Low-Temperature Studies of Electron Transfer to the M Side of YFH Reaction Centers from Rhodobacter capsulatus

Abstract

There have been extensive experimental and theoretical studies of the temperature dependence of the rates of electron transfer between the cofactors associated primarily with the L polypeptide (or A branch) in the bacterial photosynthetic reaction center (RC). The focus of this paper is to gain further insight into the temperature dependence of rate of initial electron transfer to the parallel cofactor chain associated mainly with the M polypeptide (or B branch), which is inactive in the native RC. To this end, picosecond transient absorption measurements have been carried out on RCs of the YFH mutant of the photosynthetic bacterium Rhodobacter capsulatus at 77 K. In wild-type RCs, symmetry-related residues Phe M208 and Tyr L181 flank the primary electron donor (P) and are key to initial charge separation. In the YFH mutant these residues are swapped, i.e., are Tyr M208 and Phe L181. The third mutation in YFH changes Leu M212 to His and results in replacement of the L-side bacteriopheophytin (H(L)) with a bacteriochlorophyll denoted beta. Studies were carried out at 77 K for RCs in detergent-buffer/glycerol glasses utilizing either the detergent N-lauryl-N,N-dimethylamine N-oxide (LDAO) or Deriphat 160-C. In both media, excitation of P to its lowest singlet excited state (P*) elicits complex kinetic behavior that, in the simplest phenomenological description, involves two P* cofactor-protein populations: one that is capable of charge separation (active) and one that is not (inactive). The amplitudes of the components of the P* stimulated-emission kinetic profiles, and the amplitudes and time course of accompanying P-bleaching recovery, reveal that the two P* populations, active/inactive, are in 60/40 (LDAO) or 40/60 (Deriphat) proportion. In the nonphotoactive fraction, P* decays solely via return to the ground state with its inherent lifetime (i.e., lifetime in the absence of electron transfer) of 170 ps (LDAO) or 350 ps (Deriphat). In the photoactive fraction, P* has a lifetime of 4.5 ps (LDAO) or 13 ps (Deriphat) and decays by parallel electron transfer to H(M) (30%) and beta (70%) on the M and L branches, respectively. The rate constant for P* --> P(+)H(M)(-) electron transfer is (15 ps)(-1) (LDAO) or (43 ps)(-1) (Deriphat) at 77 K. These rate constants are about 2-fold greater than those determined at 295 K in the corresponding detergent/buffer solutions. These results combined with related findings from prior work show that the primary charge-separation events on both sides of the RC are basically activationless processes. In particular, in the functionally active P* population of YFH RCs, there is little or no apparent energy barrier (e.g., involving motions of the cofactors or protein or both) for electron transfer from P* to the either side of the RC. This conclusion holds irrespective of the precise description of the complex kinetic behavior that is observed. In addition to the observations on the temperature-dependent photochemistry, the 77 K transient absorption spectra in the near-infrared resolve a bacteriopheophytin anion band at 955 nm for wild-type RCs and, for the beta-containing mutants L(M212)H and YFH, a bacteriochlorophyll anion band at 1015 nm.