Structural analyses combined with small-angle X-ray scattering reveals that the retention of heme is critical for maintaining the structure of horseradish peroxidase under denaturing conditions.

Abstract

We analyzed the structure of horseradish peroxidase (HRP) under denaturing conditions of 9M urea or 6M guanidine hydrochloride (GdnHCl). Far-UV circular dichroism (CD) spectra indicated the existence of native-like secondary structure of holo-HRP in 9M urea. In addition, slight changes in near-UV and Soret region CD spectra of holo-HRP in 9M urea suggest that the tertiary structure of holo-HRP and the binding of heme remain partially intact in this condition. A transition in the thermal unfolding transition curve of holo-HRP in 9M urea indicated the existence of a considerable amount of secondary structure. However, no secondary structure, tertiary structure, or interaction between heme and HRP were observed in holo-HRP in 6M GdnHCl. Small-angle X-ray scattering indicated that although distal and proximal domains of holo-HRP in 9M urea might be partially unfolded, the central region that contains the heme might maintain its tertiary structure. Our results suggest that retention of the heme is essential for maintenance of the structure of HRP under highly denaturing conditions.