Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate

The process of photoactivation, the assembly of the water-oxidizing complex (WOC) of photosystem II (PSII) membranes, has been examined using two major improvements in methodology. First, a new lipophilic chelator, N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene (TPDBA), has been used that permits complete extraction of both manganese and calcium and the three extrinsic WOC polypeptides while minimizing damage to the apo-PSII protein and, importantly, eliminating the need to use reductants. Second, an ultrasensitive, fast-response, polarographic cell and detection system were built. The apparatus features (a) an ultrabright red light-emitted diode (LED) for controlling the light intensity, pulse duration, and dark intervals, features critical for minimization of photoinhibition; (b) a microvolume (5 mu L) O-2 polarographic cell (Clark type) fitted with a thin silicone membrane for rapid response (100 ms); and (c) DC/AC preamplifier integrated into the microcell and interfaced to a bandpass AC amplifier. The sensitivity enables detection of similar to 5 x 10(-14) mol of O-2 per flash at a signal to noise = 5/1. These improvements permit 100-fold lower Mn concentrations to be explored. Under optimum conditions, complete recovery of O-2-evolving activity could be restored compared to that of PSII membranes depleted of the three extrinsic polypeptides (35% V-max vs intact PSII). Titration of the photoactivation steady-state O-2 yield, Y-ss, and the half-time for recovery, t(1/2), vs Mn concentration demonstrate that 4.0 Mn/P680 are cooperatively taken up at 95% restoration of Y-ss and that 1.1-1.2 Mn atoms are involved in the rate-limiting photolytic step under steady-state conditions. Due to minimization of photoihibition, this intermediate exhibits a single exponential recovery kinetic over the entire population of PSII centers. Mn atoms in excess of 4 Mn/P680 accelerate the rate of photoactivation but decrease the yield above 8-10 Mn/P680. Maxima in both Y-ss and t(1/2) are observed at similar electrochemical potentials of the medium, 380 and 340 mV, respectively. We attribute this maximum to either elimination of a recombination reaction between the redox-active tyrosine-161 of the D1 polypeptide (Y-z(+)) and an electron acceptor, possibly cytochrome b(559), or stabilization of an intermediate in photoactivation. At low Mn2+ concentrations, a new pre-steady-state kinetic intermediate which binds fewer than 4 Mn atoms can be directly observed. This early kinetic phase has a rate that depends on Mn concentration and is independent of the electron acceptor identity and concentration.