Mutational effects at the subunit interfaces of human hemoglobin: Evidence for a unique sensitivity of the T quaternary state to changes in the hinge region of the α1β2 interface

A set of variant human hemoglobins, each with an Ala or Gly substitution at a single residue, has been prepared, and the kinetics of their reactions with carbon monoxide have been measured. This reaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. The magnitudes of the effects of the mutations on CO combination vary widely, and, with the exception of βY145, the residues with the most significant effects on these kinetics are found in the hinge region of the α1β2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heine on both α or both β subunits, were prepared for βW37E, βW37A, αY140G, and αY140A, hinge region variants causing large kinetic changes, and for βY145G. Such hybrids permit measurements of the kinetics of CO binding to only the heine-containing α or β subunits within the unliganded hemoglobin tetramer. Mutations at β37 and α140 have global effects on the T state, increasing the rates of CO binding to both types of subunits. Mutation of βY145 has a large effect on the β subunits in the deoxygenated T state, but very little effect on the α subunits. Oxygen equilibria measurements on the crystalline T state of βW37E also indicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of the variant hemoglobins in solution are sensitive to numerous variables besides the properties of the deoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause the largest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the α1β2 interface.