, 298 K, 66% glycerol (v/v)]. There is a barrier to electron transfer of ≈500 meV which is mostly ∆H AB q (420 meV). Additional sub- states of the reactant, P + Q A - , have been identified. For example, at low pH the reaction freezes out more slowly than predicted, showing the presence of an active state ≈40 meV above the inactive, dark-adapted state (13). Measure- ments at high pH have characterized an unprotonated reactant † Supported by the Department of Agriculture (CRESS 2001-35318- 11190, for financial support) and by the NIH (RR03060, for mainte- nance of central facilities). * To whom correspondence should be addressed. Telephone: 212- 650-5557. Fax: 212-650-6940. E-mail: gunner@sci.ccny.cuny.edu. ‡ City College of New York. § Centre de Ge ´ne ´tique Mole ´culaire, CNRS. 1 Abbreviations: RCs, reaction centers; P, bacteriochlorophyll dimer which is the primary electron donor; QA and QB, primary and secondary quinone electron acceptors; P + QA - and P + QB - , reactant (P + QA - QB) and product (P + QAQB - ) redox states, respectively. All equilibrium and rate constants have a two-letter subscript. The first is the reactant and the second the product substate. KAB and kAB are the effective equilibrium and rate constants between all subpopulations of P + QA - and P + QB - under the conditions of measurement. 10021 Biochemistry 2002, 41, 10021-10025 10.1021/bi025573y CCC: $22.00 © 2002 American Chemical Society Published on Web 07/12/2002