The oxidation of selenocysteine is involved in the inactivation of glutathione peroxidase by nitric oxide donor

Glutathione peroxidase (GPx) was inactivated by S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitric oxide donor (Asahi, M., Fujii, J., Suzuki, K., See, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Taniguchi, N. (1995) J. Biol. Chem. 270, 21035-21039). The structural basis of the inactivation was studied. We also show that 3-morpholinosydnonimine N-ethylcarbamide, a peroxynitrite precursor, as well as synthetic peroxynitrite also inactivated bovine GPx. The degree of incorporation of a sulfhydryl reagent, n-octyldithionitrobenzoic acid, into GPx decreased after pretreatment with SNAP as evidenced by mass spectrometry. To identify the modification site of this enzyme by SNAP, both SNAP-pretreated and untreated GPxs were reacted with n-octyldithionitrobenzoic acid and digested with lysylendopeptidase, and the resulting peptides were subjected to mass spectrometry. This technique identified a bridge between two peptides, one of which contains Sec(45) at the catalytic center and Cys(74), and the other contains Cys(91). Although there are two possible combinations, selenocysteine 45 (Sec(45)) and Cys(91) or Cys(74) and Cys(91), the tertiary structure of GPx indicates that a cross-link between Sec(45) and Cys(91) is more feasible. This is consistent with the experimental evidence that SNAP specifically inactivates GPx, in which Sec(45) forms the catalytic center. Thus, we conclude that SNAP mainly oxidized Sec(45) to form a selenenyl sulfide (Se-S) with a free thiol, leading to the inactivation of the enzyme. These data suggest that nitric oxide and its derivatives directly inactivate GPx in a specific manner via the production of a selenenyl sulfide, resulting in an increase in intracellular peroxides that are responsible for cellular damage.