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The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution.

The molecular oxygen we breathe is produced from water-derived oxygen species bound to the Mn4CaO5 cluster in photosystem II (PSII). Present research points to the central oxo-bridge O5 as the 'slow exchanging substrate water (Ws)', while, in the S2 state, the terminal water ligands W2 and W3 are both discussed as the 'fast exchanging substrate water (Wf)'. A critical point for the assignment of Wf is whether or not its exchange with bulk water is limited by barriers in the channels leading to the Mn4CaO5 cluster. In this study, we measured the rates of H2 16O/H2 18O substrate water exchange in the S2 and S3 states of PSII core complexes from wild-type (WT) Synechocystis sp. PCC 6803, and from two mutants, D1-D61A and D1-E189Q, that are expected to alter water access via the Cl1/O4 channels and the O1 channel, respectively. We found that the exchange rates of Wf and Ws were unaffected by the E189Q mutation (O1 channel), but strongly perturbed by the D61A mutation (Cl1/O4 channel). It is concluded that all channels have restrictions limiting the isotopic equilibration of the inner water pool near the Mn4CaO5 cluster, and that D61 participates in one such barrier. In the D61A mutant this barrier is lowered so that Wf exchange occurs more rapidly. This finding removes the main argument against Ca-bound W3 as fast substrate water in the S2 state, namely the indifference of the rate of Wf exchange towards Ca/Sr substitution.

Photosystem II (PSII) is a multi‐subunit protein‐pigment complex with diverse redox‐active cofactors, which enabled the biological availability of O2 on Earth. The substrate specificity and the underlying redox chemistry of the Mn4CaO5 catalytic center are investigated using alternate substrates such as small molecules (ammonia and methanol) and halides (Cl‐, Br‐, I‐) instead of the natural substrate water. Changes in the kinetic profiles of steady‐state O2 evolution and of dichlorophenolindophenol (DCIP) photochemical reduction by PSII as well as the detection of modified sites by proteomic analysis implied the possibility of alternate substrate photooxidation. Of particular interest is the role of two chlorides bound close to the putative water channels in the native system. The mutation of D2‐K317 to alanine is believed to impair the binding of a catalytically relevant chloride, eliminating the chloride requirement for water oxidation catalysis. The efficiency of small molecule photooxidation by the OEC is enhanced by the mutated D2‐K317A PSII complex without the competition from chloride. These results provide insight into the role of bound chloride in native PSII as a filter for enhancing the selectivity of water oxidation. The design principles for PSII may be extended to new strategies for developing highly selective catalysts. [ABSTRACT FROM AUTHOR]

Photoinduced water oxidation at the O2-evolving complex (OEC) of photosystem II (PSII) is a complex process involving a tetramanganese-calcium cluster that is surrounded by a hydrogen-bonded network of water molecules, chloride ions, and amino acid residues. Although the structure of the OEC has remained conserved over eons of evolution, significant differences in the chloride-binding characteristics exist between cyanobacteria and higher plants. An analysis of amino acid residues in and around the OEC has identified residue 87 in the D1 subunit as the only significant difference between PSII in cyanobacteria and higher plants. We substituted the D1-Asn87 residue in the cyanobacterium Synechocystis sp. PCC 6803 (wildtype) with alanine, present in higher plants, or with aspartic acid. We studied PSII core complexes purified from D1-N87A and D1-N87D variant strains to probe the function of the D1-Asn87 residue in the water-oxidation mechanism. EPR spectra of the S2 state and flash-induced FTIR spectra of both D1-N87A and D1-N87D PSII core complexes exhibited characteristics similar to those of wildtype Synechocystis PSII core complexes. However, flash-induced O2-evolution studies revealed a decreased cycling efficiency of the D1-N87D variant, whereas the cycling efficiency of the D1-N87A PSII variant was similar to that of wildtype PSII. Steady-state O2-evolution activity assays revealed that substitution of the D1 residue at position 87 with alanine perturbs the chloride-binding site in the proton-exit channel. These findings provide new insight into the role of the D1-Asn87 site in the water-oxidation mechanism and explain the difference in the chloride-binding properties of cyanobacterial and higher-plant PSII.

The residue D1-D170 bridges Mn4 with the Ca ion in the O2-evolving Mn4CaO5 cluster of Photosystem II. Recently, the D1-D170E mutation was shown to substantially alter the Sn+1-minus-Sn FTIR difference spectra [Debus RJ (2021) Biochemistry 60:3841-3855]. The mutation was proposed to alter the equilibrium between different Jahn–Teller conformers of the S1 state such that (i) a different S1 state conformer is stabilized in D1-D170E than in wild-type and (ii) the S1 to S2 transition in D1-D170E produces a high-spin form of the S2 state rather than the low-spin form that is produced in wild-type. In this study, we employed EPR spectroscopy to test if a high-spin form of the S2 state is formed preferentially in D1-D170E PSII. Our data show that illumination of dark-adapted D1-D170E PSII core complexes does indeed produce a high-spin form of the S2 state rather than the low-spin multiline form that is produced in wild-type. This observation provides further experimental support for a change in the equilibrium between S state conformers in both the S1 and S2 states in a site-directed mutant that retains substantial O2 evolving activity. [ABSTRACT FROM AUTHOR]

The Fourier transform infrared difference spectra of the individual S state transitions in D1-D342N mutant photosystem II (PSII) particles from cyanobacterium Synechocystis sp. PCC 6803 are compared with those in wild type PSII particles in order to determine if D1-Asp342 ligates a Mn ion undergoing oxidation during one or more of the [S.sub.0] to [S.sub.1], [S.sub.1] to [S.sub.2], or [S.sub.2] to [S.sub.3] transitions. The results show no evidence, that D1-Asp342 ligates the same Mn ion that is ligated by the [alpha]-CO[O.sup.-] group of D1-Ala344.

Research has been conducted on photosystem II containing tedox-active tyrosines. The effect of cryogenic temperature on the light-induced tyrosyl radicals has been investigated as a pH function in photosystem II from cyanobacteria and plants and the details are reported.

Research has been conducted on the tetranuclear manganese cluster in photosystem II ligated by histidine residues. The legation of this photosystem is demonstrated by the electron spin echo envelope modulation study performed via isolation of the histidine-labeled photosystem II particles from a cyanobacterium.

Research has been conducted on the wild-type and mutant cells of the cyanobacterium Synechocystis sp. PCC 6803. The mid-frequency and low-frequency Fourier transform infrared spectroscopy difference spectra produced by the particles isolated from these cells has been investigated.

Glutamate 189 of the D1 polypeptide has been found to modulate the magnetic and redox properties of the manganese cluster and tyrosine Y(sub.Z) in photosystem II (PSII). PSII is a multisubunit, integral membrane protein complex. It uses light energy to oxidize water and reduce plastoquinone. Seventeen D1-Glu189 mutations have been generated in the cyanobacterium Synechocystis sp. PCC 6803 for study. Results indicate D1-GLU189 participates in a network of hydrogen bonds that change properties of both YZ and the Mn cluster. The also so suggest that D1-Glu189 positions a group that accepts a proton from D1-His190.

The ability of histidine residue to coordinate the O (sub)2-evolving Mn cluster is discussed. Results demonstrate that mutation D1-H332E alters magnetic properties of Mn cluster and redox properties of Y (sub)Z and Mn cluster.

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a highresolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation. [ABSTRACT FROM AUTHOR]

Photosystem II (PSII) of green plants and cyanobacteria produces dioxygen at the expense of light. Mutants from Synechocystis sp. PCC 6803 of the residue D1-Asp61, in the lumenal loop were studied from D1-D61, in both cells and oxygen-evolving core particles. Oxygen-release was slowed drastically in D61A and D61N in cells, although it was unchanged in D61E.

The hypothesis that His190 of the D1 polypeptide promotes electron transfer from tyrosine Y(sub Z) to P6800+ was tested to further elucidate the function of D1-His190 in the proton-coupled electron transfer reactions of photosystem II. Findings showed that D1-His190 is the immediate proton acceptor for Y(sub Z) and that the hydroxyl proton of Y(sub Z) remains bound to D1-His190 during the lifetime of Y(sub Z)*, therefore encouraging the reduction of Y(sub Z)*.

A study was conducted to examine the presence of the S1-state multiline EPR signal in Synechocystic preparations. Membranes were prepared from spinach while spinach-enriched membranes were washed in a buffer solution supporting 300 mM sucrose, 10 mM NaCl and 25 mM MES-NaOH. Results suggested that the presence of the S1-state multiline in Synechocystis core particles may be an alternative form of the manganese cluster.

Research conducted to identify amino acid residues that influence the binding of manganese or calcium to photosystem II found that His-332, Glu-333 and His-337 influence the binding of calcium and His-332, Glu-333, His-337 and Asp-332 influence the assembly and stability of the Mn cluster. These amino acid residues are located at the carboxyl-terminal region of the D1 polypeptide of photosystem II. His-332, Glu-333 and His-337 may ligate Mn, while Asp-342 may ligate manganese, calcium, or both.