| Abstract
| - 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. Thisreaction is rate-limited by the binding of the first CO to the deoxygenated T state of the protein. Themagnitudes of the effects of the mutations on CO combination vary widely, and, with the exception ofβY145, the residues with the most significant effects on these kinetics are found in the hinge region ofthe α1β2 interface. Mixed-metal hybrids, with zinc protoporphyrin IX in place of heme on both α or bothβ subunits, were prepared for βW37E, βW37A, αY140G, and αY140A, hinge region variants causinglarge kinetic changes, and for βY145G. Such hybrids permit measurements of the kinetics of CO bindingto only the heme-containing α or β subunits within the unliganded hemoglobin tetramer. Mutations atβ37 and α140 have global effects on the T state, increasing the rates of CO binding to both types ofsubunits. Mutation of βY145 has a large effect on the β subunits in the deoxygenated T state, but verylittle effect on the α subunits. Oxygen equilibria measurements on the crystalline T state of βW37E alsoindicate large affinity increases in both subunits of this variant. The overall oxygen equilibria of thevariant hemoglobins in solution are sensitive to numerous variables besides the properties of thedeoxygenated T state. In contrast to CO combination kinetics, the residues whose alterations cause thelargest changes in overall oxygen equilibria in solution are scattered seemingly randomly within the α1β2interface.
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