Plants and cyanobacteria produce atmospheric dioxygen from water, powered by sunlight and catalyzed by a manganese complex in photosystem II. A classic S-cycle model for oxygen evolution involves five states, but only four have been identified. The missing S4 state is particularly important because it is directly involved in dioxygen formation. Now progress comes from an x-ray technique that can monitor redox and structural changes in metal centers in real time with 10-microsecond resolution. We show that in the O2-formation step, an intermediate is formed--the enigmatic S4 state. Its creation is identified with a deprotonation process rather than the expected electron-transfer mechanism. Subsequent electron transfer would give an additional S4' state, thus extending the fundamental S-state cycle of dioxygen formation.

Structural and electronic changes (oxidation states) of the Mn(4)Ca complex of photosystem II (PSII) in the water oxidation cycle are of prime interest. For all four transitions between semistable S-states (S(0) --> S(1), S(1) --> S(2), S(2) --> S(3), and S(3),(4) --> S(0)), oxidation state and structural changes of the Mn complex were investigated by X-ray absorption spectroscopy (XAS) not only at 20 K but also at room temperature (RT) where water oxidation is functional. Three distinct experimental approaches were used: (1) illumination-freeze approach (XAS at 20 K), (2) flash-and-rapid-scan approach (RT), and (3) a novel time scan/sampling-XAS method (RT) facilitating particularly direct monitoring of the spectral changes in the S-state cycle. The rate of X-ray photoreduction was quantitatively assessed, and it was thus verified that the Mn ions remained in their initial oxidation state throughout the data collection period (>90%, at 20 K and at RT, for all S-states). Analysis of the complete XANES and EXAFS data sets (20 K and RT data, S(0)-S(3), XANES and EXAFS) obtained by the three approaches leads to the following conclusions. (i) In all S-states, the gross structural and electronic features of the Mn complex are similar at 20 K and room temperature. There are no indications for significant temperature-dependent variations in structure, protonation state, or charge localization. (ii) Mn-centered oxidation likely occurs on each of the three S-state transitions, leading to the S(3) state. (iii) Significant structural changes are coupled to the S(0) --> S(1) and the S(2) --> S(3) transitions which are identified as changes in the Mn-Mn bridging mode. We propose that in the S(2) --> S(3) transition a third Mn-(mu-O)(2)-Mn unit is formed, whereas the S(0) --> S(1) transition involves deprotonation of a mu-hydroxo bridge. In light of these results, the mechanism of accumulation of four oxidation equivalents by the Mn complex and possible implications for formation of the O-O bond are considered.

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Structural changes upon photoreduction caused by x-ray irradiation of the water-oxidizing tetramanganese complex of photosystem II were investigated by x-ray absorption spectroscopy at the manganese K-edge. Photoreduction was directly proportional to the x-ray dose. It was faster in the higher oxidized S 2 state than in S 1 ; seemingly the oxidizing potential of the metal site governs the rate. X-ray irradiation of the S 1 state at 15 K initially caused single-electron reduction to S 0 * accompanied by the conversion of one di--oxo bridge between manganese atoms, previously separated by ϳ2.7 Å , to a mono--oxo motif. Thereafter, manganese photoreduction was 100 times slower, and the biphasic increase in its rate between 10 and 300 K with a breakpoint at ϳ200 K suggests that protein dynamics is rate-limiting the radical chemistry. For photoreduction at similar x-ray doses as applied in protein crystallography, halfway to the final Mn II 4 state the complete loss of inter-manganese distances <3 Å was observed, even at 10 K, because of the destruction of -oxo bridges between manganese ions. These results put into question some structural attributions from recent protein crystallography data on photosystem II. It is proposed to employ controlled x-ray photoreduction in metalloprotein research for: (i) population of distinct reduced states, (ii) estimating the redox potential of buried metal centers, and (iii) research on protein dynamics.Numerous enzymes contain protein-bound metal centers forming their active site. A prominent example is the water-oxidizing manganese-calcium (Mn 4 Ca) complex of oxygenic photosynthesis bound to the D1 protein of photosystem II (PSII), 2 which is embedded in the thylakoid membrane of higher plants, green algae, and cyanobacteria (1). The manganese complex catalyzes the light-driven oxidation of two water molecules, yielding reducing equivalents, protons, and the dioxygen of the atmosphere. By the sequential absorption of four quanta of visible light by PSII that drive the stepwise abstraction of four electrons, the manganese complex cycles through four semi-stable states called S 1 , S 2 , S 3 , and S 0 , where the subscripts denote the number of accumulated oxidizing equivalents (2). The S 1 represents the dark-stable state; dioxygen is liberated only in the S 3 3 S 0 transition (for reviews see Refs. 1 and 3).Important structural information on the manganese complex has been obtained by x-ray absorption spectroscopy (XAS) (Refs. 4 -7 and the references therein) and recently also by protein crystallography (8 -11). The crystallographic results on PSII represent a long awaited breakthrough. With respect to the manganese complex, however, the question has emerged regarding to what extent the obtained structural information is invalidated by modifications caused by the numerous radicals that are inevitably created by x-ray irradiation (11-13). In all four structures (8 -11) manganese ions were found; however, there were inconsistencies in their probable number and position with respec...