Can nitrocobalamin be reduced by ascorbic acid to nitroxylcobalamin? Some surprising mechanistic findings
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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.
KeywordsNitrocobalamin Nitroxylcobalamin Nitrite Ascorbic acid Redox reactions
Nitroxylcobalamin (CblNO, formally CoIII–NO−)  is one of the most interesting forms of Vitamin B12 that was shown to be stable in biological systems [2, 3]. Under physiological conditions, CblNO can be produced in a very efficient reaction between the major intracellular form of Vitamin B12r, viz. cob(II)alamin , and nitric oxide (·NO) for which k = 7.4 × 108 M−1 s−1 and KNO ≈ 1 × 108 M−1 at 25 °C [5, 6, 7]. It has been postulated that cobalamins show the potential to eliminate excess ·NO from organisms [8, 9] since the CoIII–NO− complex can be protonated at neutral pH to form CoIII–NOH, which in turn can undergo aquation to release HNO. The latter species is known to dimerize and decompose to water and gaseous N2O in aqueous solution .
Hydroxocobalamin hydrochloride (HOCbl·HCl, ≥ 98%) was purchased from Sigma-Aldrich, sodium nitrite was purchased from LPPH and ascorbic acid was obtained from Polfa Kraków. Acetic acid (CH3COOH, \(\ge\) 99.5–99.9%) and sodium hydroxide (NaOH, \(\ge\) 98.8%) were obtained from a range of suppliers (Sigma-Aldrich, Merck, Fisher Scientific or POCH). All chemicals used throughout this study were of analytical grade or better.
All solutions were prepared in de-ionized water using a water purification system. Strictly anaerobic solutions were prepared using appropriate air-free techniques and handling the solutions in appropriate glassware. Oxygen-free argon or nitrogen was used to deoxygenate the reactant solutions. UV–Vis spectral measurements were carried out in screw-cap cuvettes equipped with a silicone septum. pH measurements were carried out at room temperature using a HI 221 (Hanna Instruments) pH-meter equipped with an AmpHel glass electrode filled with a 3 M KCl solution.
UV–Vis spectra and kinetic data were recorded on Perkin Elmer Lambda 25 spectrophotometer equipped with a thermostated (25.0 ± 0.1 °C) cell holder (Perkin Elmer PTP-6 Peltier System). All data were analyzed using Origin Lab software.
Results and discussion
A blank experiment was performed in the absence of cobalamin in which solutions of nitrite and HAsc− were mixed under exactly the same conditions as we used in the experiments with cobalamin. The absorbance maximum at 262 nm which comes from HAsc− decreases with time to reach the pre-reaction value after 20 h from the start of the reaction (Figure S2, Supporting Information). We ascribe these findings to slow side reactions between ascorbic acid and nitrite, and/or slow diffusion of oxygen into the sealed cuvettes over longer periods of time (see further discussion).
We also used FeII(EDTA) as a very efficient trap for the intermediate formation of ·NO [6, 24]. On mixing typical concentrations of nitrite and ascorbate under Ar atmosphere and allowing them to react for 2 h, the addition of FeII(edta) immediately resulted in the formation of Fe(edta)NO as shown in Figure S3 (Supporting Information). This is clear evidence for the intermediate formation of ·NO during the reduction of nitrite by ascorbate under the selected conditions of this study.
The results reported above show that it is indeed possible to observe the formation of CblNO under milder reducing conditions with ascorbic acid at pH < 5. In terms of the biological relevance of these findings, we repeated a series of measurements where the pH was systematically decreased from 7.2 to 5.0, to see where the changeover from Cbl(II) to CblNO as reaction product occurs. In these experiments a typical concentration ratio of [CblOH2]:[NO2−]:[HAsc−] = 1:5:10 was selected as done in Fig. 2. On decreasing the pH the reaction product changed from only Cbl(II) (pH 7.2, Figure S4, Supporting Information) to a mixture of Cbl(II) and CblNO (pH 5.5, Figure S5, Supporting Information), to only CblNO (pH 5.0, Figure S6, Supporting Information). It follows that as we go to milder reducing conditions by lowering the pH, only Cbl(NO) is formed at pH ≤ 5.0, which must be related to the pH dependence of the redox potential for the two-electron ascorbic acid/dehydroascorbate transformation. According to the Pourbaix diagram for this transformation , the redox potential of H2Asc at pH 0 is + 0.4 V, of H2Asc/HAsc− at pH 4.1 (pKa1) is + 0.16 V, for HAsc− at pH 7.0 (8.0) is + 0.07 (+ 0.04 V), and for HAsc−/Asc2− at pH 11.3 (pKa2) is − 0.15 V. These data clearly show the large change in redox potential to a significantly stronger reducing agent on increasing the pH of the solution.
The challenge now will be to find a biologically relevant reducing agent that under mild reaction conditions will reduce CblNO2 to CblNO at pH 7.4.
The results of this study have clearly demonstrated that it is possible to obtain stable solutions of CblNO in the presence of a reducing agent starting from CblNO2 under well-selected reaction conditions. The remaining question is how can we account for the different reaction steps observed?
The CblOH2 formed in reaction (3) is immediately reduced by HAsc− in reactions (1) and (2) and the nitroxyl product is formed in the very fast radical coupling of Cbl(II) and ·NO (k = 7.4 x 108 M−1 s−1 ), reaction (4). Thus, reactions (1) to (4) account for the observation that CblNO was formed faster and remained for longer times in the reaction mixture when higher concentrations of HAsc− were used, whereas increasing nitrite concentration slowed down the conversion of CblNO2 to CblNO. At the point where ascorbic acid is depleted, CblNO can react with excess ·NO to yield CblOH2 and N2O, similar to that found for the reaction between free HNO and ·NO to form NO2− and N2O . Subsequently, CblOH2 is converted to CblNO2, the final reaction product in the presence of excess nitrite.
In this case, the produced ·NO will react rapidly with Cbl(II) to form CblNO in reaction (4).
The suggested reaction sequence in Scheme 3 is based on a one-electron reduction process by which nitrite is reduced to ·NO by ascorbate. From the recent literature [30, 31] it is known that free ·NO can be reduced by ascorbic acid to form HNO, which in turn can react with CblOH2 to form CblNO. Brasch and coworkers  demonstrated that using Angeli’s salt (HN2O3−) as source of HNO, the formation of CblNO can occur at pH > 10.8, where the rate-determining step is the release of HNO by Angeli’s salt, such that no mechanistic details about the mechanism of the reaction between CblOH2/CblOH and HNO/NO− could be revealed. At present, it is questionable whether further reduction of ·NO to HNO and a direct reaction of CblOH2 with HNO to form CblNO can account for the results presented in this study.
All in all, our goal to find suitable reaction conditions to produce CblNO from CblNO2 in the presence of a reducing agent over a long period of time was successful and has added to the overall understanding of the complex reaction system.
The reaction of CblNO2, one of the naturally occurring forms of cobalamin, with ascorbate has been studied by UV–Vis spectroscopy. The present study provides mechanistic information on this reaction at pH < 5. Under this condition, the only product of the reaction is CblNO. However, for the reduction of CblNO2 by ascorbate, no direct evidence for the reduction of coordinated nitrite could be found. On the contrary, we showed that excess of nitrite suppressed the formation of CblNO, from which we can conclude that ascorbic acid/ascorbate cannot reduce coordinated nitrite. We suggest that the studied system is not just a simple reaction, but a rather complex chemical process. During the reaction of ascorbic acid with nitrocobalamin, the first products formed are the reduced form of Vitamin B12 (Cbl(II)) and nitric oxide (·NO) that subsequently react rapidly to form CblNO. Our results show that the studied reactions are extremely oxygen sensitive due to the reverse oxidation of both Cbl(II) and CblNO to CblOH2 and CblNO2, respectively.
The authors gratefully acknowledge financial support from the National Science Center in Poland (Grant no. DEC-2016/21/N/ST4/00178).
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