Low-Temperature Pulsed EPR Study at 34 GHz of the Triplet States of the Primary Electron Donor P₈₆₅and the Carotenoid in Native and Mutant Bacterial Reaction Centers ofRhodobacter sphaeroides

Abstract

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.

Mechanism of Triplet Energy Transfer in Photosynthetic Bacterial Reaction Centers

“…The oscillation frequency (ω isc ) influences the yield of 3 P and the relative rates of the two recombination reactions. , Past studies suggested that the singlet–triplet exchange coupling interaction in reaction centers is temperature independent. , In the above kinetic model, where the interconversion among states is much slower than the individual decay rates, the total population decay can be approximately represented by the sum of two exponential decays, k s and a rate limited by ω isc . Using the yield of 3 P (35–40%) and the observed decay rate (1/12 ns –1 ) of the total population of 1 (P + H A – )/ 3 (P + H A – ) at room temperature, one can estimate the singlet and triplet radical pair recombination rates using the equation: where Φ Recombination is the relative yield of the individual recombination reaction steps.…”

“…54 The oscillation frequency (ω isc ) influences the yield of 3 P and the relative rates of the two recombination reactions. 55,56 Past studies suggested that the singlet−triplet exchange coupling interaction in reaction centers is temperature independent. 57,58 In the above kinetic model, where the interconversion among states is much slower than the individual decay rates, the total population decay can be approximately represented by the sum of two exponential decays, k s and a rate limited by ω isc .…”

Bacteriopheophytin triplet state in Rhodobacter sphaeroides reaction centers

“…Regarding mechanism 2, it has been proposed in the literature that a triplet state may be formed on BPhe by intersystem crossing (Uchoa et al 2008 ), but on the other hand results from EPR spectroscopy have been presented that show that 3 BPhe formation via intersystem crossing is unlikely (Marchanka et al 2007 ). Although in the present study the RCs were excited at 532 nm, implying initial formation of the singlet excited states of H A /H B , expectations from the well-characterized mechanism of RC energy transduction, is that excited state energy should be passed on a subpicosecond time scale to P* (Stanley et al 1996 ; Jordanides et al 2001 ), or that charge separation should be initiated from an alternative state such as B A * (van Brederode et al 1997 ).…”

“…So far, the yield and mechanism of quenching of triplet states have been investigated in a variety of RCs with mutations near the A- and/or B-branch cofactors (Laible et al 1998 ; deWinter and Boxer 1999 ; Marchanka et al 2007 ; Gibasiewicz et al 2011 ). A previous study by Gibasiewicz and coworkers (Gibasiewicz et al 2011 ) explored the influence of point mutations around the cofactors of the A-branch on the yield of triplet formation, but did not address the localization of the triplet states or their lifetimes.…”

Temperature Dependence of Electron Transfer to the M-Side Bacteriopheophytin in Rhodobacter capsulatus Reaction Centers

“…Others have found that a signal due to electron transfer to H M seen at room temperature diminishes at low temperature and interpreted the finding in terms of perturbation of the L-branch energetics with temperature 86 or that P* f P + H M -has an activation energy. 87,88 A weak temperature dependence of electron transfer to the M branch has been reported for the H(M182)L mutant, where a bacteriopheophytin denoted φ B replaces B M . 76 In this mutant P* decays to form P + H L -and P + φ B -at both room and low temperature; electron transfer to P + H M -does not occur because P + φ B -is apparently lower in free energy.…”