NOVEL WATER-MEDIATED HYDROGEN-BONDS AS THE STRUCTURAL BASIS FOR THE LOW-OXYGEN AFFINITY OF THE BLOOD SUBSTITUTE CANDIDATE RHB(ALPHA-96VAL-]TRP)

One of the most promising approaches for the development of a syntheti c blood substitute has been the engineering of novel mutants of human hemoglobin (Hb) A which maintain cooperativity, but possess lowered ox ygen affinity. We describe here two crystal structures of one such pot ential blood substitute, recombinant (r) Hb(alpha 96Val-->Trp), refine d to 1.9 Angstrom resolution in an alpha-aquomet, beta-deoxy T-state, and to 2.5 Angstrom 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 affi nity had been attributed to a stabilization of the deoxy T-state inter face by alpha 96Trp-beta 99Asp hydrogen bonds. Difference Fourier maps of the T-state structure clearly show that alpha 96Trp is in a confor mation different from that predicted by the simulation, with its indol e side chain directed away from the interface and into the central cav ity. In this conformation, the indole nitrogen makes novel water-media ted hydrogen bends across the T-state interface with beta 101Glu. We p ropose that these water-mediated hydrogen bonds are the structural bas is for the lowered oxygen affinity of rHb(alpha 96Val-->Trp), and disc uss the implications of these findings for future molecular dynamics s tudies and the design of Hb mutants.