Hydrogen bonding to P700:: Site-directed mutagenesis of threonine A739 of photosystem I in Chlamydomonas reinhardtii

The primary electron donor P700 of photosystem I is a dimer comprised of chlorophyll a (P-B) and chlorophyll a' (P-A). P-A is involved in a hydrogen bond network with several surrounding amino acid residues and a nearby water molecule. To investigate the influence of hydrogen bond interactions on the properties of P700, the threonine at position A739, which donates a putative hydrogen bond to the 13(1)-keto group Of P-A, was replaced with valine, histidine, and tyrosine in Chlamydomonas reinhardtii using site-directed mutagenesis. Growth of the mutants was not impaired. (i) The (P700+. - P700) FTIR difference spectra of the mutants lack a negative band at 1634 cm(-1) observed in the wild-type spectrum and instead exhibit a new negative band between 1658 and 1672 cm(-1) depending on the mutation. This band can therefore be assigned to the 13(1)-keto group of P-A which is upshifted to higher frequencies upon removal of the hydrogen bond. (ii) The main bleaching band in the Q(y) region of the (P700(+.) - P700) and ((3)P700 - P700) absorption difference spectra is blue shifted for the mutants by similar to6 nm compared to that of the wild type. A blue shift is also observed for the main bleaching in the Soret region. (iii) The (P700(+.) - P700) CD difference spectrum of the wild type reveals two bands at 694 nm (positive CD) and 680 nm (negative CD) of approximately equal area. For each mutant, these two components are blue-shifted to the same extent. The results strongly suggest that a blue shift of the Qy absorption band Of P-A is responsible for a blue shift of the exciton bands. (iv) Redox titrations yielded a decrease in the midpoint potential for the oxidation of P700 by 32 mV for the exchange of Thr against Val. (v) ENDOR spectroscopy shows that the hfc of the methyl protons at position 12 of the spin-carrying Chl P-B is decreased due to the removal of the hydrogen bond to P-A. This indicates a redistribution of spin density in P700(+.) compared to that in the wild type. This gives evidence for an electronic coupling between the two halves of the dimer in the wild type and mutants.