Insights into Proton-Transfer Pathways during Water Oxidation in Photosystem II.

Water oxidation by photosystem II (PSII) involves the release of O2, electrons, and protons at the oxygen-evolving complex (OEC). These processes are facilitated by a hydrogen-bonded network of amino acid residues and waters surrounding the OEC. It is crucial to probe the proton-transfer pathways from the OEC as proton release helps to maintain the charge balance required for efficient water oxidation. In this study, we generate point mutations in the cyanobacterium Synechocystis sp. PCC 6803 at secondary-shell amino acid residues surrounding the OEC: D2-K317, D1-S169, CP43-R357, D1-D61, and D1-N181. We employ direct experimental methods to study the O2 evolution rate under varying pH ranging from 3-8. The pH dependence follows a bell-shaped curve in both wild-type and mutated PSII from which we can derive the effective acidic pKa. The effective acidic pKa provides insights into the protonation states of the amino acid residues participating in the proton-transfer process during the rate-determining step of water oxidation. The presence of an additional effective pKa in D1-S169A PSII and D2-K317A PSII indicates the possibility of multiple proton-transfer pathways during the rate-determining step of water oxidation. We also studied the O2 evolution rate in H2O and D2O with varying pL (L = H or D) to identify the amino acid residues participating in the proton-transfer process. We find that replacing the positively charged lysine with a neutral alanine in D2-K317A PSII and aspartate with alanine in D1-D61A PSII significantly enhances the kinetic solvent isotope effect (KSIE), indicating that proton transfer becomes rate-limiting at the optimal pH in these mutated PSII. However, the KSIE remains unchanged for D1-N181A, D1-S169A, and CP43-R357K PSII. Thus, perturbing the channel defined by the D1-D61 and D2-K317 residues strongly hampers the proton-transfer mechanism, and in turn, the water oxidation reaction of PSII. Hence, our study provides a direct experimental probe to identify that the D1-D61 and D2-K317 residues participate in the proton-transfer process. These results, thereby, provide us a deeper understanding of the proton-transfer processes in the water oxidation mechanism.

Energetics of the S2 State Spin Isomers of the Oxygen-Evolving Complex of Photosystem II.

The S2 redox intermediate of the oxygen-evolving complex in photosystem II is present as two spin isomers. The S = 1/2 isomer gives rise to a multiline electron paramagnetic resonance (EPR) signal at g = 2.0, whereas the S = 5/2 isomer exhibits a broad EPR signal at g = 4.1. The electronic structures of these isomers are known, but their role in the catalytic cycle of water oxidation remains unclear. We show that formation of the S = 1/2 state from the S = 5/2 state is exergonic at temperatures above 160 K. However, the S = 1/2 isomer decays to S1 more slowly than the S = 5/2 isomer. These differences support the hypotheses that the S3 state is formed via the S2 state S = 5/2 isomer and that the stabilized S2 state S = 1/2 isomer plays a role in minimizing S2QA- decay under light-limiting conditions.

Photosynthetic water oxidation: binding and activation of substrate waters for O-O bond formation.

Photosynthetic water oxidation occurs at the oxygen-evolving complex (OEC) of Photosystem II (PSII). The OEC, which contains a Mn4CaO5 inorganic cluster ligated by oxides, waters and amino-acid residues, cycles through five redox intermediates known as S(i) states (i = 0-4). The electronic and structural properties of the transient S4 intermediate that forms the O-O bond are not well understood. In order to gain insight into how water is activated for O-O bond formation in the S4 intermediate, we have performed a detailed analysis of S-state dependent substrate water binding kinetics taking into consideration data from Mn coordination complexes. This analysis supports a model in which the substrate waters are both bound as terminal ligands and react via a water-nucleophile attack mechanism.

Cation Effects on the Electron-Acceptor Side of Photosystem II.

The normal pathway of electron transfer on the electron-acceptor side of photosystem II (PSII) involves electron transfer from quinone A, QA, to quinone B, QB. It is possible to redirect electrons from QA(-) to water-soluble Co(III) complexes, which opens a new avenue for harvesting electrons from water oxidation by immobilization of PSII on electrode surfaces. Herein, the kinetics of electron transfer from QA(-) to [Co(III)(terpy)2](3+) (terpy = 2,2';6',2″-terpyridine) are investigated with a spectrophotometric assay revealing that the reaction follows Michaelis-Menten saturation kinetics, is inhibited by cations, and is not affected by variation of the QA reduction potential. A negatively charged site on the stromal surface of the PSII protein complex, composed of glutamic acid residues near QA, is hypothesized to bind cations, especially divalent cations. The cations are proposed to tune the redox properties of QA through electrostatic interactions. These observations may thus explain the molecular basis of the effect of divalent cations like Ca(2+), Sr(2+), Mg(2+), and Zn(2+) on the redox properties of the quinones in PSII, which has previously been attributed to long-range conformational changes propagated from divalent cations binding to the Ca(II)-binding site in the oxygen-evolving complex on the lumenal side of the PSII complex.

Oxygen-evolving complex of Photosystem II: an analysis of second-shell residues and hydrogen-bonding networks.

The oxygen-evolving complex (OEC) is a Mn4O5Ca cluster embedded in the Photosystem II (PSII) protein complex. As the site of water oxidation, the OEC is connected to the lumen by channels that conduct water, oxygen, and/or protons during the catalytic cycle. The hydrogen-bond networks found in these channels also serve to stabilize the oxidized intermediates, known as the S states. We review recent developments in characterizing these networks via protein mutations, molecular inhibitors, and computational modeling. On the basis of these results, we highlight regions of the PSII protein in which changes have indirect effects on the S1, S2, and S3 oxidation states of the OEC while still allowing photosynthetic activity.