Conformational states in denaturants of cytochrome c and horseradish peroxidases examined by fluorescence and circular dichroism.
Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase [CCP(MI)] and horseradish peroxidase (HRP) in their ferric forms. CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl. CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl. The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic. The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl. Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data. The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding. Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture. The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI). Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.
Tsaprailis
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Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Chan
D W
DW
English
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AM
D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006735:Horseradish Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003574:Cytochrome c Group; D011994:Recombinant Proteins; D004492:Edetic Acid; D006735:Horseradish Peroxidase; D004229:Dithiothreitol