Identification of the intermediate charge-separated state of P⁺β_L^- in a leucine M214 to histadine mutant of the Rhodobacter sphaeroides reaction center using femtosecond midinfrared spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β_L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors BL and BM), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P_L/P_M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm^-1 and 1770 cm^-1,and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P* decayed on a timescale of ~6.3 ps to P⁺B_L^-. The decay of P⁺B_L^- occurred with a lifetime of ~2 ps, ~3 times slower than that observed in wild-type and R-26 RCs (~0.7 ps). Further electron transfer to the bL BChl resulted in formation of the P⁺β_L^- state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared βspectra of P⁺B_L^- and P⁺β_L^- showed clear differences related to the different environments of the two BChls in the mutant RC.