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 lib. sphaeroides mutant bacterial reaction centers (RCs) in which the native bacteriopheophytin electron carrier BPh(L) is replaced with a bacteriochlorophyll (BChl) molecule, denoted by beta. 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/E104V, for which spectral evidence shows that a putative hydrogen-bonding interaction between beta 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 degrees with respect to the 870-nm transition of P at both 285 and 77 K for both beta-type mutants, compared to 65 +/- 3 degrees for the P(+)BPh(L)(-) 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(+)beta(-), or P(+)BChl(L)(-), or a combination of the two as the result of quantum-mechanical mixing or thermal equilibration.