Structural rearrangements preceding dioxygen formation by the water oxidation complex of photosystem II

Photosynthetic water oxidation is catalyzed by the Mn4CaO5 cluster of photosystem II. Recent studies implicate an oxo bridge atom, O5, of the Mn4CaO5 cluster, as the slowly exchanging substrate water molecule. The D1-V185N mutant is in close vicinity of O5 and known to extend the lag phase and retard the O-2 release phase (slow phase) in this critical last S-3(+) -> S-0 transition of water oxidation. The pH dependence, hydrogen/deuterium (H/D) isotope effect, and temperature dependence on the O-2 release kinetics for this mutant were studied using time-resolved O-2 polarography, and comparisons were made with WT and two mutants of the putative proton gate D1-D61. Both kinetic phases in V185N are independent of pH and buffer concentration and have weaker H/D kinetic isotope effects. Each phase is characterized by a parallel or even lower activation enthalpy but a less favorable activation entropy than the WT. The results indicate new rate-determining steps for both phases. It is concluded that the lag does not represent inhibition of proton release but rather, slowing of a previously unrecognized kinetic phase involving a structural rearrangement or tautomerism of the S-3(+) ground state as it approaches a configuration conducive to dioxygen formation. The parallel impacts on both the lag and O-2 formation phases suggest a common origin for the defects surmised to be perturbations of the H-bond network and the water cluster adjacent to O5.