Oxygen and CO binding to triply NO and asymmetric NO/CO hemoglobin hybrids

The bimolecular and geminate CO recombination kinetics have been measured for hemoglobin (Hb) with over 90% of the ligand binding sites occupied by NO. Since Hb(NO)4 with inositol hexaphosphate (IHP) at pH below 7 is thought to take on the low affinity (deoxy) conformation, the goal of the experiments was to determine whether the species IHPHb-(NO)3(CO) also exists in this quaternary structure, which would allow ligand 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 during the geminate phase is low (< 1%) in aqueous solutions, suggesting that the IHPHb(NO)3(CO) hybrid is also essentially in the deoxy conformation. By mixing stock solutions of HbCO and HbNO, the initial exchange of dimers produces asymmetric (alpha NO beta NO/alpha CO beta CO) hybrids. At low pH in the presence of IHP, this hybrid also displays 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 reflecting the differences in the alpha and beta chains. Samples of symmetric hybrids (a2NOI32Co or a2Co922NO) showed a lower (R-like) bimolecular yield and less slow phase for the CO bimolecular recombination, relative to the asymmetric hybrid or the triply NO species. The slower (T state) bimolecular rate of 7 x 104 M-1 s-1 was observed for CO rebinding to a chain.While oxygen equilibrium studies with 'HPHb(NO)3 were hampered by a high oxidation rate, it was possible to perform experiments with samples equilibrated with a mixed CO/oxygen atmosphere. Photodissociation of CO allows a temporary exposure of the binding sites to oxygen. The results confirm that IHPHb(NO)3 has a low oxygen affinity.