Functional implications of the proximal hydrogen-bonding network in myoglobin: a resonance Raman and kinetic study of Leu89, Ser92, His97, and F-helix swap mutants.

Abstract

Resonance Raman spectra have been obtained for both the equilibrium deoxy derivative and the 10 ns photoproduct of the CO derivative of several mutants of sperm whale myoglobin. The particular mutations on the F-helix were chosen to expose the role of the proximal hydrogen-bonding network in maintaining the position of the heme, the proximal histidine, and the heme-7-propionate. In each mutant, one or more hydrogen bonds are altered or eliminated. A careful comparison of the spectra from the equilibrium and transient five coordinate species indicates that the tertiary relaxation after photodissociation is nearly complete within 10 ns, as is the case in the WT protein. The iron-proximal histidine stretching mode (nu(Fe-His)) and several low-frequency propionate-sensitive modes in the Raman spectra reveal the impact of specific disruptions in the hydrogen-bonding network on the heme pocket geometry. Two categories of perturbation are observed with respect to nu(Fe-His): (1) a shift in the peak frequency without a change in line shape and (2) changes in the overall line shape which may or may not be accompanied by a frequency shift. The alterations in the nu(Fe-His) band are interpreted as arising from conformational heterogeneity and local geometrical changes within the pocket, including movement of the heme group, and are discussed in terms of changes in the population distribution as revealed via a curve-fitting analysis. None of the frequency shifts in the nu(Fe-His) band are as large as that reported for the His93Gly(imidazole) mutant, suggesting that the covalent linkage between the heme and His93 plays a crucial role in maintaining the geometry of the proximal pocket. Molecular modeling indicates that the nu(Fe-His) frequency shifts observed in the present study originate from changes in the His93 imidazole ring azimuthal angle. The systematic variations in the interactions of the heme-7-propionate in the mutants have exposed several properties of the propionate-sensitive Raman bands. The frequencies of nu9 (the 240 cm-1 shoulder on the nu(Fe-His) band) and delta(cbetacccd) at approximately 370 cm-1 appear to be correlated. A decrease in hydrogen-bond strength to this propionate in response to changes in stereochemistry or degree of disorder is associated with a decrease in the frequency of both nu9 and delta(cbetacccd). The mutations that cause a weakening of the hydrogen bonding to the heme-7-propionate also result in changes in nu(Fe-His) which are interpreted as evidence that this propionate participates in the anchoring of the heme within the heme pocket. Changes in gamma7 at approximately 300 cm-1, gamma6 at approximately 335 cm-1, and nu8 at approximately 342 cm-1 are discussed in terms of pocket disorder. A titration from pH 5.1 to 7.4 suggests that His97 is protonated in the WT protein by pH 5.1. Geminate-rebinding studies on these mutants indicate that disruption of the hydrogen-bonding network has only modest effects on ligand-binding kinetics, suggesting that the role of the hydrogen-bonding network may be one of maintaining heme pocket stability rather than of specific protein function.