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Mechanistic Studies on the Reactions of Vitamin B12 Complexes with the Nitroxyl (HNO) Donors Angeli's Salt and Piloty's Acid

Subedi, Harishchandra

Abstract Details

2014, PHD, Kent State University, College of Arts and Sciences / Department of Chemistry.
Nitric oxide (NO, •NO), the `1992 Molecule of the Year’, is a gaseous messenger molecule produced in cells from L–arginine by nitric oxide synthases. The chemical and biological properties of NO have been extensively studied for well over a decade. However, recent studies have shown that nitroxyl (nitrosyl hydride, HNO), the one–electron–reduction product of NO, may also be formed in biological systems, from nitric oxide synthase–catalyzed oxidation of L–arginine in the absence of tetrahydrobiopterin and by enzyme catalyzed reduction of NO. Much less is known about the reactivity of HNO, including its reactivity with transition metal complexes. A fundamental understanding of this is important, given that about one third of proteins are metalloproteins. In this dissertation we present detailed kinetic and mechanistic studies of the reactions of two well characterized HNO donors, Angeli’s salt (AS) and Piloty’s acid (PA), with cobalamins (vitamin B12 derivatives). Cobalamins have important role as cofactors in two B12–dependent enzymatic reactions in humans. Furthermore, cobalamins have three readily accessible oxidation states. Chapter 2 of this dissertation is concerned with studies on the reactions of reduced vitamin B12 complexes, cob(II)alamin (Cbl(II)) and cob(I)alamin (Cbl(I)–), with the HNO donor PA. Cob(II)alamin is a major intracellular form of B12 and cob(I)alamin is a short–lived precursor of the two B12 coenzyme forms methylcobalamin and adenosylcobalamin. Although it is well established that HNO reduces transition metals including transition metal centers of porphyrins and metalloproteins, oxidation of a transition metal center by HNO has yet to be reported. Kinetic studies on the reaction of Cbl(II) with PA show that PA decomposition to give HNO (and C6H5SO2–) is the rate–determining step, the reaction stoichiometry is 1:2 Cbl(II):PA, and nitroxylcobalamin (NOCbl), N2 and C6H5SO2– being formed. A mechanism is proposed in which reduction of Cbl(II) by HNO results in formation of cob(I)alamin (Cbl(I)–) and NO. Cob(I)alamin intermediate is subsequently oxidized back to Cbl(II) by a second HNO molecule, and Cbl(II) reacts rapidly with NO to form nitroxylcobalamin, NOCbl. Chapter 3 involves kinetic studies on the reaction of the oxidized form of cobalamin (Cbl(III), aquacobalamin/hydroxocobalamin; pKa(H2OCbl+ = 7.8)) with PA. Under alkaline conditions (pH > 10.00), the rate–determining–step is decomposition of deprotonated PA to give 3NO– (pKa(HNO) = 11.4). The hydroxo ligand of hydroxocobalamin is inert to substitution. It is therefore likely that 3NO– (and also possibly HNO) reduce Cbl(III) to Cbl(II), being oxidized to NO. Cbl(II) and NO rapidly combine to give the observed NOCbl product. At lower pH conditions (< pH 10), PA instead reacts directly with Cbl(III) to give NOCbl. The mechanism of this latter reaction is unclear. Chapters 4 and 5 are focused on studies of the reaction between Angeli’s salt and Cbl(II) and Cbl(III), respectively. The reaction of Cbl(II) with AS occurs via a Cbl(I)– intermediate with AS decomposition being the rate–determining–step. The reaction stoichiometry is 1:2 Cbl(II):AS and NOCbl, nitrite and N2 are the reaction products. As for the Cbl(II)/PA system, HNO reduces the Cbl(II) to Cbl(I)– and a second HNO molecule oxidize Cbl(I)– back to Cbl(II), followed by the combination of NO and Cbl(II) to give NOCbl. The reaction of AS with Cbl(III), however, undergoes via different mechanisms depending upon the pH conditions. At pH < 9.90 aquacobalamin reacts directly with the monoprotonated form of Angeli’s salt, HN2O3–, to form NOCbl and nitrite. At pH > 10.80 the reaction instead switches predominantly to a mechanism in which spontaneous decomposition of AS 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. NOCbl is formed as the cobalamin product in all of the above reactions. Furthermore it has been proposed that NOCbl is formed in vivo. The final chapter of this thesis, Chapter 6, is concerned with the mechanistic studies on the reaction of extremely air–sensitive cobalamin complex, NOCbl, with dioxygen. 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.
Nicola E. Brasch, Ph.D. (Advisor)
235 p.

Recommended Citations

Citations

  • Subedi, H. (2014). Mechanistic Studies on the Reactions of Vitamin B12 Complexes with the Nitroxyl (HNO) Donors Angeli's Salt and Piloty's Acid [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1406405910

    APA Style (7th edition)

  • Subedi, Harishchandra. Mechanistic Studies on the Reactions of Vitamin B12 Complexes with the Nitroxyl (HNO) Donors Angeli's Salt and Piloty's Acid. 2014. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1406405910.

    MLA Style (8th edition)

  • Subedi, Harishchandra. "Mechanistic Studies on the Reactions of Vitamin B12 Complexes with the Nitroxyl (HNO) Donors Angeli's Salt and Piloty's Acid." Doctoral dissertation, Kent State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=kent1406405910

    Chicago Manual of Style (17th edition)