Evidence from FTIR difference spectroscopy of an extensive network of hydrogen bonds near the oxygen-evolving Mn₄Ca 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 Å have revealed the presence of possible channels for the removal of protons from the catalytic Mn₄Ca 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 ₄Ca 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 S₂-minus-S₁ FTIR difference spectrum of PSII from the cyanobacterium Synechocystis sp. PCC 6803. On the basis of its 4 cm^-1 downshift in D₂O, this band was assigned to the carbonyl stretching vibration (C - O) of a protonated carboxylate group whose pK_a decreases during the S₁ to S₂ transition. The positive charge that forms on the Mn₄Ca cluster during the S ₁ to S₂ 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 Mn₄Ca cluster that do not ligate Mn or Ca and all are highly conserved. The 1747 cm^-1 band is present in the S₂-minus-S₁ 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 -85%. It is proposed that D1-Glu65, D2-Glu312, and D1-Glu329 participate in a common network of hydrogen bonds that includes water molecules and the carboxylate group that gives rise to the 1747 cm^-1 band. It is further proposed that the mutation of any of these three residues, or partial dehydration caused by maintaining samples at a relative humidity of -85%, disrupts the network sufficiently that the structural perturbations associated with the S₁ to S₂ transition are no longer transmitted to the carboxylate group that gives rise to the 1747 cm^-1 band. Because D1-Glu329 is located approximately 20 Å from D1-Glu65 and D2-Glu312, the postulated network of hydrogen bonds must extend for at least 20 Å across the lumenal face of the Mn₄Ca cluster. The D1-Asp61Ala, D1-Glu65Ala, and D2-Glu312Ala mutations also appear to substantially decrease the fraction of PSII reaction centers that undergo the S₃ to S₀ transition in response to a saturating flash. This behavior is consistent with D1-Asp61, D1-Glu65, and D2-Glu312 participating in a dominant proton egress channel that links the Mn₄Ca cluster with the thylakoid lumen.