STRUCTURES OF AN ENGINEERED BLOOD SUBSTITUTE AND INSIGHTS INTO HEMOGLOBIN FUNCTION. Kenneth S. Kroeger and Craig E. Kundrot. Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA

The deoxy and cyanomet structures of the potential blood substitute rHb1.1 reveal a new quaternary structure for hemoglobin and demonstrate the importance of small conformational changes far from the hemes and the allosteric interface. rHb1.1 is produced by Somatogen, Inc. and contains four changes relative to human hemoglobin A_o: a glycine that covalently joins the two [[alpha]]-chains, the naturally occuring Hb Presbyterian mutation (Asn[[beta]]108->Lys), and Val1->Met in the [[alpha]]- and [[beta]]-chains. The glycine bridge forces cyanomet rHb1.1 (determined at 2.6 Å resolution) to adopt a previously unobserved quaternary structure, bringing the number of observed ligated quaternary structures to three. The overall structure of the deoxy rHb1.1 at 2.0 Å resolution is very similar to deoxy human hemoglobin A_o. The Asn[[beta]]108->Lys mutation, however, produces a new hydrogen bond in the relatively rigid [[alpha]]₁[[beta]]₁ interface which does not form in the cyanomet structure. Thus, this mutation stabilizes the deoxy state relative to the ligated states and demonstrates the importance of small conformational changes in the [[alpha]]₁[[beta]]₁ interface which is often incorrectly regarded as rigid.

The plurality of high oxygen affinity forms of hemoglobin contrasts with the uniqueness of the low affinity form and suggests an important rule for allosteric proteins: one functional state is achieved only within a particular, well-defined structure (T for hemoglobin, high k_cat for an enzyme) while the other ("R" for hemoglobin, low k_cat for an enzyme) can be achieved by many structures. Mutations are more likely to affect the functional properties of the former state than the latter.