TY - JOUR
T1 - Reactivity of glass-embedded met hemoglobin derivatives toward external NO
T2 - Implications for nitrite-mediated production of bioactive NO
AU - Navati, Mahantesh S.
AU - Friedman, Joel M.
PY - 2009/9/2
Y1 - 2009/9/2
N2 - Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms. This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties. The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-dependent catalytic process whereby Hb generates relatively longlived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.
AB - Many protein reactions are exceedingly difficult to dissect under standard conditions due to low concentrations of reactants and intermediates. A case in point are several proposed reactions of hemoglobin with both nitrite and nitric oxide. In the present work, glassy matrices are used to dynamically control the rate at which externally introduced gaseous NO accesses and reacts with several different met Hb derivatives including the nitrite, nitrate, and aquomet forms. This novel yet general approach reveals a clear difference between nitrite and other ligands including nitrate, water, and an internal imidazole. For nitrate, water, and the internal distal imidazole, the observed spectral changes indicate that NO entering the distal heme pocket is effective in displacing these ligands from the ferric heme iron. In contrast, when the ligand is nitrite, the resulting initial spectra indicate the formation of an intermediate that has distinctly ferrous-like properties. The spectrum and the response of DAF fluorescence to the presence of the intermediate are consistent with a recently proposed nitrite anhydrase reaction. This proposed intermediate is especially significant in that it represents a pathway for a nitrite-dependent catalytic process whereby Hb generates relatively longlived bioactive forms of NO such as S-nitrosoglutathione. The failure to form this intermediate either at low pH or when the glass is extensively dried is consistent with the requirement for a specific conformation of reactants and residue side chains within the distal heme pocket.
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U2 - 10.1021/ja903364h
DO - 10.1021/ja903364h
M3 - Article
C2 - 19663497
AN - SCOPUS:69349100009
SN - 0002-7863
VL - 131
SP - 12273
EP - 12279
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 34
ER -