Elucidating the Mechanism of Photosynthetic Water Splitting

Photosynthesis has sustained life on Earth for three billion years, producing large amounts of biomass from CO2 and water, and enriching the atmosphere with O2 to allow aerobic life. Understanding the workings of photosynthesis holds the key to producing stored chemical energy (fuel) from water using sunlight, which is especially vital in today's economy. The enzyme that carries out this difficult process (eq. 1) in photosynthesis is called photosystem II (PSII), embedded in the thylakoid membranes of plants, cyanobacteria, and algae.;2H20+hv→4e -+4H++O 2 (1).;The reactions in photosystem II are initiated by absorption of light by the reaction-center chlorophylls, collectively called P680. The excited state (P680*), created as a result of light absorption, transfers an electron to a pheophytin. The pheophytin relays the electron to a primary plastoquinone, QA and then to a secondary exchangeable plastoquinone, QB. QB upon receiving two electrons, picks up two protons from the stroma and diffuses out into the plastoquinone (PQ) pool in the thylakoid lipid bilayer as QBH2. P 680+ generated upon losing an electron, accepts an electron from a redox-active tyrosine, Yz, which in turn receives it from the Oxygen-Evolving Complex (OEC). The OEC is the catalyst in PSII, comprised of a Mn4O5Ca cluster, which drives the conversion of water to O2, electrons and protons.;All the components in PSII are in sync; enabling it to carry out the difficult process of stripping multiple electrons and protons from water, transferring electrons to the stroma and protons to the lumen, while bringing two substrate water oxygens together at the OEC to make O2. In this work, we investigated all three components of the water-oxidation equation: i) electron transport, ii) proton release, and iii) O-O bond-formation chemistry to unmask the mechanism of PSII-catalyzed water splitting.;The theoretical rate of OEC turnover is more than 500 51; however, the normal steady-state PSII O2-evolution rate is about 500 s .1; The slowest step in PSI[ is the transfer of electrons from QA to QB and diffusion of QBH2 . We characterized a novel exogenous electron acceptor, [Co(III)(terpy) 2]3+ (terpy = 2,2 ;6',2"-terpyndine), demonstrating that it follows Michaelis-Menten kinetics and accepts electrons directly from Q A by binding electrostatically to a cluster of five glutamate residues located 9 A from QA. This opens new avenues for harvesting electrons from PSII before they reach the rate-limiting QA →O BH2solutionelectron-transfer steps.;We studied the contribution of proton transfer to the rate of O 2 evolution by examining site-specific PSII mutants, with amino acids that have been altered in a putative proton transfer channel. We observed that mutating a positively charged lysine to a neutral alanine in the channel, substantially shifts the pH dependence of PSII O2 evolution and increases the 2H kinetic solvent isotope effect, indicating that proton transfer has become rate-limiting in this mutant. Additionally, examining the pH dependent isotope effect of wild-type PSII, enabled us to conclude that at low pH, the water-oxidation rate becomes slower than the QA →QBH2solution electron transfer, and hence, rate-determining.;Equipped with the understanding of electron and proton transfer rate limitation on O2 evolution, we sought to probe the rate-determining step at the OEC using 18O kinetic isotope effects (KIEs). We performed 18O ME measurements, supplemented by computational analysis on a biomimetic oxomanganese complex, [Mn(III/IV)2(mu- O)2(terpy)2(OH2)2)(NO3) 3 to serve as a benchmark study for OEC 18O KIE experiments. The experimental and theoretical 18O KIEs provided evidence that the rate-limiting step during catalytic turnover involves binding of the oxidant to this complex.;We conducted PSII 18O KIEs experiments by modulating the rate-detemnining step in PSII and obtaining different 18O KIEs. High exogenous electron-acceptor concentrations and low pH was used to decrease electron-transfer rate limitation to reveal the 18O isotope effect of water oxidation. This provides the first experimental insight into the nature of the transition-state associated with the rate-determining step in PSII catalyzed water splitting. The 18O KIE, together with the above-mentioned 2H isotope effect, is consistent with a rate-limiting proton-coupled-electron transfer, which has implications for the proposed mechanisms of natural water oxidation.