Superoxide dismutase targets NO from GSNO to Cysbeta93 of oxyhemoglobin in concentrated but not dilute solutions of the protein

The role of hemoglobin (Hb) in transmitting the vasodilatory property of NO throughout the vascular system is of much current interest. NO exchange between Hb and low-molecular-weight nitrosothiols such as S-nitrosoglutathione (GSNO) has been speculated and reported in vitro. Previously, we reported that NO delivery from GSNO to Cysbeta93 of human oxyHb is prevented in the presence of the Cu chelators, neocuproine, and DTPA.(1) In the present work, 5 mM solutions of commercial human Hb were found by ICP-MS to contain approximately 20 microM Cu and Zn, suggesting the presence of Cu,Zn-superoxide dismutase (CuZnSOD), which was confirmed by Western blotting. SOD activity measurements were consistent with the presence of approximately 20 microM CuZnSOD monomer in 5 mM Hb solutions, which is the physiological concentrations of these proteins in the red blood cell. Incubation of 3.75 mM oxyHb (15 mM heme; 7.5 mM Cysbeta93) with 3.75 or 7.5 mM GSNO gave rise to 50% or 100% S-nitrosation, respectively, of Cysbeta93 as monitored by FTIR nu(SH) absorption, whereas excess GSNO over Cysbeta93 converted oxyHb to metHb due to the reaction, oxyHb + NO<==>metHb + NO(3)(-). Removal of CuZnSOD by anion-exchange chromatography yielded an oxyHb sample that was unreactive toward GSNO, and replacement with bovine CuZnSOD restored reactivity. Addition of 1 microM GSNO (Cysbeta93/GSNO = 1) to solutions diluted 10(4)-fold from physiological concentrations of oxyHb and CuZnSOD resulted largely in metHb formation. Thus, this work reports the following key findings: CuZnSOD is an efficient catalyst of NO transfer between GSNO and Cysbeta93 of oxyHb; metHb is not detected in oxyHb/GSNO incubates containing close to the physiological concentration (5 mM) of Hb and CuZnSOD when the Cysbeta93/GSNO molar ratio is 0.5 to 1.0, but metHb is detected when the total Hb concentration is low micromolar. These results suggest that erythrocyte CuZnSOD may play a critical role in preserving the biological activity of NO by targeting it from GSNO to Cysbeta93 of oxyHb rather than to its oxyheme.