Protein Dynamics from Intramolecular Electron Transfer
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conformational energy landscape can be studied with temperature-dependent measurements of the kinetics of functional processes. At low temperatures, many degrees of freedom are thermally arrested, and distributions of reaction rates reflect a heterogeneous ensemble of protein molecules frozen in different CS. At intermediate temperatures, conformational transitions occur on the time scale of the reaction, which allows one to investigate protein motions through studies of reaction kinetics. At sufficiently high temperatures, each protein molecule fluctuates among the CS on time scales shorter than that of the reaction, and fluctuational averaging leads to single-exponential kinetics. This interplay between protein dynamics and biological function has been studied extensively in ligand binding to heme proteins [9-14]. We have investigated the coupling of protein motions to long-range electron transfer (ET) in reaction centers (RC) of purple bacteria (Rhodobacter sphaeroides).In these bacteria, the photon energy absorbed by light harvesting complexes or RC cofactors is transferred to the special pair, a bacteriochlorophyll dimer (P) on the periplasmic side of the RC protein, and the electronic system is promoted to the first excited singlet state, P*. An electron is subsequently transferred from P* to a bacteriopheophytin (1) and further to the primary quinone (QA), located 25 A away from the special pair, closer to the cytoplasmic side [15]. In the absence of the secondary quinone (QB), the electron then recombines with the hole on the special pair after -100 ins, and the RC is restored to the ground state: PIQA + hv --->P*IQA --- P+[-QA---> P+IQA- --->PIQA. 337 Mat. Res. Soc. Symp. Proc. Vol. 455 01997 Materials Research Society
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The charge separation during this cycle generates a strong electric field inside the protein, and the molecule responds to this substantial perturbation with conformational motions to solvate the new charge distribution [15-18]. We have investigated the last and slowest ET step in the sequence, PQA- -4 PQA, by measuring its kinetics as a function of temperature after cooling the sample in the dark. We have also examined the influence of illumination on the ET kinetics by switching on a strong light source during cooling to keep the protein in the charge-separated state. The experiments give evidence that, after light-induced charge separation, the protein relaxes from a dark-adapted conformation to a light-adapted conformation which can be trapped at low temperatures. In this contribution, we present a qualitative discussion. A manuscript describing a detailed physical model of the interplay between protein dynamics and electron transfer in reaction centers is in preparation [19]. EXPERIMENT
Freshly isolated RCs from Rhodobacter sphaeroides were quinone-extracted to less than I % QB, placed in 0.1 % LDAO, TRIS-buffered to pH 8, and mixed to 75 % glycerol : 25 % water (vol : vol). The sample was loaded in a plastic cuvette which was kept in thermal contact with a Cu sample holder in a
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