| Abstract
| - Selenoenzymes have a central role in maintaining cellular redox potential. These enzymes haveselenenylsulfide bonds in their active sites that catalyze the reduction of peroxides, sulfoxides, and disulfides.The selenol/disufide exchange reaction is common to all of these enzymes, and the active site redox potentialreflects the ratio between the forward and reverse rates of this reaction. The preparation of enzymescontaining selenocysteine (Sec) is experimentally challenging. As a result, little is known about the kineticrole of selenols in enzyme active sites, and the redox potential of a selenenylsulfide or diselenide bond ina protein has not been experimentally determined. To fully evaluate the effects of Sec on oxidoreductaseredox potential and kinetics, glutaredoxin 3 (Grx3) and all three Sec variants of its conserved 11CXX14Cactive site were chemically synthesized. Grx3, Grx3(C11U), and Grx3(C14U) exhibited redox potentials of−194, −260, and −275 mV, respectively. The position of redox equilibrium between Grx3(C11U−C14U)(−309 mV) and thioredoxin (Trx) (−270 mV) suggests a possible role for diselenide bonds in biologicalsystems. Kinetic analysis is consistent with the hypothesis that the lower redox potentials of the Sec variantsresult primarily from the greater nucleophilicity of the active site selenium rather than its role as either aleaving group or a “central atom” in the exchange reaction. The 102−104-fold increase in the rate of Trxreduction by the seleno-Grx3 analogues demonstrates that oxidoreductases containing either selenenylsulfide or diselenide bonds can have physiologically compatible redox potentials and enhanced reductionkinetics in comparison with their sulfide counterparts.
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