The primary electron donor of photosystem I (PS1), called P700, is a heterodimer of chlorophyll (Chl) a and a′. The crystal structure of photosystem I reveals that the chlorophyll a′ (PA) could be hydrogen-bonded to the protein via a threonine residue, while the chlorophyll a (PB) does not have such a hydrogen bond. To investigate the influence of this hydrogen bond on P700, PsaA-Thr739 was converted to alanine to remove the H-bond to the 131-keto group of the chlorophyll a′ in Chlamydomonas reinhardtii. The PsaA-T739A mutant was capable of assembling active PS1. Furthermore the mutant PS1 contained approximately one chlorophyll a′ molecule per reaction center, indicating that P 700 was still a Chl a/a′ heterodimer in the mutant. However, the mutation induced several band shifts in the visible P700 + - P700 absorbance difference spectrum. Redox titration of P700 revealed a 60 mV decrease in the P700/P 700+ midpoint potential of the mutant, consistent with loss of a H-bond. Fourier transform infrared (FTIR) spectroscopy indicates that the ground state of P700 is somewhat modified by mutation of ThrA739 to alanine. Comparison of FTIR difference band shifts upon P700 + formation in WT and mutant PS1 suggests that the mutation modifies the charge distribution over the pigments in the P700+ state, with ∼14-18% of the positive charge on PB in WT being relocated onto PA in the mutant. 1H-electron-nuclear double resonance (ENDOR) analysis of the P700+ cation radical was also consistent with a slight redistribution of spin from the PB chlorophyll to PA, as well as some redistribution of spin within the PB chlorophyll. High-field electron paramagnetic resonance (EPR) spectroscopy at 330-GHz was used to resolve the g-tensor of P700+, but no significant differences from wild-type were observed, except for a slight decrease of anisotropy. The mutation did, however, provoke changes in the zero-field splitting parameters of the triplet state of P700 ( 3P700), as determined by EPR. Interestingly, the mutation-induced change in asymmetry of P700 did not cause an observable change in the directionality of electron transfer within PSI.
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