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Light-Induced Fourier Transform Infrared Difference Spectroscopy Study of the Molecular Mechanism of Photosynthetic Oxygen Evolution


Oxygenic photosynthesis produces nearly all the O2 on Earth and sustains nearly all of its biomass. This process is catalyzed by Photosystem II (PSII), a large, transmembrane protein embedded in the thylakoid membranes of plants, algae, and cyanobacteria. PSII is catalyzed by a Mn4CaO5 cluster. As light energy is absorbed by the reaction centers of PSII, the Mn4CaO5 cluster accumulates oxidizing equivalents through a discreet and precisely choreographed light-induced electron transfer. The light induced oxidations cause the catalytic cluster to cycle through five oxidation states, Sn (n=0-4) where ‘n’ refers to the number of oxidizing equivalents. After formation of the S4 state, the cluster oxidizes two water molecules, releases O2 and returns to the S0 state, the lowest oxidation state of its catalytic cycle. The oxidation of water is a thermodynamically and kinetically demanding reaction. This is managed by PSII through the careful choreography of proton and electron transfers to the Mn4CaO5 cluster throughout the catalytic cycle. The Mn4CaO5’s reactivity in each catalytic step is carefully controlled by its protein environment. Identifying key amino acid residues, determining their responsibility in the reaction, and characterizing the proton egress pathways during the individual S-state transitions are paramount in understanding the oxygen evolution mechanism.

This study interrogates and identifies the amino acid residues responsible for controlling the Mn¬4CaO5 cluster’s reactivity and determines the role of each residue through Fourier transform infrared (FTIR) difference spectroscopy. FTIR difference spectroscopy is capable of characterizing the dynamic structural rearrangements during PSII’s catalytic cycle. A combination of site-directed mutagenesis (D1-V185N, D1-S169A, D1-E189G, D1-E189S, D1-E329A), isotopic substitutions, and the substitution of Sr2+ for Ca2+ in PSII’s catalytic center were used to further delineate the dominant water access and proton egress pathways that link the catalytic cluster with the thylakoid lumen, characterize the influence of specific protein residues on substrate water molecules and on the network of hydrogen bonds in these pathways, and elucidate the potential substrates and mechanisms of the S2 to S3 transition.

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