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Here, we report results of spectrophotometric, spectrofluorimetric, and ultrafiltration studies on the reaction between cobinamides (viz. aquahydroxo-, nitro-, aquacyano- and dicyanocobinamides; Cbi) and bovine serum albumin (BSA), and reactivity of formed complexes toward cyanide. (H2O)(HO−)Cbi binds BSA at almost equimolar ratio predominantly via amino group of lysine side chains. The mechanism of the reaction involves two steps, i.e. the coordination of amino group on Co(III), and further stabilization of the generated complex. The reaction of (H2O)(NO2−)Cbi with BSA is similar with that involving (H2O)(HO−)Cbi, and both complexes bind cyanide significantly slower than free (H2O)(HO−)Cbi and (H2O)(NO2−)Cbi. (H2O)(CN−)Cbi binds BSA predominantly via aminogroup as well, however, its coordination proceeds substantially faster and less tightly than in the case of (H2O)(HO−)Cbi. Binding of (H2O)(CN−)Cbi and (H2O)(HO−)Cbi occurs at different sites of BSA as was indicated by spectrofluorimetric titration. Reaction of the complex between (H2O)(CN−)Cbi and BSA with cyanide proceeds much faster than in the case of the complex between (H2O)(HO−)Cbi and BSA. (CN−)2Cbi is partially decyanated by BSA, however, its binding by BSA is relatively low. [ABSTRACT FROM AUTHOR]

Selenocysteine (Sec) is a crucial component of mammalian thioredoxin reductase (TrxR) where it serves as a nucleophile for disulfide bond rupture in thioredoxin (Trx). Generation of the reduced state of Sec in TrxR requires consecutive two electron transfer steps, namely: (i) from NADPH to flavin adenine dinucleotide, (ii) from reduced flavin to the disulfide bond Cys59‐S‐S‐Cys64, and finally (iii) from Cys59 and Cys64 to the selenosulfide bond Cys497‐S‐Se‐Sec498. In this work, we studied the reaction between reduced riboflavin (RibH2) and selenocystine (Sec‐Sec), an oxidized form of Sec. The interaction between RibH2 and Sec‐Sec proceeded relatively slowly in comparison with its reverse reaction, that is, reduction of riboflavin (Rib) by Sec. The rate constant for the reaction between RibH2 and Sec‐Sec was (7.9 ± 0.1) × 10−2 M−1 s−1 (pH 7.0, 25.0°C). The reaction between Rib and Sec proceeded via two steps, namely, a rapid reversible binding of Rib to Sec having a protonated selenol group to form a Sec‐Rib complex, followed by nucleophilic attack of Sec‐Rib by a second Sec molecule harboring a deprotonated selenol group. The equilibrium constant for the overall reduction process of Rib by Sec is (1.2 ± 0.1) × 106 M−1 (25.0°C). The finding that the interaction of RibH2 with oxidized selenol is reversible with its equilibrium favored toward the reverse reaction provides an additional explanation for the exceptional mechanism of the mammalian Trx/TrxR system involving transient reduction of a disulfide bond. [ABSTRACT FROM AUTHOR]

Here, we report the results of kinetic and mechanistic studies of the first step of reaction between thiols (viz., cysteine, N-acetylcysteine, mercaptoethanol, thioglycolic acid and 5-thio-2-nitrobenzoic acid (TNB)) and selenite producing thiol-S-Selenites (RS-SeO2−) in aqueous solutions of various acidity. In this set of aliphatic thiols, their reactivity toward selenite in neutral medium is directly dependent on the acidity of thiol group, whereas the stability of RS-SeO2− is affected by the interaction with groups adjacent to S–Se bond. In the case of TNB, the reaction with selenite in neutral medium proceeds much slower than processes involving aliphatic thiols due to low nucleophilicity of TNB thiol group. Using Ellman's reagent, we demonstrated that selenite binds substantially weaker to thiol group of cysteine residue in bovine serum albumin than to thiol group of free cysteine. [ABSTRACT FROM AUTHOR]

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 β-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. [ABSTRACT FROM AUTHOR]

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 (H 2 OCbl) and bovine serum albumin (BSA, the bovine counterpart of HSA) using Ultraviolet-Visible and fluorescent spectroscopy, and electron paramagnetic resonance. The reaction between H 2 OCbl 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 β-axial position of the cobalamin may be occupied by an amino acid residue from the protein. Reaction of BSA containing reduced disulfide bonds with H 2 OCbl 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 H 2 OCbl bound to BSA. These data suggest that the two tetrapyrroles interact with BSA in different binding pockets.