Kinetic and Mechanistic Studies on the Reactions of the Reduced Vitamin B₁₂ Complex Cob(I)alamin with Nitrite and Nitrate

Abstract

Abstract The kinetics of the reactions between cob(I)alamin [Cbl(I)] and nitrite and nitrate have been studied by UV/Vis and stopped‐flow spectroscopy. Enzyme‐bound Cbl(I) is an important transient species in several B 12 ‐catalyzed enzyme reactions. Levels of nitrite and nitrate are elevated during oxidative stress, as a consequence of elevated nitric oxide levels. Although nitrite and nitrate are generally considered to be benign species, our studies show that nitrate and especially nitrite react rapidly with Cbl(I) at neutral pH conditions ( k app = 6.5 × 10 –3 and 1.7 × 10 3 M –1 s –1 , respectively, at pH 7, 25.0 °C). A reaction pathway is postulated for the reaction between Cbl(I) and (H)NO 2 involving a 2e – rate‐determining step to form Cbl(III) and HNO. The latter species reacts further with Cbl(I), ultimately resulting in the oxidation of 4Cbl(I) by HNO 2 to yield 4Cbl(II) and NH 2 OH. The reaction between Cbl(I) and (H)NO 3 results in the oxidation of 8Cbl(I) by (H)NO 3 to give 8Cbl(II) and NH 4 + (pH 5–7).