Abstract
Nitrobindins (Nbs) are all-β-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO(2), and promoting peroxynitrite isomerization to NO(3)(-). 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., k(on)) for the reduction of NO(2)(-) 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 × 10(1) M(-1) s(-1), and 5.8 M(-1) s(-1), respectively. The values of k(on) increased linearly with decreasing pH, thus indicating that the NO(2)(-)-based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO(2) reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-β-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-β-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.