Ligation of D1-His332 and D1-Asp170 to the Manganese Cluster of Photosystem II from Synechocystis Assessed by Multifrequency Pulse EPR Spectroscopy

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S-2 state. Spectra of natural isotopic abundance PSII (N-14-PSII), uniformly N-15-labeled PSII (N-15-PSII), and N-15-PSII containing N-14-histidine (N-14-His/N-15-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional K-a- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new N-14-based interactions.