Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants

Abstract

The effect of 1R-camphor on the conformational stability of the heme active site of cytochrome P450cam has been investigated. The absorption spectra of the heme moiety showed the presence of two hitherto unknown intermediates formed at low urea concentrations or during small temperature perturbations. The corresponding thermodynamic parameters were obtained by global fitting of the experimental data to a generalized sequential unfolding model at different wavelengths, which showed that the active conformation of the enzyme is stabilized by binding of the substrate at the active site. Circular-dichroism spectra of the enzyme in the visible- and far-UV region were studied to identify the critical range of denaturant concentration and the temperature at which the tertiary structure around the heme center was affected with almost no change in the secondary structure of the enzyme. This critical range of urea concentration was 0-2.8 M in the presence of camphor and 0-1.5 M in the absence of camphor. The tertiary structure of the enzyme was found to undergo conformational change in the temperature range 20-60 degrees C in the presence of the substrate and 20-47 degrees C in its absence. The spectral assignments of the intermediate species of the heme active site with the intact secondary structure of the enzyme were made by deconvolution of the Soret absorption spectra, and the results were analyzed to determine stabilization of the heme active-site geometry by 1R-camphor. Results showed that subtle conformational changes due to melting of the tertiary contacts in the active site lead to formation of intermediates which are coordinatively similar to the native enzyme. Analogous intermediate species might be responsible for leakage in the redox catalytic cycle of the enzyme.

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

“…[10][11][12][13] In the denaturing process, the native cysteine coordination environment around the heme is changed, which is characterized by the conversion of P450 to its P420 form. The P420 form is termed according to the fact that its ferrous-CO absorbance maximizes at ca.…”

“…It is also assumed that the cytochrome P420 is an altered form of P450, in which one of the axial ligands is replaced by another chemical compound or amino acid residue, but the heme is still attached to the apoprotein. 10,[14][15][16] However, the P420 forms are not characterized by uniform and well-defined protein structures under different conditions. 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. The β-bulge and its flanking region consist of 31 amino acid residues and is long enough for flexible movement in interaction with heme.…”

“…Although sharing a common overall fold and topology, 6 the amino acids sequences of mammalian P450s are very different from those of bacterial species, especially the sequences of the active site cavity and the β-bulge region where the axial ligand cysteine locates. 10,13,20,[22][23][24] Hence, the coordination structures of the mammalian P420 forms may have their unique traits.…”

“…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.…”