STRUCTURAL CHARACTERIZATION OF SELENOSUBTILISIN BY SE-77 NMR-SPECTROSCOPY

The semisynthetic selenoenzyme selenosubtilisin catalyzes the reduction of alkyl hydroperoxides with concomitant oxidation of 3-carboxy-4-nitrobenzenethiol. To gain insight into the mechanism of this process, three oxidation states of the enzyme that are relevant to the redox cycle have been characterized by Se-77 NMR spectroscopy. Oxidation of selenosubtilisin with hydrogen peroxide in the absence of thiol yields a species whose spectrum consists of two peaks at 1188 and 1190 ppm, consistent with the selenium prosthetic group being in the seleninic acid oxidation state. The observation of two signals suggests that Met222 in the active site may have been partially oxidized during preparation of the sample and illustrates the sensitivity of the selenium Probe to its electronic microenvironment. The pK(a) value of the enzyme-bound seleninic acid is at least 1.5 pH units lower than that of simple alkane seleninic acids, as determined by the pH dependence of its NMR signals. This fact indicates significant stabilization of the conjugate base of the seleninic acid by proximal hydrogen bonding groups within the active site and accounts for the relatively sluggish rate at which oxidized selenosubtilisin reacts with thiols. Reduction of the oxidized enzyme with 3 equiv of 3-carboxy-4-nitrobenzenethiol cleanly produces the cognate disulfide and an enzyme-bound selenenyl sulfide derivative (Se-77 389 ppm). No other intermediates at the selenenic acid oxidation state were detected spectroscopically, including either the selenenic acid itself or a cyclic selenamide in which the selenium is covalently connected to the protein backbone. Treatment of the enzyme with excess dithiothreitol at neutral pH, on the other hand, reduces the prosthetic group to the selenol, as judged by the single broad "Se resonance observed at -215 ppm. Like the seleninic acid, the selenol has an unusually low pK(a) (<4) and is deprotonated at all accessible pHs. In analogy to the chemistry of glutathione peroxidase, an important naturally occurring selenoenzyme, both the selenenyl sulfide and selenol derivatives of selenosubtilisin are believed to be key intermediates in the peroxidase action of the enzyme, while the seleninic acid is most likely a side product that is formed at high peroxide concentrations. This study thus demonstrates the feasibility of using Se-77 NMR spectroscopy to characterize an active selenoenzyme's catalytic center. The enormous chemical shift range and the sensitivity of this technique to subtle variations in chemical environment promise to be invaluable for probing structure-function relationships in this and related selenium-containing proteins.