One of the most promising approaches for the development of a synthetic blood substitute has been the engineering of novel mutants of human hemoglobin (Hb) A which maintain cooperativity, but two cry structures one possess lowered oxygen affinity. We describe here two crystal structures of such potential blood substitute, recombinant (r) Hb(α96Val→Trp), refined to 1.9 resolution in an α-aquomet, β-deoxy T-state, and to 2.5 Å resolution in a carbonmonoxy R-state. On the basis of molecular dynamics simulations, a particular conformation had been predicted for the engineered Trp residue, and the lowered oxygen affinity had been attributed to a stabilization of the deoxy T-state interface by α96Trp-β99Asp hydrogen bonds. Difference Fourier maps of the T-state structure clearly show that α96Trp is in a conformation different from that predicted by the simulation, with its indole side chain directed away from the interface and into the central cavity. In this conformation, the indole nitrogen makes novel water-mediated hydrogen bonds across the T-state interface with β101Glu. We propose that these water- mediated hydrogen bonds are the structural basis for the lowered oxygen affinity of rHb(α96Val→Trp), and discuss the implications of these findings for future molecular dynamics studies and the design of Hb mutants.
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