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

The primary quinone acceptor radical anion Q(A)· (a menaquinone-9) is studied in reaction centers (RCs) of Rhodopseudomonas viridis in which the high-spin non-heme Fe is replaced by diamagnetic Zn. The procedure for the iron substitution, which follows the work of Debus et al. [Debus, R. J., Feher, G., and Okamura, M. Y. (1986) Biochemistry 25, 2276-2287], is described. In Rps. viridis an exchange rate of the iron of ~50% ± 10% is achieved. Time-resolved optical spectroscopy shows that the ZnRCs are fully competent in charge separation and that the charge recombination times are similar to those of native RCs. The g tensor of Q(A)· in the ZnRCs is determined by a simulation of the EPR at 34 GHz yielding g(x) = 2.00597 (5), g(y) = 2.00492 (5), and g(z) = 2.00216 (5). Comparison with a menaquinone anion radical (MQ·) dissolved in 2-propanol identifies Q(A)· as a naphthoquinone and shows that only one tensor component (g(x)) is predominantly changed in the RC. This is attributed to interaction with the protein environment. Electron-nuclear double resonance (ENDOR) experiments at 9 GHz reveal a shift of the spin density distribution of Q(A)· in the RC as compared with MQ· in alcoholic solution. This is ascribed to an asymmetry of the Q(A) binding 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 envelope modulation (ESEEM) techniques performed on Q(A)· in the ZnRCs, two N nuclear quadrupole tensors are determined that arise from the surrounding amino acids. One nitrogen coupling is assigned to a N(δ(1))-H of a histidine and the other to a polypeptide backbone N-H by comparison with the nuclear quadrupole couplings of respective model systems. Inspection of the X-ray structure of Rps. viridis RCs shows that His(M217) and Ala(M258) are likely candidates for the respective amino acids. The quinone should therefore be bound by two H bonds to the protein that could, however, be of different strength. An asymmetric H-bond situation has also been found for Q(A)· in the RC of Rhodobacter sphaeroides. Time-resolved electron paramagnetic resonance (EPR) experiments are performed on the radical pair state P·Q(A)· in ZnRCs of Rps. viridis that were treated with o-phenanthroline to block electron transfer to Q(B). The orientations of the two radicals in the radical pair obtained from transient EPR and their distance deduced from pulsed EPR (out-of-phase ESEEM) are very similar to the geometry observed for the ground state PQ(A) in the X-ray structure [Lancaster, R., Michel, H. (1997) Structure 5, 1339]