OXYGEN AND CO BINDING TO TRIPLY NO AND ASYMMETRIC NO CO HEMOGLOBIN HYBRIDS/

The bimolecular and geminate CO recombination kinetics have been measu red for hemoglobin (Hb) with over 90% of the ligand binding sites occu pied by NO. Since Hb(N)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the go al of the experiments was to determine whether the species (IHP)Hb(NO) 3(CO) also exists in this quaternary structure, which would allow liga nd binding studies to tetramers in the deoxy conformation. For samples at pH 6.6 in the presence of IHP, the bimolecular kinetics show only a slow phase with rate 7 x 10(4) M-1 s-1, characteristic of CO binding to deoxy Hb, indicating that the triply NO tetramers are in the deoxy conformation. Unlike Hb(CO)4, the fraction recombination occurring du ring the geminate phase is low (<1%) in aqueous solutions, suggesting that the (IHP)Hb(NO)3(CO) hybrid is also essentially in the deoxy conf ormation. By mixing stock solutions of HbCO and HbNO, the initial exch ange of dimers produces asymmetric (alpha(NO)beta(NO)/alpha(CO)beta(CO )) hybrids. At low pH in the presence of IHP, this hybrid also display s a high bimolecular quantum yield and a large fraction of slow (deoxy -like) CO recombination; the slow bimolecular kinetics show components of equal amplitude with rates 7 and 20 x 10(4) M-1 s-1, probably refl ecting the differences in the alpha and beta chains. Samples of symmet ric hybrids (alpha2(NO)beta2CO or alpha2(CO)beta2NO) showed a lower (R -like) bimolecular yield and less slow phase for the CO bimolecular re combination, relative to the asymmetric hybrid or the triply NO specie s. The slower (T state) bimolecular rate of 7 x 10(4) M-1 s-1 was obse rved for CO rebinding to a beta chain. While oxygen equilibrium studie s with (IHF)Hb(NO)3 were hampered by a high oxidation rate, it was pos sible to perform experiments with samples equilibrated with a mixed CO /oxygen atmosphere. Photodissociation of CO allows a temporary exposur e of the binding sites to oxygen. The results confirm that (IHP)Hb(NO) 3 has a low oxygen affinity.