Electron paramagnetic resonance studies of zinc-substituted reaction centers from Rhodopseudomonas viridis

The primary quinone acceptor radical anion QA(-.) (a menaquinone-9) is stud ied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high -spin non-heme Fe2+ is replaced by diamagnetic Zn2+. The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Fe her, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is describe d. In Rps. viridis an exchange rate of the iron of similar to 50% +/- 10% i s achieved. Time-resolved optical spectroscopy shows that the ZnRCs are ful ly competent in charge separation and that the charge recombination times a re similar to those of native RCs. The g tensor of QA(-.) in the ZnRCs is d etermined by a simulation of the EPR at 34 GHz yielding g, = 2.00597 (5), g , = 2.00492 (5), and g, = 2.00216 (5). Comparison with a menaquinone anion radical (MQ4(-.)) dissolved in 2-propanol identifies QA(-.) as a naphthoqui none and shows that only one tenser component (g) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at g GHz reveal a shi ft of the spin density distribution of QA(-.) in the RC as compared with MQ (4)(-) in alcoholic solution. This is ascribed to an asymmetry of the QA bi nding site. Furthermore, a hyperfine coupling constant from an exchangeable proton is deduced and assigned to a proton in a hydrogen bond between the quinone oxygen and surrounding amino acid residues. By electron spin-echo e nvelope modulation (ESEEM) techniques performed on QA(-.) in the ZnRCs, two N-14 nuclear quadrupole tensors are determined that arise from the surroun ding amino acids. One nitrogen coupling is assigned to a N-delta(1)-H of a histidine and the other to a polypeptide backbone N-H by comparison with th e nuclear quadrupole couplings of respective model systems. Inspection of t he X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) a re likely candidates for the respective amino acids. The quinone should the refore be bound by two H bonds to the protein that could, however, be of di fferent strength. An asymmetric H-bond situation has also been found for QA (-.) in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagne tic resonance (EPR) experiments are performed on the radical pair state P-9 60(+) (.)QA(-.) in ZnRCs of Rps. viridis that were treated with o-phenanthr oline to block electron transfer to QB. The orientations of the two radical s in the radical pair obtained from transient EPR and their distance deduce d from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry obs erved for the ground state P(960)Q(A) in the X-ray structure [Lancaster, R. , Michel, H. (2997) Structure 5, 1339].