Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study.

Nitrobindins (Nbs) are all-ß-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3-. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2- to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M-1 s-1, 9.3 M-1 s-1, 1.4 × 101 M-1 s-1, and 5.8 M-1 s-1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2--based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-ß-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-ß-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.

Hydroxylamine-induced oxidation of ferrous nitrobindins.

Hemoglobin and myoglobin are generally taken as molecular models of all-α-helical heme-proteins. On the other hand, nitrophorins and nitrobindins (Nb), which are arranged in 8 and 10 ß-strands, respectively, represent the molecular models of all-ß-barrel heme-proteins. Here, kinetics of the hydroxylamine- (HA-) mediated oxidation of ferrous Mycobacterium tuberculosis, Arabidopsis thaliana, and Homo sapiens nitrobindins (Mt-Nb(II), At-Nb(II), and Hs-Nb(II), respectively), at pH 7.0 and 20.0 °C, are reported. Of note, HA displays antibacterial properties and is a good candidate for the treatment and/or prevention of reactive nitrogen species- (RNS-) linked aging-related pathologies, such as macular degeneration. Under anaerobic conditions, mixing the Mt-Nb(II), At-Nb(II), and Hs-Nb(II) solutions with the HA solutions brings about absorbance spectral changes reflecting the formation of the ferric derivative (i.e., Mt-Nb(III), At-Nb(III), and Hs-Nb(III), respectively). Values of the second order rate constant for the HA-mediated oxidation of Mt-Nb(II), At-Nb(II), and Hs-Nb(II) are 1.1 × 104 M-1 s-1, 6.5 × 104 M-1 s-1, and 2.2 × 104 M-1 s-1, respectively. Moreover, the HA:Nb(II) stoichiometry is 1:2 as reported for ferrous deoxygenated and carbonylated all-α-helical heme-proteins. A comparative look of the HA reduction kinetics by several ferrous heme-proteins suggests that an important role might be played by residues (such as His or Tyr) in the proximity of the heme-Fe atom either coordinating it or not. In this respect, Nbs seem to exploit somewhat different structural aspects, indicating that redox mechanisms for the heme-Fe(II)-to-heme-Fe(III) conversion might differ between all-α-helical and all-ß-barrel heme-proteins.

Oxygen-mediated oxidation of ferrous nitrosylated nitrobindins.

The O2-mediated oxidation of all-ß-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 kon = (6.3 ±â€¯0.8) × 103 M-1 s-1, (1.4 ±â€¯0.2) × 103 M-1 s-1, and (3.9 ±â€¯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.