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 O-2 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 N-2 is favored. The diazene intermediate reacts with water via a previously unknown 2-flash O-2 evolution catalytic cycle: [(S-2)NHNH] + 2H(2)O -> [NH2NH2 + (S-2)H2O2] -> [S0 (NHNH)] + 1/2 O-2 + H2O. A lower redox state forms (S-3) by direct reduction and is 7 reducing equivalents (electrons) below the O-2-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 O-2-evolving S-4 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.