NITRIC-OXIDE RECOMBINATION TO DOUBLE MUTANTS OF MYOGLOBIN - ROLE OF LIGAND DIFFUSION IN A FLUCTUATING HEME POCKET

Picosecond recombination of nitric oxide to the double mutants of myog lobin, His64Gly.Val68Ala and His64Gly.Val68Ile, at E7 and E11, has bee n studied experimentally and by computation. It is shown that distal r esidues have a profound effect on NO recombination. Recombination in t he mutants may be explained in terms of fluctuating free volume and st ructure of the heme pocket. The double mutants provide insight into th e effects of free volume and steric hindrance on rates of ligand rebin ding following photolysis. Water molecules of the first solvation shel l replace surface residues deleted by mutation and can block apparent holes in the protein structure. Thus, water molecules extend the time required for ligands to escape significantly to a nanosecond time scal e, which is much longer than would be expected for an open heme pocket . Both nearly exponential (G64A68) and markedly nonexponential (native and G64I68) kinetics are observed, a result at variance with expectat ion from the model of Petrich et al. [Petrich, J. W., Lambry, J. C., K uczera, K., Karplus, M., Poyart, C., and Martin, J. L. (1991) Biochemi stry 30, 3975-3987], which attributes nonexponential kinetics to proxi mal effects.