FTIR spectroscopy of Synechocystis 6803 mutants affected on the hydrogen bonds to the carbonyl groups of the PsaA chlorophyll of P700 supports an extensive delocalization of the charge in P700

P700, the primary electron donor of photosystem I is an asymmetric dimer made of one molecule of chlorophyll a' (P-A) and one of chlorophyll a (P-B). While the carbonyl groups of P-A are involved in hydrogen-bonding interactions with several surrounding amino acid side chains and a water molecule, P-B does not engage in hydrogen bonding with the protein. Light-induced FTIR difference spectroscopy of the photooxidation of P700 has been combined with site-directed mutagenesis in Synechocystis sp. PCC 6803 to investigate the influence of these hydrogen bonds on the structure of P700 and P700(+). When the residue Thr A739, which donates a hydrogen bond to the 9-keto C=O group of P-A, is changed to Phe, a differential signal at 1653(+)/1638(-) cm(-1) in the P700(+)/P700 FTIR difference spectrum upshifts by similar to30-40 cm(-1), as expected for the rupture of the hydrogen bond or, at least, a strong decrease of its strength. The same upshift is also observed in the FTIR spectrum of a triple mutant in which the residues involved in the three main hydrogen bonds to the 9-keto and 10a-carbomethoxy groups of P-A have been changed to the symmetry-related side chains present around P-B. In addition, the spectrum of the triple mutant shows a decrease of a differential signal around 1735 cm-1 and the appearance of a new signal around 1760 cm(-1). This is consistent with the perturbation of a bound 10a-ester C=O group that becomes free in the triple mutant. All of these observations support the assignment scheme proposed previously for the carbonyls of P700 and P700(+) [Breton, J., Nabedryk, E., and Leibl, W. (1999) Biochemistry 38, 11585-11592] and therefore reinforce our previous conclusions that the positive charge in P700(+) is largely delocalized over P-A and P-B.