Carbonmonoxy horseradish peroxidase as a function of pH and substrate: Influence of local electric fields on the optical and infrared spectra

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were mo nitored as a function of pH and substrate binding. The analyses of experime ntal results together with semiempirical calculations show that the CO-porp hyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external pertur bations with different symmetry dependencies. In this way, the complex is n onisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6 .0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrat ional stretch frequency is at 1903 cm(-1). Addition of the substrates benzo hydroxamic acid or naphthohydroxamic acid produces a split of similar to 32 0 cm(-1) in the Q(0,0) absorption band that is clearly evident at <100 K an d shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also ca uses a split in the Q(0,0) optical band and elicits a shift in <nu>(CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local elec tric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the dis tal histidine, although other effects are also considered. The larger effec t on the Q(0,0) band with substrate at low pH and the shift of nu (CO) at h igh pH can be rationalized by the directionality of the field and the orien tation dependence of dipolar interactions.