FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O.
Fourier transform infrared (FTIR) spectroscopy was employed to examine the thermal denaturation of the Fe(III), Fe(II), and Fe(II)-CO forms of cytochrome c peroxidase and horseradish peroxidase in phosphate buffer at pD 7.0. The amide I' regions of the deconvolved spectra are consistent with predominantly alpha-helical secondary structure around room temperature, but the alpha-helical absorption of the two peroxidases differs significantly; bands assigned to alpha-helical components occur at 1659 and 1649 cm-1 in horseradish peroxidase and at 1652 and 1637 cm-1 in cytochrome c peroxidase. The thermal denaturation mechanisms of the peroxidases also vary. All three forms of cytochrome c peroxidase retain their secondary structure up to 50 degrees C, when bands characteristic of aggregation (1616 and 1684 cm-1) appear in the amide I' region, and above 55 degrees C rapid loss of secondary structure is accompanied by enhanced aggregation. In horseradish peroxidase, on the other hand, the Fe(III) and Fe(II) states exhibit dissimilar denaturation mechanisms. Slow, gradual alteration of secondary structure is observed for Fe(III) horseradish peroxidase on heating, and polypeptide unfolding appears to be complete around 90 degrees C, without aggregation. In Fe(II) and Fe(II)-CO horseradish peroxidase, aggregation bands appear at approximately 55 degrees C, signaling the onset of denaturation. Frequency shifts in the v(CO) bands above room temperature reveal the conformational changes in the heme cavity precede global conformational changes in cytochrome c peroxidase but not in horseradish peroxidase. The reduction in amide II intensities, due to peptide H-D exchange on heating the peroxidases in D2O, indicates the formation above room temperature of partially unfolded states with increased solvent accessibility but intact secondary structures.
Holzbaur
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Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
English
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Ismail
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D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D003903:Deuterium; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006358:Hot Temperature; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins; D017550:Spectroscopy, Fourier Transform Infrared
D005290:Ferric Compounds; D005296:Ferrous Compounds; D011994:Recombinant Proteins; D003903:Deuterium; D006735:Horseradish Peroxidase; D003578:Cytochrome-c Peroxidase