Abstract

There is evidence that binding of the vasorelaxant, nitric oxide (NO), to CysÝ93 of human adult hemoglobin (Hb) plays a role in blood pressure regulation. S-Nitrosation of CysÝ93 by low-molecular-weight S-nitrosothiols (RSNOs) such as S-nitrosoglutathione (GSNO) was directly probed by FTIR by monitoring the p(SH) vibrations of Hb and by ESI-MS. The results indicate that CysÝ93 is S-nitrosated when oxyHb (HbFe II O 2 ), but not deoxyHb (HbFe II ), is exposed to GSNO (1:1 heme/GSNO). No FTIR or ESI-MS evidence was obtained for oxyHb S-nitrosation in the presence of DTPA (a Cu II chelator) and neocuproine (a Cu I chelator). Monitoring the heme Soret and visible bands revealed that oxyHb was converted to metHb (HbFe III ) in the presence of excess GSNO over CysÝ93 and that this was inhibited on addition of neocuproine. The combined data are consistent with a mechanism involving Cu II -catalysis of CysÝ93 S-nitrosation by free NO generated by Cu I -catalysis of GSNO breakdown but not with trans-S-nitrosation (NO + transfer between thiols). Reductive cleavage of GSNO by deoxyHb to release free NO that binds to additional deoxyHb to give nitrosylHb was recently reported [Spencer, N. Y., Zeng, H., Patel, R. P., and Hogg, N. (2000) J. Biol. Chem. 275(47), 36562]. The reaction between GSNO and deoxyHb was reexamined in the presence of both neocuproine and DTPA using a copper-depleted Hb solution prepared by exhaustive dialysis vs. EDTA. Direct spectroscopic and mass spectral analyses of the deoxyHb/GSNO incubates show that nitrosylHb formation was significantly decelerated in presence of neocuproine. Deceleration was less in the presence of DTPA and in Hb solutions dialyzed vs. EDTA. NitrosylHb formation was almost totally inhibited when neocuproine was added to dialyzed deoxyHb plus GSNO. Extensive GSSG formation was observed by mass spectrometry in deoxyHb/GSNO incubates in the absence of metal chelators where deoxyHb nitrosylation was complete in <5 min. The data presented are inconsistent with direct reduction of GSNO by deoxyHb but indicate a mechanism involving free NO generated by Cu I catalysis of GSNO breakdown and Cu II reduction by GSH. The Cu and Zn content in commercial human Hb samples was determined by ICP-MS, and V20 oM of each metal was present in 5 mM Hb solutions. (Abstract shortened by UMI.)