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 4MnII + 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 MnIII(MnIV)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.