Zwitterion Modulation of O₂-Evolving Activity of Cyanobacterial Photosystem II

Photosystem II (PSII) is the only enzyme in nature that can catalyze the challenging catalytic photooxidation of H2O into four protons, four electrons, and O2. Slowing down turnover of the O2-evolving complex (OEC) is a plausible approach to gain mechanistic information on the reaction. However, modulating the kinetics of the reaction without perturbing the active site is a challenge. In this study, it is shown that the steady-state activity of cyanobacterial PSII is inhibited by small zwitterions, such as glycine betaine and β-alanine. We show that the binding of zwitterions is nondenaturing, is highly reversible, and results in the decrease of the rate of catalytic turnover by ∼50% in the presence of excess zwitterion. Control measurements of photoinduced electron transfer in O2-inactive PSII show that the inhibition by zwitterions is the result of a specific decrease in the rate of catalytic turnover of the OEC. Recovery of activity upon addition of an exogenous proton carrier (HCO3−) provides evidence that proton-transfer pathways, thought to be essential for the relay of protons from the OEC to the lumen, are affected. Interestingly, no inhibition is observed for spinach PSII, suggesting that zwitterions act specifically by binding to the extrinsic proteins on the lumenal side of PSII, which differ significantly between plants and cyanobacteria, to slow proton transfer on the electron donor side of PSII

Investigation of the Inhibitory Effect of Nitrite on Photosystem II

The role of chloride in photosystem II (PSII) is unclear. Several monovalent anions compete for the Cl^– site(s) in PSII, and some even support activity. NO₂^– has been reported to be an activator in Cl^–-depleted PSII membranes. In this paper, we report a detailed investigation of the chemistry of NO₂^– with PSII. NO₂^– is shown to inhibit PSII activity, and the effects on the donor side as well as the acceptor side are characterized using steady-state O₂-evolution assays, electron paramagnetic resonance (EPR) spectroscopy, electron-transfer assays, and flash-induced polarographic O₂ yield measurements. Enzyme kinetics analysis shows multiple sites of NO₂^– inhibition in PSII with significant inhibition of oxygen evolution at <5 mM NO₂^–. By EPR spectroscopy, the yield of the S₂ state remains unchanged up to 15 mM NO₂^–. However, the S₂-state <i>g</i> = 4.1 signal is favored over the <i>g</i> = 2 multiline signal with increasing NO₂^– concentrations. This could indicate competition of NO₂^– for the Cl^– site at higher NO₂^– concentrations. In addition to the donor-side chemistry, there is clear evidence of an acceptor-side effect of NO₂^–. The <i>g</i> = 1.9 Fe­(II)-Q_A^–• signal is replaced by a broad <i>g</i> = 1.6 signal in the presence of NO₂^–. Additionally, a <i>g</i> = 1.8 Fe­(II)-Q^–• signal is present in the dark, indicating the formation of a NO₂^–-bound Fe­(II)-Q_B^–• species in the dark. Electron-transfer assays suggest that the inhibitory effect of NO₂^– on the activity of PSII is largely due to the donor-side chemistry of NO₂^–. UV–visible spectroscopy and flash-induced polarographic O₂ yield measurements indicate that NO₂^– is oxidized by the oxygen-evolving complex in the higher S states, contributing to the donor-side inhibition by NO₂^–

Redirecting Electron Transfer in Photosystem II from Water to Redox-Active Metal Complexes

A negatively charged region on the surface of photosystem II (PSII) near QA has been identified as a docking site for cationic exogenous electron acceptors. Oxygen evolution activity, which is inhibited in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is recovered by adding CoIII complexes. Thus, a new electron-transfer pathway is created with CoIII as the new terminal electron acceptor from QA–. This binding site is saturated at ∼2.5 mM [CoIII], which is consistent with the existence of low-affinity interactions with a solvent-exposed surface. This is the first example of a higher plant PSII in which the electron-transfer pathway has been redirected from the normal membrane-associated quinone electron acceptors to water-soluble electron acceptors. The proposed CoIII binding site may enable efficient collection of electrons generated from photochemical water oxidation by PSII immobilized on an electrode surface

Redirecting Electron Transfer in Photosystem II from Water to Redox-Active Metal Complexes

A negatively charged region on the surface of photosystem II (PSII) near QA has been identified as a docking site for cationic exogenous electron acceptors. Oxygen evolution activity, which is inhibited in the presence of the herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), is recovered by adding CoIII complexes. Thus, a new electron-transfer pathway is created with CoIII as the new terminal electron acceptor from QA–. This binding site is saturated at ∼2.5 mM [CoIII], which is consistent with the existence of low-affinity interactions with a solvent-exposed surface. This is the first example of a higher plant PSII in which the electron-transfer pathway has been redirected from the normal membrane-associated quinone electron acceptors to water-soluble electron acceptors. The proposed CoIII binding site may enable efficient collection of electrons generated from photochemical water oxidation by PSII immobilized on an electrode surface

Multiple Redox-Active Chlorophylls in the Secondary Electron-Transfer Pathways of Oxygen-Evolving Photosystem II

Photosystem II (PS II) is unique among photosynthetic reaction centers in having secondary electron donors that compete with the primary electron donors for reduction of P680+. We have characterized the photooxidation and dark decay of the redox-active accessory chlorophylls (Chl) and β-carotenes (Car) in oxygen-evolving PS II core complexes by near-IR absorbance and EPR spectroscopies at cryogenic temperatures. In contrast to previous results for Mn-depleted PS II, multiple near-IR absorption bands are resolved in the light-minus-dark difference spectra of oxygen-evolving PS II core complexes including two fast-decaying bands at 793 and 814 nm and three slow-decaying bands at 810, 825, and 840 nm. We assign these bands to chlorophyll cation radicals (Chl+). The fast-decaying bands observed after illumination at 20 K could be generated again by reilluminating the sample. Quantization by EPR gives a yield of 0.85 radicals per PS II, and the yield of oxidized cytochrome b559 by optical difference spectroscopy is 0.15 per PS II. Potential locations of Chl+ and Car+ species, and the pathways of secondary electron transfer based on the rates of their formation and decay, are discussed. This is the first evidence that Chls in the light-harvesting proteins CP43 and CP47 are oxidized by P680+ and may have a role in Chl fluorescence quenching. We also suggest that a possible role for negatively charged lipids (phosphatidyldiacylglycerol and sulfoquinovosyldiacylglycerol identified in the PS II structure) could be to decrease the redox potential of specific Chl and Car cofactors. These results provide new insight into the alternate electron-donation pathways to P680+

Insights into Substrate Binding to the Oxygen-Evolving Complex of Photosystem II from Ammonia Inhibition Studies

Water oxidation in Photosystem II occurs at the oxygen-evolving complex (OEC), which cycles through distinct intermediates, S₀–S₄. The inhibitor ammonia selectively binds to the S₂ state at an unresolved site that is not competitive with substrate water. By monitoring the yields of flash-induced oxygen production, we show that ammonia decreases the net efficiency of OEC turnover and slows the decay kinetics of S₂ to S₁. The temperature dependence of biphasic S₂ decay kinetics provides activation energies that do not vary in control and ammonia conditions. We interpret our data in the broader context of previous studies by introducing a kinetic model for both the formation and decay of ammonia-bound S₂. The model predicts ammonia binds to S₂ rapidly (<i>t</i>_1/2 = 1 ms) with a large equilibrium constant. This finding implies that ammonia decreases the reduction potential of S₂ by at least 2.7 kcal mol^–1 (>120 mV), which is not consistent with ammonia substitution of a terminal water ligand of Mn­(IV). Instead, these data support the proposal that ammonia binds as a bridging ligand between two Mn atoms. Implications for the mechanism of O–O bond formation are discussed

Evidence against Bicarbonate Bound in the O₂-Evolving Complex of Photosystem II

The oxidation of water to molecular oxygen by photosystem II (PSII) is inhibited in bicarbonate-depleted media. One contribution to the inhibition is the binding of bicarbonate to the non-heme iron, which is required for efficient electron transfer on the electron-acceptor side of PSII. There are also proposals that bicarbonate is required for formation of O2 by the manganese-containing O2-evolving complex (OEC). Previous work indicates that a bicarbonate ion does not bind reversibly close to the OEC, but it remains possible that bicarbonate is bound sufficiently tightly to the OEC that it cannot readily exchange with bicarbonate in solution. In this study, we have used NH2OH to destroy the OEC, which would release any tightly bound bicarbonate ions from the active site, and mass spectrometry to detect any released bicarbonate as CO2. The amount of CO2 per PSII released by the NH2OH treatment is observed to be comparable to the background level, although N2O, a product of the reaction of NH2OH with the OEC, is detected in good yield. These results strongly argue against tightly bound bicarbonate ions in the OEC

High Turnover Remote Catalytic Oxygenation of Alkyl Groups: How Steric Exclusion of Unbound Substrate Contributes to High Molecular Recognition Selectivity

H-bonding mediated molecular recognition between substrate and ligand −COOH groups orients the substrate so that remote, catalyzed oxygenation of an alkyl C−H bond by a Mn-oxo active site can occur with very high (>98%) regio- and stereoselectivity. This paper identifies steric exclusionexclusion of non H-bonded substrate molecules from the active siteas one requirement for high selectivity, along with the entropic advantage of intramolecularity. If unbound substrate molecules were able to reach the active site, they would react unselectively, degrading the observed selectivity. Both of the faces of the catalyst are blocked by two ligand molecules each with a −COOH group. The acid p-tBuC6H4COOH binds to the ligand −COOH recognition site but is not oxidized and merely blocks approach of the substrate therefore acting as an effective inhibitor for ibuprofen oxidation in both free acid and ibuprofen ester form. Dixon plots show that inhibition is competitive for the free acid ibuprofen substrate, no doubt because this substrate can compete with the inhibitor for binding to the recognition site. In contrast, inhibition is uncompetitive for the ibuprofen-ester substrate, consistent with this ester substrate no longer being able to bind to the recognition site. Inhibition can be reversed with MeCOOH, an acid that can competitively bind to the recognition site but, being sterically small, no longer blocks access to the active site

O₂ Evolution and Permanganate Formation from High-Valent Manganese Complexes

O2 Evolution and Permanganate Formation from High-Valent Manganese Complexe

Study of Proton Coupled Electron Transfer in a Biomimetic Dimanganese Water Oxidation Catalyst with Terminal Water Ligands

The oxomanganese complex [H2O(terpy)MnIII(μ-O)2MnIV(terpy)H2O]3+ (1, terpy = 2,2′:6-2′′-terpyridine) is a biomimetic model of the oxygen-evolving complex of photosystem II with terminal water ligands. When bound to TiO2 surfaces, 1 is activated by primary oxidants (e.g., Ce4+(aq) or oxone in acetate buffers) to catalyze the oxidation of water yielding O2 evolution [G. Li et al. Energy Environ. Sci. 2009, 2, 230−238]. The activation is thought to involve oxidation of the inorganic core [MnIII(μ-O)2MnIV]3+ to generate the [MnIV(μ-O)2MnIV]4+ state 1ox first and then the highly reactive Mn oxyl species MnIVO• through proton coupled electron transfer (PCET). Here, we investigate the step 1 → 1ox as compared to the analogous conversion in an oxomanganese complex without terminal water ligands, the [(bpy)2 MnIII(μ-O)2 MnIV (bpy)2]3+ complex (2, bpy = 2,2′-bipyridyl). We characterize the oxidation in terms of free energy calculations of redox potentials and pKa’s as directly compared to cyclic voltammogram measurements. We find that the pKa’s of terminal water ligands depend strongly on the oxidation states of the Mn centers, changing by ∼13 pH units (i.e., from 14 to 1) during the III,IV → IV,IV transition. Furthermore, we find that the oxidation potential of 1 is strongly dependent on pH (in contrast to the pH-independent redox potential of 2) as well as by coordination of Lewis base moieties (e.g., carboxylate groups) that competitively bind to Mn by exchange with terminal water ligands. The reported analysis of ligand binding free energies, pKa’s, and redox potentials indicates that the III,IV → IV,IV oxidation of 1 in the presence of acetate (AcO−) involves the following PCET: [H2O(terpy)MnIII(μ-O)2MnIV(terpy)AcO]2+ → [HO(terpy)MnIV(μ-O)2MnIV(terpy)AcO]2+ + H+ + e−