Specific isotopic labeling and photooxidation-linked structural changes in the manganese-stabilizing subunit of photosystem II

Photosystem II (PSII) oxidizes water to molecular oxygen; the catalytic site is a cluster of four manganese ions. The catalytic site undergoes four sequential light-driven oxidation steps to form oxygen; these sequentially oxidized states are referred to as the S-n states, where n refers to the number of oxidizing equivalents stored. The extrinsic manganese stabilizing protein (MSP) of PSII influences the efficiency and stability of the manganese cluster, as well as the rates of the S state transitions. To understand how MSP influences photosynthetic water oxidation, we have employed isotope editing and difference Fourier transform infrared spectroscopy. MSP was expressed in Escherichia coli under conditions in which MSP aspartic and glutamic acid residues label at yields of 65 and 41%, respectively. Asparagine and glutamine were also labeled by this approach. GC/MS analysis was consistent with minimal scrambling of label into other amino acid residues and with no significant scrambling into the peptide bond. Selectively labeled MSP was then reconstituted to PSII, which had been stripped of native MSP. Difference Fourier transform infrared spectroscopy was used to probe the S(1)Q(A) to S(2)Q(A)(-) transition at 200 K, as well as the S(1)Q(B) to S(2)Q(B)(-) transition at 277 K. These experiments show that aspargine, glutamine, and glutamate residues in MSP are perturbed by photooxidation of manganese during the S-1 to S-2 transition.