Kinetic and mechanistic studies of the NO center dot-mediated oxidation ofoxymyoglobin and oxyhemoglobin

The second-order rate constants for the reactions between nitrogen monoxide and oxymyoglobin or oxyhemoglobin, determined by stopped-flow spectroscopy , increase with increasing pH. At pH 7.0 the rates are (43.6 +/- 0.5) x 10( 6) M-1 s(-1) for oxymyoglobin and (89 +/- 3) x 10(6) M-1 s(-1) for oxyhemog lobin (per heme), whereas at pH 9.5 they are (97 +/- 3) x 10(6) M-1 s(-1) a nd (144 +/- 3) x 10(6) M-1 s(-1), respectively. The rate constants for the reaction between oxyhemoglobin and NO. depend neither on the association gr ade of the protein (dimer/tetramer) nor on the concentration of the phospha te buffer (100-1 mM). The nitrogen monoxide-mediated oxidations of oxymyogl obin and oxyhemoglobin proceed via intermediate peroxynitrito complexes whi ch were characterized by rapid scan UV/vis spectroscopy. The two complexes MbFe(III)OONO and HbFe(III)OONO display very similar spectra with absorptio n maxima around 500 and 635 nm. These species can be observed at alkaline p H but rapidly decay to the met-form of the proteins under neutral or acidic conditions. The rate of decay of MbFe(III)OONO increases with decreasing p H and is significantly larger than those of the analogous complexes of the two subunits of hemoglobin. No free peroxynitrite is formed during these re actions, and nitrate is formed quantitatively, at both pH 7.0 and 9.0. This result indicates that, as confirmed from protein analysis after reacting t he proteins with NO. for 10 times, when peroxynitrite is coordinated to the heme of myoglobin or hemoglobin it rapidly isomerizes to nitrate without n itrating the globins in physiologically significant amounts.