In studies of enzyme intermediates and metalloprotein coordination, a moiety amenable to optical, vibrational, or magnetic resonance spectroscopic techniques may or may not be naturally present to enable experimental methods. Selenocysteine, a naturally occurring selenoamino acid analog of cysteine, can be genetically or synthetically substituted for cysteine in an enzyme of interest as a powerful X-ray spectroscopic probe. This atomic substitution allows for characterization at Se K-edge X-ray energies and can yield insights into electronic environment, protonation state, and metal-Se covalency at a particular atomic position. This technique has yielded valuable insights into a variety of metalloprotein mechanisms, particularly in those with multiple thiolate positions of interest.
This talk will present our applications of Se K-edge high energy resolution fluorescence-detected X-ray absorption near edge structure (HERFD-XANES) to selenocysteine-containing proteins without transition metal cofactors. Our interest is in directly characterizing oxidation and protonation states to determine selenocysteine speciation throughout enzyme catalysis. Two enzyme systems under investigation are 1) pyruvate formate lyase, in which cysteine-to-selenocysteine mutations have been introduced into positions central to a proposed thiyl radical mechanism and 2) human iodothyronine deiodinases, which are thought to use a catalytically essential selenocysteine residue to dehalogenate aryl iodine substituents.