Nitrous oxide degradation by cobalamin-dependent methionine synthase: characterization of the reactants and products in the inactivation reaction.
Cobalamin-dependent methionine synthase catalyzes the remethylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. The enzyme is susceptible to inactivation by the anaesthetic gas, nitrous oxide, through either short-term exposure to high levels or chronic exposure to low levels of this agent. We have studied the chemical reaction wherein the bound cobalamin prosthetic group of the enzyme from Escherichia coli catalyzes the degradation of nitrous oxide. By poising the enzyme at low ambient potentials in an electrochemical cell, the concentration of enzyme in the highly reactive cob(I)alamin state can be controlled, and the observed rate of inactivation is directly dependent upon the amount of enzyme in this redox state. The inactivation consumes both nitrous oxide and electrons, while nitrogen gas is evolved. The inactivation process is also directly dependent upon the proton concentration, but the effect of pH was found to be on the enzyme, revealing a redox-linked base presumed to be on or near the cobalamin. The chemical reaction between enzyme-bound cob(I)alamin and nitrous oxide that leads to inactivation is therefore independent of pH. In a single turnover experiment, where enzyme-bound cob(I)alamin is generated from methylcobalamin by methyl transfer to the substrate homocysteine, the cob(I)alamin decays to cob(II)alamin, suggesting that the degradation of nitrous oxide involves one-electron reduction of nitrous oxide. We propose that the inactivation chemistry is likely to be a one-electron reduction of nitrous oxide, which leads to the formation of a highly reactive oxidant, such as hydroxyl radical, and subsequent enzyme damage.
Drummond
J T
JT
Biophysics Research Division, University of Michigan, Ann Arbor 48109.
Matthews
R G
RG
D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004563:Electrochemistry; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006710:Homocysteine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000378:metabolism; D008745:Methylation; D009609:Nitrous Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D013053:Spectrophotometry; D014805:Vitamin B 12 / Q000494:pharmacology*
D006710:Homocysteine; D008715:Methionine; D013766:5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; D009609:Nitrous Oxide; D014805:Vitamin B 12; D010100:Oxygen