The nature and dynamics of the charge-separated intermediate in reaction centers in which bacteriochlorophyll replaces the photoactive bacteriopheophytin. 1. Spectral characterization of the transient state

We report studies aimed at elucidating the nature of the charge-separated intermediate P⁺I^- in two Rb. sphaeroides mutant bacterial reaction centers (RCs) in which the native bacteriopheophytin electron carrier BPh_L is replaced with a bacteriochlorophyll (BChl) molecule, denoted by β. One mutant is the previously reported (M)L214H, which yields the pigment change via introduction of a His residue at position 214 on the M polypeptide. The second is a new double mutant, (M)L214H/(L)E104V, for which spectral evidence shows that a putative hydrogen-bonding interaction between β and the native glutamic acid at L104 is removed. Time-resolved photodichroism measurements of the anion band of the charge-separated intermediate P⁺I^- yield an angle of 46 ± 3° with respect to the 870-nm transition of P at both 285 and 77 K for both beta-type mutants, compared to 65 ± 3° for the P⁺BPhL^- intermediate in wild-type RCs and the (L)E104V single mutant. These results and other spectral data are discussed in conjunction with models in which P⁺I^- in the beta-type mutants is P⁺β^-, or P⁺BChl_L^-, or a combination of the two as the result of quantum-mechanical mixing or thermal equilibration.