Refolding Processes of Cytochrome P450cam from Ferric and Ferrous Acid Forms to the Native Conformation

Abstract

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.

Novel Conformational Transitions of Human Cytochrome P450 2C8 during Thermal and Acid‐induced Unfolding

“…The absorption peaks of the Fe 3＋ -P420 2C8 acidic form (λ max , 383, 542 and 640 nm) are not typical of Cys -, His, Met, or Pro-ligated ferric hemes, 17,48 instead they are similar to the absorption spectrum of free hemin chloride dissolved in acetone (λ max , 382, 512, 540 and 640 nm). 50 Furthermore, the intensity of the 383 nm absorption band at pH 3.0 was half of 416 nm band's intensity at pH 7.4, while the extinction coefficient of the hemin (ε 385 nm , 58.4 mL•mol -1 •cm -1 ) was also half of the native Fe 3＋ -P450 2C8's at 416 nm (ε 416 nm , 108 mL•mol -1 •cm -1 ).…”

“…In contrast, the protein could successfully refold in the pH jump experiment of substrate-binding P450 cam . 17 Therefore, we suggested that the escape of the heme from the heme pocket might be a reason of irreversibility of the Fe 3＋ -P420 2C8 acidic form.…”

“…Egawa et al 17 have suggested that the unfolded P450 cam is capable of returning to its native coordination state by forming the folding intermediates of non-native coordination. Such intermediates might be necessary for the folding of P450 cam in vivo.…”

“…Structural details of the P420 heme pockets may be different. 10,[17][18][19] Previous studies suggested that the heme axial ligand cysteine in the P420 cam form was replaced by another amino acid, maybe a histidine residue (His) located at the same loop region (the β-bulge). 10,13,20,21 Actually, P450 cam has a long loop prior to the β-bulge.…”

“…Most of previous unfolding/refolding studies were carried out on P450 cam , a soluble bacterial P450 comprising 414 amino acid residues. 10,11,[14][15][16][17][25][26][27] Some studies also used mammalian P450s (microsomal P450), 13,[28][29][30][31][32] however, the molecular details of these transitions had not been discussed in detail. In this paper, we studied human P450 2C8 to explore the P450 to P420 transition and its reversibility.…”