Refolding processes of cytochrome P450cam from ferric and ferrous acid forms to the native conformation. Formations of folding intermediates with non-native heme coordination state.
Changes in heme coordination state and protein conformation of cytochrome P450(cam) (P450(cam)), a b-type heme protein, were investigated by employing pH jump experiments coupled with time-resolved optical absorption, fluorescence, circular dichroism, and resonance Raman techniques. We found a partially unfolded form (acid form) of ferric P450(cam) at pH 2.5, in which a Cys(-)-heme coordination bond in the native conformation was ruptured. When the pH was raised to pH 7.5, the acid form refolded to the native conformation through a distinctive intermediate. Formations of similar acid and intermediate forms were also observed for ferrous P450(cam). Both the ferric and ferrous forms of the intermediate were found to have an unidentified axial ligand of the heme at the 6th coordination sphere, which is vacant in the high spin ferric and ferrous forms at the native conformation. For the ferrous form, it was also indicated that the 5th axial ligand is different from the native cysteinate. The folding intermediates identified in this study demonstrate occurrences of non-native coordination state of heme during the refolding processes of the large b-type heme protein, being akin to the well known folding intermediates of cytochromes c, in which c-type heme is covalently attached to a smaller protein.
Egawa
Tsuyoshi
T
Department of Biochemistry, School of Medicine, Keio University, Shinanomachi, Shinjuku-ku, Tokyo 160-8582. tegawaw@aecom.yu.edu
Hishiki
Takako
T
Ichikawa
Yusuke
Y
Kanamori
Yasukazu
Y
Shimada
Hideo
H
Takahashi
Satoshi
S
Kitagawa
Teizo
T
Ishimura
Yuzuru
Y
D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays
D008024:Ligands; D006418:Heme; D007501:Iron; D019475:Camphor 5-Monooxygenase; D003545:Cysteine