STRUCTURAL AND FUNCTIONAL-EFFECTS OF APOLAR MUTATIONS OF THE DISTAL VALINE IN MYOGLOBIN

High-resolution structures of the aquomet, deoxy: and CO forms of Ala6 8, Ile68, Leu68, and Phe68 sperm whale myoglobins have been determined by X-ray crystallography. These 12 new structures, plus those of wild -type myoglobin, have been used to interpret the effects of mutations at position 68 and the effects of cobalt substitution on the kinetics of O-2, CO, and NO binding. Molecular dynamics simulations based on cr ystal structures have provided information about the time-dependent be havior of photolyzed ligands for comparison with picosecond geminate r ecombination studies. The Val68-->Ala mutation has little effect on th e structure and function of myoglobin. In Ala68 deoxymyoglobin, as in the wild-type protein, a water molecule hydrogen-bonded to the N-epsil on atom of the distal histidine restricts ligand binding and appears t o be more important in regulating the function of myoglobin than direc t steric interactions between the Ligand and the C-gamma atoms of the native valine side-chain. This distal pocket water molecule is displac ed by the larger side-chains at position 68 in the crystal structures of Leu68 and Ile68 deoxymyoglobins. The Leu68 side-chain can rotate ab out its C-alpha-C-beta and C-beta-C-gamma bonds to better accommodate bound ligands, resulting in net increases in overall association rate constants and affinities due to the absence of the distal pocket water molecule. However, the flexibility of Leu68 makes simulation of picos econd NO recombination difficult since multiple starting conformations are possible. In the case of Ile68, rotation of the substituted side- chain is restricted due to branching at the beta carbon, and as a resu lt, the delta methyl group is located close to the iron atom in both t he deoxy and liganded structures. The favorable effect of displacing t he distal pocket water molecule is offset by direct steric hindrance b etween the bound ligand and the terminal carbon atom of the isoleucine side-chain, resulting in net decreases in affinity for all three liga nds and inhibition of geminate recombination which is reproduced in th e molecular dynamics simulations. In Phe68 myoglobin, the benzyl side- chain is pointed away from the ligand binding site, occupying a region in the back of the distal pocket. As in wild-type and Ala68 myoglobin s, a well-defined water molecule is found hydrogen bonded to the dista l histidine in Phe68 deoxymyoglobin. This water molecule, in combinati on with the large size of the benzyl side-chain, markedly reduces the speed and extent of ligand movement into the distal pocket. Ligand mov ement away from the iron atom is also reduced dramatically by the Phe6 8 side-chain, causing large and rapid geminate recombination phases fo r all Ligands.