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...