We report the first studies on the reaction between an HNO donor compound and vitamin B12 complexes. Kinetic and mechanistic studies have been carried out on the reaction between the vitamin B12 derivative aquacobalamin (H2OCbl(+)/HOCbl; pKa = 7.8) and the HNO donor Angeli's salt. Studies were carried out with aquacobalamin in excess, since nitrite also reacts with aquacobalamin to form nitrocobalamin (NO2Cbl). At pH <9.90 aquacobalamin reacts directly with the monoprotonated form of Angeli's salt, HN2O3(-), to form nitroxylcobalamin (NO(-)-Cbl(III); NOCbl) and nitrite. At pH >10.80 the reaction instead switches predominantly to a mechanism in which spontaneous decomposition of Angeli's salt to give HNO and nitrite becomes the rate-determining step, followed by the rapid reaction between aquacobalamin and HNO/NO(-) to again give NOCbl. Both reactions proceed with a 1:1 stoichiometry and formation of nitrite is confirmed using the Griess assay.

Although it is well established that nitrosyl hydride (nitroxyl, HNO) reduces transition metals including transition-metal centers of porphyrins and metalloproteins, oxidation of a metal center by HNO has yet to be reported. Kinetic and mechanistic studies on the Co II vitamin B 12 form, cob(II)alamin [Cbl(II)], with the widely used HNO donor Angeli's salt (AS) have been carried out. The stoichiometry of the reaction is Cbl(II)/AS = 1:2, and AS decomposition to give HNO and

There is accumulating evidence for the existence of HNO in biological systems. Compared with NO (˙NO), much less is known about the chemical and biochemical reactivity of HNO. Kinetic and mechanistic studies have been carried out on the reaction between the vitamin B12-derived radical complex cob(II)alamin (Cbl(II)˙, Cbl(II)) with the widely used HNO donor Piloty's acid (PA). A stoichiometry of 1 : 2 Cbl(II) : PA was obtained and PA decomposition to HNO and benzenesulfinate (C6H5SO2(-)) is the rate-determining step. No evidence was found for nitrite (Griess assay), ammonia (Nessler's test) or NH2OH (indooxine test) in the product solution, and it is likely that HNO is instead reduced to N2. A mechanism is proposed in which reduction of Cbl(II) by (H)NO results in formation of cob(I)alamin (Cbl(I)(-)) and ˙NO. The Cbl(I)(-) intermediate is subsequently oxidized back to Cbl(II) by a second (H)NO molecule, and Cbl(II) reacts rapidly with ˙NO to form nitroxylcobalamin (NOCbl). Separate studies on the reaction between Cbl(I)(-) and PA shows that this system involves an additional step in which Cbl(I)(-) is first oxidized by (H)NO to Cbl(II), which reacts further with (H)NO to form NOCbl, with an overall stoichiometry of 1 : 3 Cbl(I)(-) : PA. Experiments in the presence of nitrite for both systems support the involvement of a Cbl(I)(-) intermediate in the Cbl(II)/PA reaction. These systems provide the second example of oxidation of cob(I)alamin by (H)NO.

Studies by others suggest that the reduced vitamin B12 complex, cob(II)alamin, scavenges nitric oxide to form air-sensitive nitroxylcobalamin (NO(-)-Cbl(III); NOCbl) in vivo. The fate of newly formed NOCbl is not known. A detailed mechanistic investigation of the oxidation of NOCbl by oxygen is presented. Only base-on NOCbl reacts with O2, and the reaction proceeds via an associative mechanism involving a peroxynitritocob(III)alamin intermediate, Co(III)-N(O)OO(-). The intermediate undergoes O-O bond homolysis and ligand isomerization to ultimately yield NO2Cbl and H2OCbl(+)/HOCbl, respectively. Ligand isomerization may potentially occur independent of O-O bond homolysis. Formation of (•)OH and (•)NO2 intermediates from O-O bond homolysis is demonstrated using phenol and tyrosine radical traps and the characterization of small amounts of a corrinoid product with minor modifications to the corrin ring.

Detailed kinetic and mechanistic studies have been carried out on the reaction between aquacobalamin/hydroxocobalamin (CblOH2 +/CblOH) and nitroxyl (HNO) generated by Piloty’s acid (PA, N-hydroxybenzenesulfonamide) over a wide pH range (3.5–13). The resulting data showed that in a basic solution HNO can react with hydroxocobalamin to form nitrosylcobalamin despite the inert nature of CblOH. It was shown that at low PA concentrations the rate-determining step is the decomposition of PhSO2NHO– to release HNO, whereas the reaction between CblOH and HNO becomes the rate-determining step at high PA concentrations. Data from kinetic studies on the reaction of CblOH with an excess of HNO enabled us to experimentally determine the pK a(HNO) value from initial rate data as a function of pH, giving pK a(HNO) = 11.47 ± 0.04. An especially interesting observation was made in the neutral pH range, where PA is stable and does not produce HNO. Under such conditions, rapid formation of CblNO was observed in the studied system. The obtained data suggest that CblOH2 + reacts directly with PA to form a Piloty’s acid-bound cobalamin intermediate, which deprotonates rapidly at neutral pH followed by rate-determining S–N bond cleavage to give CblNO and release PhSO2 –.

Thiolatocobalamins (RSCbls), or vitamin B12 derivatives with a thiolato ligand coordinated at the β‐axial site of the cobalamin, have been shown to have superior antioxidant properties relative to the currently available therapeutic forms of cobalamin. It has also recently been shown that small amounts of glutathionylcobalamin are isolable from mammalian cells in the presence of a ligand trap. Kinetic studies are now presented for four representative thiolatocobalamins of glutathione, N‐acetylcysteine, homocysteine, and captopril, which show that RSCbls spontaneously decompose to aquacobalamin (H2OCbl+) at pH < 3. A mechanism is proposed in which rapid protonation at the thiol sulfur of RSCbl precedes rate‐determining decomposition. These results indicate that protection of RSCbls from acidic environments such as that found in the stomach is essential if RSCbls are to remain intact.

The N-hydroxysulphonamide Piloty’s acid, PhSO2NHOH, was first reported in the scientific literature over a century ago. N-hydroxysulphonamides have recently attracted considerable attention, due to their propensity to spontaneously decompose to cleanly release nitroxyl (HNO) upon deprotonation of the parent molecule. Like its well-established cousin nitric oxide (NO), HNO is a biological signaling molecule with a rich redox chemistry. Furthermore therapeutics that generate HNO in vivo show considerable promise in treating cardiovascular disease including congestive heart failure, through mechanisms unique to HNO. HNO reacts primarily with metal centres and thiols (protein thiol residues and small molecular weight thiols) in biological systems. The cobalt-containing vitamin B12 macrocycle is a cofactor for numerous enzymes in humans and bacteria. Detailed mechanistic studies on the reactions of Piloty’s acid with all three oxidation states of vitamin B12 – Co(I), Co(II) and Co(III) – will be presented, and reveal a complex and rich coordination chemistry for this simple triatomic molecule.