Evidence from time resolved studies of the P700(.+)/A1(.-) radical pair for photosynthetic electron transfer on both the PsaA and PsaB branches of the photosystem I reaction centre.

Kinetic analysis using pulsed electron paramagnetic resonance (EPR) of photosynthetic electron transfer in the photosystem I reaction centres of Synechocystis 6803, in wild-type Chlamydomonas reinhardtii, and in site directed mutants of the phylloquinone binding sites in C. reinhardtii, indicates that electron transfer from the reaction centre primary electron donor, P700, to the iron-sulphur centres, Fe-S(X/A/B), can occur through either the PsaA or PsaB side phylloquinone. At low temperature reaction centres are frozen in states which allow electron transfer on one side of the reaction centre only. A fraction always donates electrons to the PsaA side quinone, the remainder to the PsaB side.

Site-directed mutagenesis of PsaA residue W693 affects phylloquinone binding and function in the photosystem I reaction center of Chlamydomonas reinhardtii.

To investigate the environment of the phylloquinone secondary electron acceptor A(1) within the photosystem I reaction center, we have carried out site-directed mutagenesis of two tryptophan residues (W693 and W702) in the PsaA subunit of Chlamydomonas reinhardtii. One of these conserved tryptophans (W693) is predicted to be close to the phylloquinone and has been implicated in the interaction of A(1) with an aromatic residue through pi--pi stacking. We find that replacement of W702 with either histidine or leucine has no effect on the electronic structure of A(1)(*-) or on forward electron transfer from A(1)(*-) to the iron--sulfur center F(x). In contrast, the same mutations of W693 alter the electronic structure of the photoaccumulated A(1)(*-) and slow forward electron transfer as measured by the decay of the electron spin-polarized signal arising from the P700(*+)/A(1)(*-) radical pair. These results provide support for the hypothesis that W693 has a role in poising the redox potential of A(1)/A(1)(*-) so it can reduce F(x), and they indirectly provide evidence for electron transfer along the PsaA-side branch of cofactors in PSI.

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 signals in anaerobically prepared membranes from Chlorobium limicola and Heliobacterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A1*-) photoaccumulated in photosystem I. We have used ENDOR and special TRIPLE resonance spectroscopy to demonstrate conclusively that these signals arise from menasemiquinone electron acceptors reduced by photoaccumulation. Hyperfine couplings to two protons H-bonded to the semiquinone oxygens have been 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 the semiquinone have also been assigned. The electronic structure of these menasemiquinones in these reaction centers is very similar to that of phyllosemiquinone in PSI, and shows a distorted electron spin density distribution relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the effect of detergents and oxygen on membranes of C. limicola. Triton X-100 and oxygen affect the menaquinone binding site, but n-dodecyl beta-D-maltoside preparations exhibit a relatively unaltered special TRIPLE spectrum for the photoaccumulated menasemiquinone.