Influence of iron-removal procedures on sequential electron transfer in photosynthetic bacterial reaction centers studied by transient EPR spectroscopy.

Abstract

Electron spin polarized electron paramagentic resonance (ESP EPR) spectra were obtained with deuterated iron-removed photosynthetic bacterial reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and H-subunit content on the observed P865+QA- charge-separated state. Fe-removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (kQ) from the intermediate acceptor H- to the primary acceptor QA of (200 ps)-1 vs (3-6 ns)-1 were observed. Correlation of the transient EPR and optical results shows that the observed slow kQ rate in Fe-removed RCs is H-subunit-independent, and, in some cases, independent of Fe-site occupancy as Zn2+ substitution does not ensure retention of the native kQ. In addition, shifts in the optical spectrum of P865 and differences in the high-field region of the Q-band ESP spectrum for Fe-removed RCs with slow kQ indicate possible structural changes near P865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where kQ is at least 15-fold slower at room temperature than the (200 ps)-1 rate observed for native Fe-containing RCs have different relative amplitudes and small g-value shifts compared to the spectra of Zn-RCs which have a kQ unchanged from native RCs. These differences reflect the trends in polarization predicted from the sequential electron transfer polarization (SETP) model [Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al. (1996) Chem. Phys. Lett. 253, 293-298]. Thus, SETP modeling of these highly resolved ESP spectra obtained with well-characterized proteins will provide definitive information about any light-induced structural changes of P865, H, and QA that occur upon formation of the P865+QA- charge-separated state.