ENDOR and special TRIPLE resonance spectroscopy of photoaccumulated semiquinone electron acceptors in the reaction centers of green sulfur bacteria and heliobacteria

Photoaccumulation at 205 K in the presence of dithionite produces EPR signa ls in anaerobically prepared membranes from Chlorobium limicola and Helioba cterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A(1)(. -)) photoaccumulated in photosystem I, We have used ENDOR and special TRIPL E resonance spectroscopy to demonstrate conclusively that these signals ari se from menasemiquinone electron accepters reduced by photoaccumulation. Hy perfine couplings to two protons H-bonded to the semiquinone oxygens have b een identified by exchange of H, chlorum into D2O, and hyperfine couplings to the methyl group, and the methylene group of the phytyl side chain, of t he semiquinone have also been assigned. The electronic structure of these m enasemiquinones in these reaction centers is very similar to that of phyllo semiquinone in PSI, and shows a distorted electron spin density distributio n relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the effect of detergents and oxyg en on membranes of C, limicola. Triton X-100 and oxygen affect the menaquin one binding site, but n-dodecyl beta-D-maltoside preparations exhibit a rel atively unaltered special TRIPLE spectrum for the photoaccumulated menasemi quinone.