Hydroxylamine-induced oxidation of ferrous nitrobindins

Area of Science:

• Biochemistry
• Biophysics
• Protein Chemistry

Background:

• Heme-proteins are crucial biological molecules, with all-α-helical (e.g., hemoglobin) and all-β-barrel (e.g., nitrobindins) structures representing distinct classes.
• Nitrobindins (Nb) are all-β-barrel heme-proteins, contrasting with the more commonly studied all-α-helical heme-proteins.
• Hydroxylamine (HA) exhibits antibacterial properties and is being explored for treating reactive nitrogen species (RNS)-linked aging pathologies like macular degeneration.

Purpose of the Study:

• To determine the kinetics of hydroxylamine-mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II)).
• To compare the oxidation mechanisms of all-β-barrel nitrobindins with those of all-α-helical heme-proteins.
• To elucidate the role of heme-proximal residues in the redox conversion of heme-iron.

Main Methods:

• Spectrophotometric monitoring of the oxidation of ferrous nitrobindins (Mt-Nb(II), At-Nb(II), Hs-Nb(II)) by hydroxylamine under anaerobic conditions at pH 7.0 and 20.0 °C.
• Kinetic analysis to determine the second-order rate constants for the oxidation reactions.
• Stoichiometric analysis of the hydroxylamine-nitrobindin interaction.

Main Results:

• The second-order rate constants for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) were determined as 1.1 × 10⁴ M⁻¹s⁻¹, 6.5 × 10⁴ M⁻¹s⁻¹, and 2.2 × 10⁴ M⁻¹s⁻¹, respectively.
• The stoichiometry of the reaction was found to be 1:2 (HA:Nb(II)), consistent with other heme-proteins.
• Spectral changes indicated the formation of ferric nitrobindin derivatives (Mt-Nb(III), At-Nb(III), Hs-Nb(III)).

Conclusions:

• Nitrobindins undergo HA-mediated oxidation with distinct kinetics compared to other heme-proteins.
• The stoichiometry of HA binding to nitrobindins is similar to that observed in all-α-helical heme-proteins.
• Structural differences, particularly the role of heme-proximal residues, likely account for variations in redox mechanisms between all-α-helical and all-β-barrel heme-proteins.