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 in terms of structure-function relationships. Evidence is presented that the Soret band shift does not depend primarily on the covalent linkage of the heme iron to the protein by using the mutation H93G(L) in which 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 nature of the exogenous ligand L in H93G(L), the magnitude and time evolution of the Soret band shift in a viscous buffer on the nanosecond time scale are hardly perturbed in all cases studied. Comparison of the Soret band and charge transfer Band III shifts demonstrates that both have a similar time dependence on the nanosecond to microsecond time scale following flash photolysis in viscous solvents. We conclude that the nonexponential kinetics of protein relaxation probed by transient absorption band position shifts involves primarily distal coordinates prior to ligand escape. This result agrees with earlier measurements of Soret band shifts in distal pocket mutants of myoglobin (1). We suggest that the band shifts are primarily a response to changes in the electrostatic field around the heme (a transient Stark shift) associated with changes in protein structure that occur following ligand photodissociation.