EPR AND ENDOR STUDIES OF PROTEIN DI-IRON-OXO CLUSTERS

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The transition-metal ions that are found in the active centers of many proteins are essential for fundamental processes of life such as respiration and replication. The pivotal role that proteins containing dinuclear iron-oxo clusters have in these processes is being established as more occurrences of these metalloproteins are discovered. The aim of this proposal is to apply and develop new methods that can probe the active sites of proteins, and thus give information concerning the origin of the functional diversity exhibited by diiron-oxo proteins. Until recently, EPR studies of metalloproteins have been limited to those exhibiting half-integer spin (S = 1/2, 3/2, ...), but recent developments promise much wider applicability of the technique. Specifically, the neglected metalloproteins with integer spin (S = 1, 2, ...) can now be studied with EPR. The study proposed here will apply integer spin EPR spectroscopy to characterize the dioxygen active iron clusters of diiron-oxo proteins. In addition, structural aspects of the active sites (iron ligation, substrate effects and accessibility) will be determined from the magnitudes of hyperfine couplings of nuclei close to the irons. The hyperfine couplings are measured with ENDOR spectroscopy. Few previous ENDOR studies of diiron-oxo clusters exist, thus the work proposed here will establish a firm basis for the interpretation of ENDOR spectra of these types of metal clusters.