NITROUS-OXIDE INACTIVATION OF COBALAMIN-DEPENDENT METHIONINE SYNTHASEFROM ESCHERICHIA-COLI - CHARACTERIZATION OF THE DAMAGE TO THE ENZYME AND PROSTHETIC GROUP

Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivat es cobalamin-dependent methionine synthase. This enzyme uses the highl y reactive, enzyme-bound cob(I)alamin oxidation state of the prostheti c group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)ala min is capable of reductively degrading nitrous oxide, and here we cha racterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inacti vated on a milligram scale by equilibrating enzyme containing bound co b(II)alamin with a reduced electrochemical mediator to give the reacti ve cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylme thionine (AdoMet), and inactive enzyme can no longer be reductively me thylated using AdoMet. The damage is oxidative, and it includes the co valent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link betwee n peptides containing the only two cysteines within this domain. Spect rally, the prosthetic group bound to inactive enzyme resembles cob(II) alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [Co-57]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, bu t two products derived from the cobalamin were also observed. We inter pret the finding of oxidatively modified products as strong evidence t hat reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modi fying sites proximal to the cobalamin.