B-side electron transfer promoted by absorbance of multiple photons in Rhodobacter sphaeroides R-26 reaction centers

Femtosecond transient absorbance spectra of quinone-depleted Rhodobacter sphaeroides R-26 reaction centers in the Q_X transition region have been measured at 15 K under various excitation conditions. This study focuses on the excitation wavelength dependence and excitation intensity dependence of the formation of charge-separated states on the A- and B-side of the reaction center, judging from the bleaching of the 533 nm (B-side) and 544 nm (A-side) ground-state transitions of the reaction center bacteriopheophytins (H_A and H_B). Upon low-intensity selective excitation directly into the bacteriopheophytin Q_Y transitions (near 760 nm), bleaching of both ground-state bacteriopheophytin Q_X transitions (533 and 544 nm) appeared immediately, showing that initially either the A- or B-side bacteriopheophytin could be excited. However, both excited states ultimately resulted in P⁺H_A^- formation under these conditions. Low-intensity excitation at any of the various wavelengths (595, 750, 760, and 880 nm) showed no difference in the kinetics of the A-side charge separation forming P⁺H_A^- and no substantial formation of the B-side charge-separated state, P⁺H_B^-. In contrast, high-intensity 595 nm excitation resulted in substantial long-lived bleaching of the B-side bacteriopheophytin ground-state transition at 533 nm. This 533 nm bleaching was formed with essentially the same time constant as the bleaching at 544 nm due to A-side charge separation. Both bleaching bands persisted at the longest times measured (hundreds of picoseconds) in quinone-removed reaction centers. The long-lived bleaching at 533 nm using high-intensity excitation most likely represents the formation of P⁺H_B^- with a relative yield (percent of total charge separate state) of nearly 40%. One possible mechanism for B-side electron transfer is that two-photon excitation of the reaction center resulting in the state P*B_B* makes P⁺B_B^- thermodynamically accessible.