Oxygen-mediated oxidation of ferrous nitrosylated nitrobindins

The O₂-mediated oxidation of all-β-barrel ferrous nitrosylated nitrobindin from Arabidopsis thaliana (At-Nb(II)-NO), Mycobacterium tuberculosis (Mt-Nb(II)-NO), and Homo sapiens (Hs-Nb(II)-NO) to ferric derivative (At-Nb(III), Mt-Nb(III), and Hs-Nb(III), respectively) has been investigated at pH 7.0 and 20.0 °C. Unlike ferrous nitrosylated horse myoglobin, human serum heme-albumin and human hemoglobin, the process in Nb(II)-NO is mono-exponential and linearly dependent on the O₂ concentration, displaying a bimolecular behavior, characterized by k_on = (6.3 ± 0.8) × 10³ M^-1 s^-1, (1.4 ± 0.2) × 10³ M^-1 s^-1, and (3.9 ± 0.5) × 10³ M^-1 s^-1 for At-Nb(II)-NO, Mt-Nb(II)-NO, and Hs-Nb(II)-NO, respectively. No intermediate is detected, indicating that the O₂ reaction with Nb(II)-NO is the rate-limiting step and that the subsequent conversion of the heme-Fe(III)-N(O)OO^- species (i.e., N-bound peroxynitrite to heme-Fe(III)) to heme-Fe(III) and NO₃^- is much faster. A similar mechanism can be invoked for ferrous nitrosylated human neuroglobin and rabbit hemopexin, in which the heme-Fe(III)-N(O)OO^- species is formed as well, although the rate-limiting step seems represented by the reshaping of the six-coordinated heme-Fe(III) complex. Although At-Nb(II)-NO and Mt-Nb(II)-NO are partially (while Hs-Nb(II)-NO is almost completely) penta-coordinated, density functional theory (DFT) calculations rule out that the cleavage of the proximal heme-Fe-His bond in Nb(II)-NO is responsible for the more stable heme-Fe(III)-N(O)OO^- species. Moreover, the oxidation of the penta-coordinated heme-Fe(II)-NO adduct does not depend on O₂ binding at the proximal side of the metal center. These features may instead reflect the peculiarity of Nb folding and of the heme environment, with a reduced steric constraint for the formation of the heme-Fe(III)-N(O)OO^- complex.