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

Abstract

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.