Mechanistic studies of the reactions of the reduced vitamin B12 derivatives with the HNO donor Piloty's acid: further evidence for oxidation of cob(I)alamin by (H)NO

There is accumulating evidence for the existence of HNO in biological systems. Compared with NO ((NO)-N-center dot), 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 B-12-derived radical complex cob(II)alamin (Cbl(II)(center dot), 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 N-2. A mechanism is proposed in which reduction of Cbl(II) by (H)NO results in formation of cob(I)alamin (Cbl(I)(-)) and (NO)-N-center dot. The Cbl(I)(-) intermediate is subsequently oxidized back to Cbl(II) by a second (H)NO molecule, and Cbl(II) reacts rapidly with (NO)-N-center dot 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.