The nitrite reductase activity of ferrous human hemoglobin:haptoglobin 1-1 and 2-2 complexes

Abstract

Haptoglobin (Hp) binding to hemoglobin (Hb) is crucial to prevent extra-erythrocytic Hb-induced damage; in turn, Hp:Hb complexes display heme-based reactivity. Here, the nitrite reductase activity of ferrous human Hb (Hb(II)) complexed with the human Hp phenotypes 1-1 and 2-2 (Hp1-1:Hb(II) and Hp2-2:Hb(II), respectively) is reported to highlight the reactivity of the αβ-dimers of Hb(II) bound to Hp. In the presence of dithionite, values of the apparent second-order rate constant for the NO2−-mediated nitrosylation of Hp1-1:Hb(II) and Hp2-2:Hb(II) (i.e., kon = 7.3 M−1 s−1 and 1.2 × 101 M−1 s−1, respectively; pH 7.3 and 20.0 °C) agree with those of sperm whale myoglobin (Mb(II)), of horse heart Mb(II), and of the R-state of Hb(II) (kon = 6.0 M−1 s−1, 2.9 M−1 s−1, and 6.0 M−1 s−1, respectively; pH 7.4 and 25.0 °C); in turn, these kon values are higher than that of the T-state of Hb(II) (kon = 1.2 × 10−1 M−1 s−1; pH 7.4 and 25.0 °C). Further, the values of kon for the nitrite reductase activity of Hp1-1:Hb(II) and Hp2-2:Hb(II) increase linearly on lowering pH from 7.5 to 5.6 (at 20.0 °C); the value of the slope of Log konversus pH is −1.10 ± 0.10, reflecting the involvement of one proton in the NO2−-mediated nitrosylation of Hp1-1:Hb(II) and Hp2-2:Hb(II). These results indicate that the conformation of the Hb αβ-dimers bound to Hp1-1 and Hp2-2 matches that of the R-state of the Hb tetramer.