Five-coordinate Mn^IV intermediate in the activation of nature's water splitting cofactor

Nature's water splitting cofactor passes through a series of catalytic intermediates (S₀-S₄) before O-O bond formation and O₂ release. In the second last transition (S₂ to S₃) cofactor oxidation is coupled to water molecule binding to Mn1. It is this activated, water-enriched all Mn^IV form of the cofactor that goes on to form the O-O bond, after the next light-induced oxidation to S₄ How cofactor activation proceeds remains an open question. Here, we report a so far not described intermediate (S₃') in which cofactor oxidation has occurred without water insertion. This intermediate can be trapped in a significant fraction of centers (>50%) in (i) chemical-modified cofactors in which Ca²⁺ is exchanged with Sr²⁺; the Mn₄O₅Sr cofactor remains active, but the S₂-S₃ and S₃-S₀ transitions are slower than for the Mn₄O₅Ca cofactor; and (ii) upon addition of 3% vol/vol methanol; methanol is thought to act as a substrate water analog. The S₃' electron paramagnetic resonance (EPR) signal is significantly broader than the untreated S₃ signal (2.5 T vs. 1.5 T), indicating the cofactor still contains a 5-coordinate Mn ion, as seen in the preceding S₂ state. Magnetic double resonance data extend these findings revealing the electronic connectivity of the S₃' cofactor is similar to the high spin form of the preceding S₂ state, which contains a cuboidal Mn₃O₄Ca unit tethered to an external, 5-coordinate Mn ion (Mn4). These results demonstrate that cofactor oxidation regulates water molecule insertion via binding to Mn4. The interaction of ammonia with the cofactor is also discussed.