Photosynthetic oxygen evolution is not reversed at high oxygen pressures: mechanistic consequences for the water-oxidizing complex.

Oxygenic photosynthesis is the most successful form of biological energy metabolism on Earth today. In terms of biomass, it dominates our planet, being represented by all species of higher plants, algae and cyanobacteria. Recent publications (1-3), letters (4-6) have addressed a fundamental question of energy metabolism in these organisms: Is the production of O2 by photosynthesis slowed or prevented at atmospheric pressures of O2 that are or were formerly present on Earth? If the answer is yes, the consequences for the evolution of life on earth are significant. Moreover, this question addresses the fundamental understanding of the chemistry of water oxidation by photosynthesis, which serves as model for creating biomimetic catalysts for solar-to-chemical energy conversion. Clausen et al. (1, 2), have given recent evidence that the last step in the four-step water oxidation reaction has an intrinsically low thermodynamic driving force (ΔG) and thus, can be prevented by application of low O2 pressure (50% suppression at 2.3 bar with an observable suppression beginning at atmospheric pressure). A consequence of their conclusion is that the rate of photosynthetic energy storage (proton-driven ATP production) would slow even at partial solar flux owing to charge recombination in O2-blocked centers. This postulated loss of solar energy conversion efficiency could have slowed the evolution of oxygenic photosynthesis on early Earth (7). Other lines of historical evidence, which have been recently reviewed, have concluded that atmospheric O2 has little if any effect on photosynthetic productivity under current atmospheric conditions (3). In contrast, inhibition of photosynthetic biomass accumulation due to illumination at elevated O2 pressure does occur in photobioreactors (8). Although, the origin of this loss is believed to be dominated by O2-sensitive photoinactivation rather than blocking of O2 evolution (9). To understand how O2 pressure might possibly influence the chemistry of O2 production, we must examine the catalytic site, the O2-evolving complex (OEC), located within a domain of the Photosystem II reaction center protein complex (PSII). In the overall reaction, four electrons and four protons are stripped from two water molecules to produce O2. It has been known for 36 years that O2 production requires a catalyst that accumulates four electron vacancies (oxidation equivalents or holes) formed upon four sequential light-driven charge separation reactions (10, 11). The yield of O2 formed by the OEC upon exposure to individual short flashes is described by the standard Kok model. This model is a simple Markov chain that passes through five sequential oxidation states (S0, S1, S2, S3, S4) that are populated by photon absorption. After S4 forms, it spontaneously decays in the dark to form the initial state, S0, by releasing O2 (Fig. 1A). The Kok model is shown in Fig. 1A and includes two additional postulated intermediates between S3 and S0 that have been proposed recently (see below). Figure 1 The S-state cycle and how to monitor it Recent evidence suggests that the terminal step in O2 evolution in which the O-O bond forms, S4 → S0 + n H+ + O2, may proceed through an intermediate (5, 12). Time-resolved “pump-probe” X-ray absorption spectroscopy (XAS) capable of tracking Mn X-ray fluorescence changes with a time resolution of 10 μs has detected a transient intermediate that forms after photoexcitation of S3 and before O-O bond formation (12). This intermediate was proposed to be a deprotonated S3-state (denoted S4’). It is important to note that the XAS data that supports the existence of this intermediate at ambient pressure does not show evidence for or against a second intermediate (12), which was recently a point of contention (4-6). Clausen and Junge proposed the formation of a different intermediate during S4 → S0, an S2-peroxo intermediate denoted S2*(H2O2), in which the substrate water has been partially oxidized by two electrons (1, 2). This was postulated to form at elevated O2 pressure and unlike native S4, was speculated to decay to the native S2 state with release of H2O2, e.g., S4 → S2*(H2O2) → S2 + H2O2. This hypothesis was based on a decrease in the manganese UV absorbance change following photo-generation of S4 under elevated O2 pressures. To explain this result, it was proposed that an S2*(H2O2) intermediate builds up at elevated O2 pressure as a result of a shift in the equilibrium of the terminal reaction. It was also speculated that the S2*(H2O2) intermediate could decay to S2 upon liberation of H2O2 (1). Formation and decay of an intermediate was later supported by delayed fluorescence work done by Clausen et al. (2), although H2O2 was not detected as a product. Both of these possibilities have been included in the modified S-state cycle model represented in Fig. 1A. The observed effect reached 50% of maximum at 2.3 bar and saturated between 14 and 30 bar. Their study was performed with detergent-extracted PSII core complexes from the cyanobacterium Synechocystis sp. PCC6803, and was supported by independent measurements of delayed chlorophyll-a fluorescence using detergent-extracted PSII-enriched thylakoid membranes isolated from spinach (2). The formation of a native peroxide-like intermediate at S2 has been postulated before (13-16) and H2O2 production has been observed from blocked PSII centers incapable of O2 production or normal S-state turnover (17, 18). However, there has been no direct experimental support for a peroxo intermediate or O2-bound product following S4 in any PSII complex until the reports by Clausen et al. In the present study, we have investigated O2 production in all three major branches of oxygen-producing phototrophs at elevated O2 pressures up to 10.3 bar (and up to 43 bar in A. maxima) using a non-invasive method that monitors chlorophyll fluorescence from intact cells (microalgae and cyanobacteria), plant leaves, and isolated thylakoid membranes. Our results show that none of the reactions of water oxidation, especially the terminal step (S4 → S0 + O2 + nH+), are mechanistically affected by elevated O2 pressures up to 50-fold greater than atmospheric conditions. Thus, the terminal reaction has no thermodynamically accessible peroxo or O2-bound intermediate that can be populated by reversing the O2 release step using elevated levels of O2. The consequences for the mechanism of water oxidation are examined.