Oxidation of either methionine 351 or methionine 358 in α₁-antitrypsin causes loss of anti-neutrophil elastase activity

Hydrogen peroxide is a component of cigarette smoke known to be essential for inactivation of α₁-antitrypsin, the primary inhibitor of neutrophil elastase. To establish the molecular basis of the inactivation of α₁-antitrypsin, we determined the sites oxidized by hydrogen peroxide. Two of the nine methionines were particularly susceptible to oxidation. One was methionine 358, whose oxidation was known to cause loss of anti-elastase activity. The other, methionine 351, was as susceptible to oxidation as methionine 358. Its oxidation also resulted in loss of anti-elastase activity, an effect not previously recognized. The equal susceptibility of methionine 358 and methionine 351 to oxidation was confirmed by mass spectrometry. To verify this finding, we produced recombinant α₁-antitrypsins in which one or both of the susceptible methionines were mutated to valine. M351V and M358V were not as rapidly inactirated as wild-type α1-antitrypsin, but only the double mutant M351V/M358V was markedly resistant to oxidative inactivation. We suggest that inactivation of α₁-antitrypsin by oxidation of either methionine 351 or 358 provides a mechanism for regulation of its activity at sites of inflammation.