STABILIZING BOUND O-2 IN MYOGLOBIN BY VALINE(68) (E11) TO ASPARAGINE SUBSTITUTION

The isopropyl side chain of valine(68) in myoglobin has been replaced by the acetamide side chain of asparagine in an attempt to engineer hi gher oxygen affinity. The asparagine replacement introduces a second h ydrogen bond donor group into the distal heme pocket which could furth er stabilize bound oxygen. The Val(68) to Asn substitution leads to si milar to 3-fold increases in oxygen affinity and 4-6-fold decreases in CO affinity. As a result, the M-value (K-CO/K-O2) is lowered 15-20-fo ld to a value close to unity. An even larger enhancement of Oz affinit y is seen when asparagine(68) is inserted into H64L sperm whale myoglo bin which lacks a distal histidine. The overall rate constants for oxy gen and carbon monoxide binding to the single V68N myoglobin mutants a re uniformly lower than those for the wad-type protein. In contrast, t he overall rate constant for NO association is unchanged. Analyses of time courses monitoring the geminate recombination of ligands followin g nanosecond and picosecond flash photolysis of MbNO and MbO(2) indica te that the barrier to ligand binding from within the heme pocket has been raised with little effect on the barrier to diffusion of the liga nd into the pocket from the solvent. The crystal structures of the aqu omet, deoxy, oxy, and carbon monoxy forms of the V68N mutant have been determined to resolutions ranging from 1.75 to 2.2 Angstrom at 150 K. The overall structures are very similar to those of the wild-type pro tein with the principal alterations taking place within and around the distal heme pocket. In all four structures the asparagine68 side chai n lies almost parallel to the plane of the heme with its amide group d irected toward the back of the distal heme pocket. The coordinated wat er molecule in the aquomet form and the bound oxygen in the oxy form c an form hydrogen-bonding interactions with both the Asn(68) amide grou p and the imidazole side chain of His(64). Surprisingly, in the carbon monoxy form of the V68N mutant, the histidine(64) side chain has swun g completely out the distal pocket, its place being taken by two order ed water molecules. Overall, these functional and structural results s how that the asparagine68 side chain (i) forms a strong hydrogen bond with bound oxygen through its -NH2 group but (ii) sterically hinders t he approach of ligands to the iron from within the distal heme pocket.