Evidence from FTIR difference spectroscopy of an extensive network of hydrogen bonds near the oxygen-evolving Mn(4)Ca cluster of photosystem II involving D1-Glu65, D2-Glu312, and D1-Glu329.

Analyses of the refined X-ray crystallographic structures of photosystem II (PSII) at 2.9-3.5 A have revealed the presence of possible channels for the removal of protons from the catalytic Mn(4)Ca cluster during the water-splitting reaction. As an initial attempt to verify these channels experimentally, the presence of a network of hydrogen bonds near the Mn(4)Ca cluster was probed with FTIR difference spectroscopy in a spectral region sensitive to the protonation states of carboxylate residues and, in particular, with a negative band at 1747 cm(-1) that is often observed in the S(2)-minus-S(1) FTIR difference spectrum of PSII from the cyanobacterium Synechocystis sp. PCC 6803. On the basis of its 4 cm(-1) downshift in D(2)O, this band was assigned to the carbonyl stretching vibration (C horizontal lineO) of a protonated carboxylate group whose pK(a) decreases during the S(1) to S(2) transition. The positive charge that forms on the Mn(4)Ca cluster during the S(1) to S(2) transition presumably causes structural perturbations that are transmitted to this carboxylate group via electrostatic interactions and/or an extended network of hydrogen bonds. In an attempt to identify the carboxylate group that gives rise to this band, the FTIR difference spectra of PSII core complexes from the mutants D1-Asp61Ala, D1-Glu65Ala, D1-Glu329Gln, and D2-Glu312Ala were examined. In the X-ray crystallographic models, these are the closest carboxylate residues to the Mn(4)Ca cluster that do not ligate Mn or Ca and all are highly conserved. The 1747 cm(-1) band is present in the S(2)-minus-S(1) FTIR difference spectrum of D1-Asp61Ala but absent from the corresponding spectra of D1-Glu65Ala, D2-Glu312Ala, and D1-Glu329Gln. The band is also sharply diminished in magnitude in the wild type when samples are maintained at a relative humidity of