INFLUENCE OF IRON-REMOVAL PROCEDURES ON SEQUENTIAL ELECTRON-TRANSFER IN PHOTOSYNTHETIC BACTERIAL REACTION CENTERS STUDIED BY TRANSIENT EPR SPECTROSCOPY

Electron spin polarized electron paramagentic resonance (ESP EPR) spec tra were obtained with deuterated iron-removed photosynthetic bacteria l reaction centers (RCs) to specifically investigate the effect of the rate of primary charge separation, metal-site occupancy, and W-subuni t content on the observed P(865)(+)Q(A)(-) charge-separated state. Fe- removed and Zn-substituted RCs from Rb. sphaeroides R-26 were prepared by refined procedures, and specific electron transfer rates (k(Q)) fr om the intermediate acceptor H- to the primary acceptor Q(A) of (200 p s)(-1) vs (3-6 ns)(-1) were observed, Correlation of the transient EPR and optical results shows that the observed slow k(Q) rate in Fe-remo ved RCs is H-subunit-independent, and, in some cases, independent of F e-site occupancy as Zn2+ substitution does not ensure retention of the native k(Q). In addition, shifts in the optical spectrum of P-865 and differences in the high-field region of the Q-band ESP spectrum for F e-removed RCs with slow k(Q) indicate possible structural changes near P-865. The experimental X-band and Q-band spin-polarized EPR spectra for deuterated Fe-removed RCs where k(Q) is at least 15-fold slower at room temperature than the (200 ps)(-1) rate observed for native Fe-co ntaining RCs have different relative amplitudes and small g-value shif ts compared to the spectra of Zn-RCs which have a k(Q) unchanged from native RCs. These differences reflect the trends in polarization predi cted From the sequential electron transfer polarization (SETP) model [ Morris et al. (1995) J. Phys. Chem. 99, 3854-3866; Tang et al.(1996) C hem, Phys. Lett. 253, 293-298], Thus, SETP modeling of these highly re solved ESP spectra obtained with well-characterized proteins will prov ide definitive information about any light-induced structural changes of P-865, H, and Q(A) that occur upon formation of The P(865)(+)Q(A)(- ) charge-separated state.