A MYOGLOBIN MUTANT DESIGNED TO MIMIC THE OXYGEN-AVID ASCARIS-SUUM HEMOGLOBIN - ELUCIDATION OF THE DISTAL HYDROGEN-BONDING NETWORK BY SOLUTION MMR

The solution H-1 NMR structure of the active site and ligand dissociat ion rate for the cyanomet complex have been determined for a sperm wha le myoglobin triple mutant Leu29(B10) --> Tyr, His64(E7) --> Gln, Thr6 7(E10) --> Arg that mimics the distal residue configuration of the oxy gen-avid hemoglobin from Ascaris suum. A double mutant that retains Le u29(B10) was similarly investigated. Two-dimensional NMR analysis of t he iron-induced dipolar shifts, together with the conserved proximal s ide structure for the two mutants, allowed the determination of the or ientations of the Paramagnetic susceptibility tensor for each complex. The resulting magnetic axes, together with paramagnetic relaxation an d steady-state NOEs, led to a quantitative description of the distal r esidue orientations. The distal Tyr29(B10) in the triple mutant provid es a strong hydrogen bond to the bound cyanide comparable to that prov ided by His64(E7) in wild-type myoglobin. The distal Gln64(E7) in the triple mutant is sufficiently close to the bound cyanide to serve as a hydrogen bond donor, but the angle is not consistent with a strong hy drogen bond. Dipolar contacts between the Arg67(E10) guanidinium group and the Gln64(E7) side chain in both mutants support a hydrogen-bond to the Gln64(E7) carbonyl group. The much lower oxygen affinity of thi s triple mutant relative to that of Ascaris hemoglobin is concluded to arise from side-chain orientations that do not allow hydrogen bonds b etween the Gln64(E7) side-chain NHs and both the ligand and Tyr29(B10) hydroxyl oxygen. Cyanide dissociation rates for the reduced cyanide c omplexes are virtually unaffected by the mutations and are consistent with a model of the rate-determining step as the intrinsically slow Fe -C bond breaking that is largely independent of any hydrogen bonds to the cyanide nitrogen.