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

Abstract

Despite detailed studies on nitroxylcobalamin (CblNO) formation, the possible intracellular generation of CblNO via reduction of nitrocobalamin (CblNO2) remains questionable. To study this further, spectroscopic studies on the reaction of CblNO2 with the intracellular antioxidant ascorbic acid (HAsc−) were performed in aqueous solution at pH < 5.0. It was found that nitroxylcobalamin is the final product of this interaction, which is not just a simple reaction but a rather complex chemical process. We clearly show that an excess of nitrite suppresses the formation of CblNO, from which it follows that ascorbic acid cannot reduce coordinated nitrite. We propose that under the influence of ascorbic acid, nitrocobalamin is reduced to Cbl(II) and nitric oxide (·NO), which can subsequently react rapidly to form CblNO. It was further shown that this system requires anaerobic conditions as a result of the rapid oxidation of both Cbl(II) and CblNO.Graphical Abstract Electronic supplementary materialThe online version of this article (10.1007/s00775-018-1540-1) contains supplementary material, which is available to authorized users.

Can Particulate Matter and Nano Metal Oxide Particles Affect the Redox Cycling of Nitrosylcobalamin in Weakly Acidic Aqueous Solution?

“…CblNO 2 (λ max = 354, 413 and 532 nm) [11,12] is converted to CblNO (λ max = 316, 344 and 475 nm) [7] with isosbestic points at 335, 373 and 490 nm. [10] A plot of absorbance at 532 nm versus time is shown in Figure 1b; the data fitted well to a first-order rate equation with an observed rate constant of k obs = (5.8 � 0.1) × 10 À 4 s À 1 at 25.0°C, following an initial zero-order rate process as observed before. [10,11] In order to investigate the role of inorganic compounds present in PM on the reaction described above, selected nanoparticles of redox-active metal oxides (Fe 2 O 3 , Fe 3 O 4 with and without stabilizer, CuO and Mn 3 O 4 ) and non-redox-active metal oxides (SiO 2 and Al 2 O 3 ) were added to the reaction mixture, shortly before the initiation of the reaction between CblNO 2 and Asc (see Experimental Section).…”

“…[10] A plot of absorbance at 532 nm versus time is shown in Figure 1b; the data fitted well to a first-order rate equation with an observed rate constant of k obs = (5.8 � 0.1) × 10 À 4 s À 1 at 25.0°C, following an initial zero-order rate process as observed before. [10,11] In order to investigate the role of inorganic compounds present in PM on the reaction described above, selected nanoparticles of redox-active metal oxides (Fe 2 O 3 , Fe 3 O 4 with and without stabilizer, CuO and Mn 3 O 4 ) and non-redox-active metal oxides (SiO 2 and Al 2 O 3 ) were added to the reaction mixture, shortly before the initiation of the reaction between CblNO 2 and Asc (see Experimental Section). The reactions were monitored by recording UV-Vis spectra, which clearly demonstrated that the addition of nanoparticles do not affect the formation of the final reaction product, i. e. CblNO 2 was still converted to CblNO with isosbestic points at 335, 373 and 490 nm.…”

“…Our recent studies [10,11] on the reaction between nitrocobalamin (CblNO 2 ) and ascorbic acid (Asc) allowed us to conclude that, depending on the reducing properties of protonated/deprotonated ascorbic acid at different pH values, the product formed is either the reduced form of Vitamin B 12a (Co II ) at pH 7 [11] or nitrosylcobalamin (CblNO, i. e. Co III À NO À ) at pH < 5. [10] It was also shown that formation of CblNO at pH < 5 is a rather complex process that involves a few reaction steps that are controlled by the concentrations of free nitrite and ascorbic acid in the reaction mixture. Since the process of CblNO formation described above seems to be of biological significance, we decided to use the system in further detailed investigations within the APARIC project.…”

“…According to our present understanding, the mechanism of the reaction involves reduction of traces of CblOH 2 by ascorbic acid (1), subsequent oxidation of Cbl(II) by free nitrite to form CblOH 2 and * NO (2), and finally a very fast reaction between Cbl(II) and * NO to form Cbl(III)-NO À (CblNO) (3). [10] CblOH…”

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.…”