Pulsed EPR Studies of Biological Manganese Clusters

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We will employ advanced EPR methods to study the structure and function of the tetranuclear manganese cluster of the Photosystem II oxygen evolving complex. Multifrequency CW and pulsed EPR and ENDOR experiments will be used to characterize the physical and electronic structures of the S state intermediates (S0, S1, S2, S3) of the oxygen evolving cycle. ENDOR and ESEEM experiments using PSII particles incorporating 2H- and 19F-labelled tyrosine will determine the distance between the YZ tyrosine and the Mn cluster in oxygen-evolving PSII particles. ESEEM and multifrequency ENDOR experiments will investigate details of substrate, substrate analog, and inhibitor binding to the Mn cluster at different S states, as well as the proximity of essential cofactors Ca2+ and Cl-. The role of the 17, 23, and 33kDa extrinsic polypeptides in limiting access of larger substrate analogs and inhibitors to the Mn cluster will be explored. Other ESEEM and multifrequency ENDOR experiments will investigate the amino acid ligation of the cluster at various S states. Parallel mode and high frequency EPR, along with ESEEM and ENDOR, will be used to detect and characterize signals from Mn cluster assembly intermediates and cluster reduction products. Studies of synthetic Mn model clusters of known structure will allow us to correlate EPR/ENDOR derived properties with known structural elements. We will also characterize the structures of interesting Mn complexes that serve as "spectral models" for the PSII Mn cluster. Finally, we will target the mononuclear Mn enzymes, Mn superoxide dismutase and Mn lipoxygenase, and the dinuclear Mn enzyme, Mn catalase, for study with our advanced EPR methods.