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Nitrite and NO Processing by CblC: A Human B 12 Trafficking Chaperone
Author(s) -
Mascarenhas Romila N,
Gherasim Carmen,
Banerjee Ruma
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.633.19
Subject(s) - cobalamin , chemistry , biochemistry , methionine synthase , nitrite reductase , cysteine , methionine , stereochemistry , enzyme , vitamin b12 , amino acid , nitrate reductase
Cobalamin (Cbl) or vitamin B 12 derivatives are processed and escorted to the two known B 12 ‐dependent enzymes: cytosolic methionine synthase (MS) and mitochondrial methylmalonyl CoA mutase (MCM). Mutations in the B 12 trafficking genes lead to inborn errors of cobalamin metabolism belonging to the cbl A‐G, J and mut complementation groups. Of these, cbl C is the most common locus for mutations and patients present with both homocystinuria and methylmalonic aciduria. Functionally, CblC is involved in the early steps of the cytosolic B 12 trafficking pathway and catalyzes divergent chemical reactions. Cobalamins entering the cytoplasm with various upper ligands are processed to a common cob(II)alamin intermediate by the CblC protein before being allocated to the methycobalamin or 5′‐deoxyadenosylcobalamin branches. Alkylcobalamins are cleaved via a nucleophilic displacement mechanism in the presence of glutathione (GSH) producing GSR and cob(I)alamin, which is subsequently oxidized to cob(II)alamin, and cyanocobalamin is cleaved via reductive elimination forming cob(II)alamin and cyanide. Herein, we report the discovery of a new activity catalyzed by CblC, i.e. nitrite reductase. The interactions between nitrite and NO with cobalamins have been characterized in solution, but the biological relevance of these reactions is largely unknown. It was previously shown that nitrosylcobalamin (NOCbl) has antitumor activity that preferentially targets cancer cells in which cobalamin receptor expression (TCII) is upregulated. The mechanism of action of NOCbl has been ascribed to its ability to release nitric oxide (NO • ). However, the ready oxidation of NOCbl to nitritocobalamin (NO 2 Cbl) was not taken into consideration. In this study, we have investigated the reactivity of human CblC towards NOCbl and NO 2 Cbl. We show that CblC binds NOCbl with a k on of 0.07 ± 0.01 mM −1 s −1 . The cobalamin is bound in a base‐off conformation as are the natural B 12 derivatives and this is likely facilitated by the strongly σ‐donating NO‐ligand that promotes the dissociation of the coordinating dimethylbenzimidazole group. Surprisingly, neither thiols nor reductants remove the nitrosylgroup from CblC‐bound NOCbl. Addition of thiols to CblC‐bound NO 2 Cbl generates nitrite and cob(II)alamin (anaerobically) or aquocobalamin (aerobically). The nitrite that is release can potentially serve as an NO • source via the activity of cellular nitrite reductases. Collectively, our results show that the promiscuity of CblC towards cobalamins can be exploited to deliver small molecules or to inhibit intracellular B 12 metabolism. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .