Reduction-induced inhibition and Mn(II) release from the photosystem II oxygen-evolving complex by hydroquinone or NH2OH are consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) oxidation state for the dark-adapted enzyme.

Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen-evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III)(2)/Mn(IV)(2) for the dark stable S(1) state of the O(2)-evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S(1) --> S(-)(1) conversion of intermediate probability, a S(-)(1) --> S(-)(3) reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn(II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S(-)(1) can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant.