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

Authors
Type
Published Article
Journal
Biochemistry
Publication Date
Volume
40
Issue
12
Pages
3483–3491
Identifiers
PMID: 11297414
Source
Medline
License
Unknown

Abstract

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, 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 vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of approximately 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at < 100 K and shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also causes 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 electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu(CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions.

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