Proton nuclear magnetic resonance investigation of the allosteric transition in ligated and unligated carp hemoglobin. Evidence for structural heterogeneity in the heme pocket.

The proton nuclear magnetic resonance spectra of carp hemoglobin (Hb) in the unligated deoxy and ligated met-cyano and met-azido forms have been recorded as a function of pH and upon addition of inositol hexaphosphate. All protein derivatives yield spectra that are consistent with appreciable molecular heterogeneity in the heme cavity. The pattern of heme methyl hyperfine shifts in carp met-cyano Hb indicates that this heterogeneity arises from the presence of heme rotational disorder, as found in native myoglobin. In carp deoxy Hb, the T----R transition manifests itself in nuclear magnetic resonance spectral changes similar to those found in modified human Hb species; namely, a decrease in heme methyl and an increase in proximal histidyl imidazole ring NH hyperfine shifts indicative of a strengthening of the iron-histidine bond. The met-cyano complex exhibits heme methyl hyperfine shifts similar to the analogous R state complex of Hb A; addition of inositol hexaphosphate did not give evidence for a quaternary structural change. Carp met-azido Hb in the R state also closely resembles the electronic structure of the HbA complex. Addition of inositol hexaphosphate appeared to effect at least a partial conversion to a T state with larger high-spin content than that observed for T state human metHbN3.