Catalytic effect of riboflavin on electron transfer from NADH to aquacobalamin.

Reduction of cobalamin by non-dedicated cellular reductases has been reported in earlier work, however, the sources of reducing power and the mechanisms are unknown. This study reports results of kinetic and mechanistic investigation of the reaction between aquacobalamin, H2OCbl, and reduced ß-nicotinamide adenine dinucleotide, NADH. This interaction leads to the formation of one-electron reduced cobalamin, cob(II)alamin, and proceeds via water substitution on aquacobalamin by NADH and further decomposition of NADH-Co(III) complex to cob(II)alamin and NADH·+. Riboflavin catalyzes the reduction of aquacobalamin by NADH both in free form and with aquacobalamin bound to the cobalamin processing enzyme CblC. The rate-determining step of this catalytic reaction is the interaction between riboflavin and NADH to produce a charge transfer complex that reacts with aquacobalamin. Aquacobalamin quenches the fluorescence of NADH and riboflavin predominantly via a static mechanism.

Characterization of the complex between native and reduced bovine serum albumin with aquacobalamin and evidence of dual tetrapyrrole binding.

Serum albumin binds to a variety of endogenous ligands and drugs. Human serum albumin (HSA) binds to heme via hydrophobic interactions and axial coordination of the iron center by protein residue Tyr161. Human serum albumin binds to another tetrapyrrole, cobalamin (Cbl), but the structural and functional properties of this complex are poorly understood. Herein, we investigate the reaction between aquacobalamin (H2OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H2OCbl and BSA led to the formation of a BSA-Cbl(III) complex consistent with N-axial ligation (amino). Prior to the formation of this complex, the reactants participate in an additional binding event that has been examined by fluorescence spectroscopy. Binding of BSA to Cbl(III) reduced complex formation between the bound cobalamin and free cyanide to form cyanocobalamin (CNCbl), suggesting that the ß-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H2OCbl produces cob(II)alamin and disulfide with intermediate formation of thiolate Cbl(III)-BSA complex and its decomposition. Finally, in vitro studies showed that cobalamin binds to BSA only in the presence of an excess of protein, which is in contrast to heme binding to BSA that involves a 1:1 stoichiometry. In vitro formation of BSA-Cbl(III) complex does not preclude subsequent heme binding, which occurs without displacement of H2OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.