Background: Potential blood substitutes can be based on hemoglobin. Two problems must be overcome with acellular hemoglobin-based blood substitutes, however; the oxygen affinity of purified human hemoglobin is too high for it to deliver oxygen to tissues, and hemoglobin tetramers dissociate into αβ dimers that can cause kidney damage. A modified form of hemoglobin, rHb1.1, has reduced oxygen affinity as the result of an Asnβ108→Lys mutation, and dimerization is prevented by the insertion of a glycine residue between the sequences of the normal α chains to produce one covalently continuous di-α-chain. Determination of the structure of rHb1.1 would provide structure-based explanations for the altered properties of rHb1.1. Results: We determined the structures of the deoxy form of rHb1.1 at 2.0 Å resolution and of cyanomet-rHb1.1 at 2.6 Å resolution. Deoxy-rHb1.1 adopts the classic 'T state' quaternary structure, but cyanomet-rHb1.1 adopts a novel quaternary structure, the B state. The most striking feature of the tertiary structures is a charged hydrogen bond involving Lysβ108 that is broken in the T→B state transition. The glycine bridge within the di-α-chain is well defined in both structures and appears to cause adoption of the B state instead of the previously observed ligand-bound quaternary structures R or Y/R2. Conclusions: A charged hydrogen bond between Lysβ108 and Tyrβ35 is broken in the transition between the deoxy and ligand-bound forms of rHb1.1. This structural change reduces the oxygen affinity of rHb1.1 by changing the relative stability of deoxy and ligand-bound states. Furthermore, our observations highlight the importance of small conformational changes in allosteric proteins, even in their most rigid domains. Three ligand-bound quaternary structures of hemoglobin (R, Y/R2 and B) have now been described. In contrast, only one quaternary structure has been observed for deoxyhemoglobin (T). The structural degeneracy of the high oxygen affinity form of hemoglobin is an important reminder that allosteric proteins may have multiple quaternary structures that are functionally very similar. This degeneracy of quaternary structures has important implications for the regulation of allosteric proteins, because different quaternary structures may be stabilized by different allosteric effectors.
Kroeger, K. S., & Kundrot, C. E. (1997). Structures of a hemoglobin-based blood substitute: Insights into the function of allosteric proteins. Structure, 5(2), 227–237. https://doi.org/10.1016/S0969-2126(97)00181-0