title: Assembly of the tetra-Mn site of photosynthetic water oxidation by photoactivation: Mn stoichiometry and detection of a new intermediate.
abstract: 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'-tetrapropionate-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 microL) O2 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 approximately 5 x 10(-14) mol of O2 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 O2-evolving activity could be restored compared to that of PSII membranes depleted of the three extrinsic polypeptides (35% Vmax vs intact PSII). Titration of the photoactivation steady-state O2 yield, Yss, and the half-time for recovery, t1/2, vs Mn concentration demonstrate that 4.0 Mn/P680 are cooperatively taken up at 95% restoration of Yss 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 Yss and t1/2 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 b559, 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.
authors.affiliation: Hoyt Laboratory, Department of Chemistry, Princeton University, New Jersey 08544, USA.
authors.forename: G M
authors.initials: GM
authors.lastname: Ananyev
authors.forename: G C
authors.initials: GC
authors.lastname: Dismukes
mesh_terms: D001596:Benzylamines / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D005292:Ferricyanides / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008345:Manganese / Q000378:metabolism*; D010084:Oxidation-Reduction; D010788:Photosynthesis; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D011422:Propionates / Q000494:pharmacology; D018724:Spinacia oleracea / Q000378:metabolism; D013997:Time Factors; D014867:Water / Q000378:metabolism
chemical_list: D001596:Benzylamines; D002614:Chelating Agents; D005292:Ferricyanides; C099990:N,N,N',N'-tetrapropionato-1,3-bis(aminomethyl)benzene; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D011422:Propionates; D014867:Water; D008345:Manganese; D002118:Calcium; C028033:potassium ferricyanide