What are the oxidation states of manganese required to catalyze photosynthetic water oxidation?

Photosynthetic O₂ production from water is catalyzed by a cluster of four manganese ions and a tyrosine residue that comprise the redox-active components of the water-oxidizing complex (WOC) of photosystem II (PSII) in all known oxygenic phototrophs. Knowledge of the oxidation states is indispensable for understanding the fundamental principles of catalysis by PSII and the catalytic mechanism of the WOC. Previous spectroscopic studies and redox titrations predicted the net oxidation state of the S₀ state to be (Mn^III)₃Mn^IV. We have refined a previously developed photoassembly procedure that directly determines the number of oxidizing equivalents needed to assemble the Mn₄Ca core of WOC during photoassembly, starting from free Mn^II and the Mn-depleted apo-WOC complex. This experiment entails counting the number of light flashes required to produce the first O₂ molecules during photoassembly. Unlike spectroscopic methods, this process does not require reference to synthetic model complexes. We find the number of photoassembly intermediates required to reach the lowest oxidation state of the WOC, S₀, to be three, indicating a net oxidation state three equivalents above four Mn^II, formally (Mn^III)₃Mn^II, whereas the O ₂ releasing state, S₄, corresponds formally to (Mn ^IV)₃Mn^III. The results from this study have major implications for proposed mechanisms of photosynthetic water oxidation.