Site-directed mutagenesis of Thr A739 of photosystem I in Chlamydomonas reinhardtii alters significantly the excitonic and electronic coupling of the primary electron donor P700

The primary electron donor P700 of photosystem I is a dimer comprised of one chlorophyll (Chl) a (PB) and one Chl a´ (PA). To investigate the influence of protein-cofactor interactions on the properties of P700, we constructed a series of site-directed mutants in the surrounding of P700. The most interesting effects were obtained for the replacement of Thr A739, a possible hydrogen bond donor to the 9-keto group of PA, against Val. The low-energy exciton absorption band of P700 observed as a shoulder at about 700 nm in the absorption spectrum of PS I complexes from wild type is not visible in the mutant TV A739. The main bleaching band in the (P700+-P700) and (3P700-P700) absorption difference spectra is blue shifted by 9 nm. Both results imply that the excitonic coupling of P700 is severely disturbed. A similar blue shift is observed for the main bleaching in the Soret region. Redox titrations yielded a decrease of the midpoint potential for the oxidation of P700 by 32 mV. ENDOR spectroscopy revealed a change of the electron spin densitiy distribution of P700+. The data provide evidence that P700 is a Chl-dimer with an asymmetric spin/charge density distribution