Kinetic and Mechanistic Studies on the Reaction of the Vitamin B 12 Complex Aquacobalamin with the HNO Donor Angeli’s Salt: Angeli’s Salt and HNO React with Aquacobalamin Harishchandra Subedi, † Hanaa A. Hassanin, †,‡ and Nicola E. Brasch* ,†,§ † Department of Chemistry and Biochemistry and § School of Biomedical Sciences, Kent State University, Kent, Ohio 44242, United States ‡ Department of Chemistry, Ain Shams University, Abbassia Square, Cairo, 11566, Egypt * S Supporting Information ABSTRACT: We report the first studies on the reaction between an HNO donor compound and vitamin B 12 complexes. Kinetic and mechanistic studies have been carried out on the reaction between the vitamin B 12 derivative aquacobalamin (H 2 OCbl + / HOCbl; pK a = 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 (NO 2 Cbl). At pH <9.90 aquacobalamin reacts directly with the monoprotonated form of Angeli’s salt, HN 2 O 3 - , 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. ■ INTRODUCTION There is currently much interest in the chemical and biochemical reactivity of the reactive nitrogen species nitrosyl hydride (nitroxyl, HNO). 1-8 HNO is generated in vivo by nitric oxide synthases from L-arginine in the absence of the tetrahydrobiopterin cofactor or by the oxidation of N-hydroxy- L-arginine. 9-11 Reduction of NO by enzymes such as ferrocytochrome c 12 and superoxide dismutase 13 leads to HNO generation. Spontaneous decomposition of S-nitro- sothiols or their reactions with thiols may also generate HNO. 10,11,14 Like NO, elevated HNO levels are associated with damage to cellular components leading to nitrosative stress. 15 Thus far HNO formation in biological systems has not been unequivocally demonstrated, and multiple groups are currently focused on designing molecules which efficiently trap HNO. 16-21 An interesting aspect of HNO-associated chemistry is the spin state change which occurs upon deprotonation of HNO in alkaline solution (the pK a for deprotonation of 1 HNO to give 3 NO - (+H + ) is ∼11.4). 10,22-24 Furthermore, HNO has a unique chemical and biological reactivity distinct from NO. 9,25-27 For example, HNO reacts directly with thiols while NO does not. 9 HNO inhibits aldehyde dehydrogenase, and as such may be useful in treating alcoholism. 28 Like NO, HNO is also a vasorelaxant 29 and shows potential in treating cardiovascular disease and preventing ischemia/reperfusion injury and congestive heart failure. 9,25,30 However, one major challenge in elucidating the chemical and biochemical reactivity of HNO stems from its rapid, spontaneous dimerization and subsequent decomposition to nitrous oxide and water in aqueous solution, eq 1. 31 + → ⎯ → ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ + =× − − HNO HNO HNO NO H O k 2 2 2 8 10 M s 2 2 6 1 1 (1) Owing to its inherent instability, HNO donor compounds are therefore required to generate HNO in situ, and indeed the design of molecules that release HNO within a useful time frame is an active area of research. 32-34 Currently, sodium trioxodinitrate or Angeli’s salt (Na 2 N 2 O 3 , AS), first synthesized by Angeli in 1903, 35 is the most widely used HNO donor in chemical and biochemical studies of HNO reactivity. 36 AS (HN 2 O 3 - ,pK a (HN 2 O 3 - ) = 9.70; pK a (H 2 N 2 O 3 ) = 2.51 37 ) decomposes rapidly to give HNO and NO 2 - at pH 4-8, eq 2, whereas at pH <4, AS is primarily a NO donor. 36,38 + → + − + − − − H I ooooooooooooo NO H HN O HNO NO K k 2 3 2 p (HN O ) 2 3 2 a 2 3 L (2) Vitamin B 12 derivatives (also known as cobalamins; Cbls) are a class of redox active cobalt containing complexes belonging to the corrinoid family which are synthesized by a number of microorganisms. 39 Cobalamins are essential coenzymes in all mammalian cells. The two B 12 -dependent enzyme reactions in humans require either adenosylcobalamin (AdoCbl, X = 5′- deoxy-5′-adenosyl (Ado), Figure 1) or methylcobalamin Received: October 16, 2013 Published: January 17, 2014 Article pubs.acs.org/IC © 2014 American Chemical Society 1570 dx.doi.org/10.1021/ic402613z | Inorg. Chem. 2014, 53, 1570-1577