Antioxidative Pseudoenzymatic Mechanism of NAD(P)H Coexisting with Oxyhemoglobin for Suppressed Methemoglobin Formation.

Abstract

Oxyhemoglobin (HbO2) coexisting with equimolar NADH retards autoxidation and oxidant-induced metHb formation based on the pseudocatalase (CAT) and pseudosuperoxide dismutase (SOD) activities. In this work, we compared the effects of NADH with those of NADPH and estimated the binding site of NAD(P)H to HbO2 to elucidate the antioxidative mechanisms. The results clarified that pseudo-CAT and pseudo-SOD activities of HbO2 coexisting with NADPH were similar to activities obtained with NADH. Prompt MetHb formation (<40 min) facilitated by oxidants (H2O2, NO, and NaNO2) was hindered by NADPH. These effects were similar to those of NADH. However, we found that NADPH is thermally unstable compared to NADH and that NADPH cannot sustain antioxidative effects for a long period of autoxidation to metHb such as 24 h. Lineweaver-Burk plots clarified that the Michaelis constants of these pseudoenzymatic activities are in the millimolar range. Addition of inositol hexaphosphate (IHP) and 2,3-diphosphoglycerate (DPG), which are known to bind not only with deoxyHb but also weakly with HbO2, showed competitive inhibition of pseudoenzymatic activities. These results suggest that the binding site of NADH and NADPH on HbO2 is the same as those of IHP and DPG. 31P nuclear magnetic resonance definitively showed 1:1 stoichiometric binding of NADH to HbO2. High-performance liquid chromatography analysis showed that NADH preferentially inhibited autoxidation of α-subunit heme. Docking simulations also predicted that the binding site of relaxed-state HbO2 with NAD(P)H is the same as those with IHP and DPG. Collectively, the pseudoenzymatic activities of HbO2 coexisting with NAD(P)H are induced by the 1:1 stoichiometric binding of NAD(P)H to HbO2.