Mechanistic information on the nitrite-controlled reduction of aquacob(III)alamin by ascorbate at physiological pH

Abstract

The interaction with nitric oxide (NO) is an important aspect of the biological activity of vitamin B12 (Cbl). Whereas the formation of nitroxylcobalamin (CblNO) via the binding of NO to reduced CblCo(II) has been studied in detail before, the possible intracellular formation of CblNO via reduction of nitrocobalamin (CblNO2) is still questionable. To study this further, spectroscopic and kinetic studies on the reaction of CblNO2 with the intracellular antioxidant ascorbic acid (Asc) were performed in aqueous solution at the physiological pH of 7.2. It was found that the redox pathway of this reaction requires anaerobic conditions as a result of the rapid re-oxidation of reduced CblCo(II). In the studied system, both CblOH2 and CblNO2 are reduced to CblCo(II) by ascorbate at pH 7.2, the CblOH2 complex being two orders of magnitude more reactive than CblNO2. Clear evidence for redox cycling between CblOH2/CblNO2 and CblCo(II) under aerobic conditions was observed as an induction period during which all oxygen was used prior to the formation of CblCo(II) in the presence of an excess of ascorbate. No evidence for the intermediate formation of CblNO or NO radicals during the reduction of CblNO2 could be found. Nitrocob(III)alamin can be reduced by ascorbic acid under physiological conditions. The products of the reaction are cob(II)alamin and nitrite ion. This reaction is ca. 200 times slower than the one involving aquacob(III)alamin.

Can nitrocobalamin be reduced by ascorbic acid to nitroxylcobalamin? Some surprising mechanistic findings

“…The kinetic data could be fitted best with a zero-order process for the initial changes in absorbance with time, and the slopes of such plots as a function of CblOH 2 concentration show typical saturation kinetics (see Figure S1e). From the value of K = 1 × 10 5 M −1 (see Scheme 2 ) reported at 25 °C [ 17 ], it can be estimated that the concentration of CblOH 2 in solution decreases from 1.95 × 10 −6 to 4.97 × 10 −7 M on increasing the nitrite concentration from 4.3 × 10 −4 to 1.72 × 10 −3 M for the experiments reported in Figure S1. This clearly demonstrates the effect of the nitrite concentration on the rate of formation of CblNO.…”

“…Under these conditions, mixtures of H 2 Asc and HAsc − have significantly weaker reducing properties than at pH ~ 7, where ascorbic acid is mainly present as HAsc − with traces of the stronger reducing agent Asc 2− [ 18 – 23 ]. UV–Vis spectra recorded during the reaction between CblNO 2 and HAsc − at pH < 5 (0.1 M acetate buffer, 25 °C, Ar atmosphere) indicated that CblNO 2 ( λ max = 354, 413 and 532 nm) is not converted to Cbl(II) as before [ 17 ], but appears to be converted directly to nitoxylcobalamin, CblNO ( λ max = 316, 344 and 475 nm) [ 6 ] with isosbestic points at 335, 373, 490 nm (Fig. 1 a).…”

“…Our recent study [ 17 ] showed that at neutral pH (pH 7.2, 0.1 M Tris buffer, 25 °C), CblNO 2 can react with another biological reducing agent, ascorbic acid (HAsc − ), to form reduced cobalamin (Cbl(II)) as product, but not CblNO as was hoped. Kinetic data showed that both CblNO 2 and CblOH 2 present in an equilibrium mixture react with HAsc − , but CblOH 2 reacts ca.…”

“…Thus, on addition of ascorbic acid to the equilibrium mixture of CblOH 2 and CblNO 2 , it reacts rapidly with CblOH 2 which in turn is reformed through the aquation of CblNO 2 ( k 1 = 1.4 × 10 −2 s −1 at 25 °C) and so represents the major reactive species in solution. Furthermore, in the presence of a large excess of nitrite the reduction of CblOH 2 can be drastically inhibited due to the formation of CblNO 2 [ 17 ]. Recent work in our laboratories indicated that this system shows a more interesting behavior under slightly different experimental conditions.…”

Nitrosyl- versus nitroxyl-cobalamin?

“…We received the following comment from a reader of JBIC concerning an apparent error made in two recent papers published by our group in JBIC [ 1 , 2 ]: ‘The authors use the term nitroxyl to describe the coordination of NO to the cobalt of cobalamin. This term is not correct nomenclature.…”