MECHANISM OF NO-INDUCED OXIDATION OF MYOGLOBIN AND HEMOGLOBIN

Nitric oxide (NO) has been implicated as mediator in a variety of phys iological functions, including neurotransmission, platelet aggregation , macrophage function, and vasodilation. The consumption of NO by extr acellular hemoglobin and subsequent vasoconstriction have been suggest ed to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O-2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue, In order to d etermine the mechanism of this key reaction, we have measured the kine tics of NO-induced oxidation of a variety of different recombinant spe rm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rate s depend linearly on [NO] but show no dependence on [O-2]. The bimolec ular rate constants for NO-induced oxidation of MbO(2) and HbO(2) are large (k'(ox,NO)=30-50 mu M(-1) s(-1) for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb an d Hb, Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitri c oxide with the iron atom to produce Fe2+-N=O or with Fe2+-O-O-delta- to produce Fe3+-OH2 and nitrate, Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the di stal pocket which normally hinder NO entry into the protein. Decreasin g the volume of the distal pocket by replacing Leu(B10) and Val(E11) w ith aromatic amino acids markedly inhibits NO-induced oxidation of MbO (2). The latter results provide a protein engineering strategy for red ucing hypertensive events caused by extracellular hemoglobin-based O-2 carriers, This approach has been explored by examining the effects of Phc(B10) and Phe(E11) substitutions on the rates of NO-induced oxidat ion of the alpha and beta subunits in recombinant human hemoglobin.