ON THE ORIGIN OF HEME ABSORPTION-BAND SHIFTS AND ASSOCIATED PROTEIN STRUCTURAL RELAXATION IN MYOGLOBIN FOLLOWING FLASH-PHOTOLYSIS

The role of the protein structural change monitored by absorption band shifts following flash photolysis of CO from myoglobin is discussed i n terms of structure-function relationships. Evidence is presented tha t the Soret band shift does not depend primarily on the covalent linka ge of the heme iron to the protein by using the mutation H93G(L) in wh ich the proximal histidine 93 is replaced by glycine and an exogenous ligand L, which coordinates the heme iron but is not covalently bound to the globin. While CO rebinding kinetics depend strongly on the natu re of the exogenous ligand L in H93G(L), the magnitude and time evolut ion of the Soret band shift in a viscous buffer on the nanosecond time scale are hardly perturbed in all cases studied. Comparison of the So ret band and charge transfer Band III shifts demonstrates that both ha ve a similar time dependence on the nanosecond to microsecond time sca le following flash photolysis in viscous solvents. We conclude that th e nonexponential kinetics of protein relaxation probed by transient ab sorption band position shifts involves primarily distal coordinates pr ior to ligand escape. This result agrees with earlier measurements of Soret band shifts in distal pocket mutants of myoglobin (1). We sugges t that the band shifts are primarily a response to changes in the elec trostatic field around the heme (a transient Stark shift) associated w ith changes in protein structure that occur following ligand photodiss ociation.