A POSSIBLE ALLOSTERIC COMMUNICATION PATHWAY IDENTIFIED THROUGH A RESONANCE RAMAN-STUDY OF 4 BETA-37 MUTANTS OF HUMAN HEMOGLOBIN-A

The highly conserved tryptophan at position beta 37 occupies a key loc us at the hinge region within the alpha(1) beta(2) interface of the ma mmalian hemoglobins. This residue is thought to play an important role in mediating the heme-heme interaction associated with the cooperativ e binding of oxygen; however, its explicit function is unclear. In thi s study, the proximal heme environments of several beta 37 mutants of adult human hemoglobin (HbA) are probed using visible (Soret band enha nced) resonance Raman spectroscopy. In the equilibrium deoxy derivativ es of these mutants, a systematic variation in proximal strain, as ref lected in the iron-proximal histidine (F8) stretching frequency, nu(Fe -His), is seen upon mutation of the beta 37 residue. The variation in proximal strain correlates with both the ligand binding rates [Kwiatko wski et al. (1998) Biochemistry 37, 4325-4335] and conformational chan ges observed at the FG corner through X-ray crystallography [Kavanaugh et al. (1998) Biochemistry 37, 4358-4373]. The results from the deoxy samples indicate a plasticity of the tertiary structure within the T quaternary state. The correlation between the X-ray data and the Raman supports the idea that the proximal strain at the heme within the T s tate can be modulated by a combination of forces including those arisi ng from the hinge region of the alpha(1) beta(2) interface, from the b inding of allosteric effecters, and from the degree of iron displaceme nt from the heme plane. Each of these contributors appears to operate through a shifting of the F helix either away from or toward the FG co rner. The Raman spectra obtained from the 10 ns CO photoproduct of the beta 37 mutant Hb's indicate that these mutants contain an altered co upling between the R state alpha(1) beta(2) interface and the proximal heme environment. This altered coupling could be due to either dissoc iation of the ligated mutant tetramers into dimers or the formation of an R state tetramer with significantly weakened hydrogen bonds and va n der Waals contacts between the alpha(1) and beta(2) subunits at the interface. In either case, the results reveal a clear-cut structural b asis for the quaternary enhancement effect in which the normal R state quaternary structure produces a higher affinity ligand binding site t han that which occurs in the corresponding dimeric form of the protein . The normal R state interface is shown to be important for stabilizin g a favorable ligand binding environment that persists long enough aft er laser photolysis to enhance the geminate rebinding process within t he photoproduct. The addition of IHP to the solution of mutant COHb pr oteins results in photoproduct spectra that are all identical and are consistent with the ligand-bound derivatives having either a T state s tructure or a very strained and anomalous R state structure.