Converting the 4-Flash Photosynthetic O₂ Evolution Cycle to a 2-Flash Catalytic Cycle with a Simple Cocatalyst: Counting Electrons and Holes Directly and Transparently

We apply a direct electron-counting method and two classes of reducing agents capable of single- vs multielectron H atom transfer reactions (nH) to probe the oxidation states of manganese in ultrapure Photosystem II (PSII) microcrystals (PSIIX) during the 4-flash catalytic cycle of O₂ evolution. Flash oximetry and Fourier analysis reveal that the former class of reductants (NH₂OH, H) forms two intermediates in the water oxidation center [WOC] via sequential 3H and 1H reactions, while the multi-H atom transfer class (NH₂NH₂, Hydrazine) operates via sequential 2H/2H reactions forming a postulated diazene intermediate (NHNH). At higher HZ concentration, a concerted 4H reaction to form N₂ is favored. The diazene intermediate reacts with water via a previously unknown 2-flash O₂ evolution catalytic cycle: [(S₂)NHNH] + 2H₂O → [NH₂NH₂ + (S₂)H₂O₂] → [S₀ (NHNH)] + 1/2 O₂ + H₂O. A lower redox state forms (S_-3) by direct reduction and is 7 reducing equivalents (electrons) below the O₂-evolving state (S₄). This unstable state disassembles to form 4Mn^II + apo-WOC-PSIIX. Starting from this inactive state, an active PSIIX reforms by reconstituting the inorganic cofactors of the WOC using 7 single turnover flashes, restoring the 4-flash catalytic cycle. These results corroborate previous photoassembly studies of various PSII complexes showing that the O₂-evolving S₄ state is formally comprised of Mn^III(Mn^IV)₃, referred to as the low oxidation paradigm (LO). We assess incompatible interpretations of earlier spectroscopic data that assign higher oxidation states of manganese in the 4-flash catalytic cycle. We distinguish between direct and indirect experimental methods and assess their limits of applicability for assigning Mn oxidation states.