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In the 1.9 Å structural model of photosystem II (PDB: 3ARC), the amino acid residue Glu333 of the D1 polypeptide coordinates to the oxygen-evolving Mn4CaO5 cluster. This residue appears to be highly significant in that it bridges the two Mn ions (MnB3 and the "dangling" MnA4) that are also bridged by the oxygen atom O5. This oxygen atom has been proposed to be derived from one of two substrate water molecules and to become incorporated into the product dioxygen molecule during the final step in the catalytic cycle. In addition, the backbone nitrogen of D1-Glu333 interacts directly with a nearby Cl- atom. To further explore the influence of this structurally unique residue on the properties of the Mn4CaO5 cluster, the D1-E333Q, mutant of the cyano bacterium Synechocystis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography, EPR, X-ray absorption, and FTIR difference spectroscopy. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild-type. In addition, the oxygen flash yields exhibited normal period-four oscillations having normal S state parameters, although the yields were lower, indicative of the mutant's lower steady-state dioxygen evolution rate of approximately 30% compared to that of the wild-type. The S1 state Mn-XANES and Mn-EXAFS and S2 state multiline EPR signals of purified D1-E333Q, PSU core complexes closely resembled those of wild-type, aside from having lower amplitudes. The Sn+1,-minus-Sn FTIR difference spectra showed only minor alterations to the carbonyl, amide, and carboxylate stretching regions. However, the mutation eliminated a negative peak at 3663 cm-1 in the weakly H-bonding O-H stretching region of the S2-minus-S1 FTIR difference spectrum and caused an approximately 9 cm-1 downshift of the negative feature in this region of the S1-minus-S0 FTIR difference spectrum. We conclude that fully functional Mn4CaO5 clusters assemble in the presence of the D1-E333Q mutarion but that the mutation decreases the yield of assembled clusters and alters the H-bonding properties of one or more water molecules or hydroxide groups that are located on or near the Mn4CaO5 cluster and that either deprotonate or farm stronger hydrogen bonds during the S0 to S1 and S1 to S2 transitions. [ABSTRACT FROM AUTHOR]

In the current X-ray crystallographic structural models of photosystem II. Glu354 of the CP43 polypeptide is the only amino acid ligand of the oxygen-evolving Mn4Ca cluster that is not provided by the D1 polypeptide. To further explore the influence of this structurally unique residue on the properties of the Mn4Ca cluster, the CP43-E354Q mutant of the cyanobacterium Synechocvstis sp. PCC 6803 was characterized with a variety of biophysical and spectroscopic methods, including polarography. EPR. X-ray absorption, FTIR. and mass spectrometry. The kinetics of oxygen release in the mutant were essentially unchanged from those in wild type. In addition, the oxygen flash yields exhibited normal period four oscillations having normal S state parameters, although the yields were lower, correlating with the mutant's lower steady-state rate (approximately 20% compared to wild type). Experiments conducted with H218O showed that the fast and slow phases of substrate water exchange in CP43-E354Q thylakoid membranes were accelerated 8.5- and 1.8-fold, respectively, in the S3 state compared to wild type. Purified oxygen-evolving CP43-E354Q PSII core complexes exhibited a slightly altered S1 state Mn-EXAFS spectrum, a slightly altered S2 state multiline EPR signal, a substantially altered S2-minus-S1 FTIR difference spectrum, and an unusually long lifetime for the S2 state (> 10 h) in a substantial fraction of reaction centers. In contrast, the S1 state Mn-EXAFS spectrum was nearly indistinguishable from that of wild type. The S2-minus-S1 FTIR difference spectrum showed alterations throughout the amide and carboxylate stretching regions. Global labeling with 15N and specific labeling with L-[1-13C]alanine revealed that the mutation perturbs both amide II and carboxylate stretching modes and shifts the symmetric carboxylate stretching modes of the α-COO group of D1-A1a344 (the C-terminus of the D1 polypeptide) to higher frequencies by 3-4 cm-1 in both the S1 and S1 states. The EPR and FTIR data implied that 76-82% of CP43-E354Q PSII centers can achieve the S2 state and that most of these can achieve the S3 state, but no evidence for advancement beyond the S2 state was observed in the FTIR data, at least not in a majority of PSII centers. Although the X-ray absorption and EPR data showed that the CP43-E354Q mutation only subtly perturbs the structure and spin state of the Mn4Ca cluster in the S4 state, the FTIR and H218O exchange data show that the mutation strongly influences other properties of the Mn4Ca cluster, altering the response of numerous carboxylate and amide groups to the increased positive charge that develops on the cluster during the S1 to S2transition and weakening the binding of both substrate water molecules (or water-derived ligands), especially the one that exchanges rapidly in the S3 state. The FTIR data provide evidence that CP43-G1u354 coordinates to the Mn4Ca cluster in the S1 state as a bridging ligand between two metal ions but provide no compelling evidence that this residue changes its coordination mode during the S1 to S2 transition. The H218O exchange data provide evidence that CP43-Glu354 interacts with the Mn ion that ligates the substrate water molecule (or water-derived ligand) that is in rapid exchange in the S3 state. [ABSTRACT FROM AUTHOR]