Kinetic characterization of the interaction of NO with the S2 and S3-states of the oxygen-evolving complex of Photosystem II

The reactivity of the S3 and S2 states towards NO and NH2OH was studied and compared using the period-4 oscillations in the F0-value induced by a train of single turnover Xenon flashes spaced 100 ms apart to monitor the reaction kinetics. The flash frequency also determined the time resolution of the assay, i.e. 100 ms. The S2 and S3-states were created by one and two single turnover pre-flashes, respectively. The NO-concentration-dependence of the S3-decay indicated that at low NO-concentrations an S2-state was formed as an intermediate, whereas at higher concentrations a seemingly monophasic decay to the S1-state was observed. The sigmoidal concentration dependence indicated that a fast interaction of the S3-state with (at least) two NO-molecules is necessary for the fast S3 to S1 decay ({tau} ~0.4 s at 1.2 mM NO). The pH-dependence of the S3-decay suggests that a protonation-reaction (pK ~6.9) is involved in the S3 to S1 decay. At intermediate NO-concentrations the protonation is only partially rate limiting, since the pH effect is more pronounced at high compared to intermediate NO-concentrations. A comparison of the reactivity of NO and hydroxylamine suggests that hydroxylamine reacts more efficiently with the S1 and S2 states, whereas NO reacts more efficiently with the S3-state. Based on our present knowledge of the oxygen evolving complex a possible reaction mechanism is proposed for the interaction between NO and the S3 state.