Role of substrate on the conformational stability of the heme active site of cytochrome P450cam: effect of temperature and low concentrations of denaturants.
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.
Murugan
R
R
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba, 400005, Mumbai, India.
Mazumdar
Shyamalava
S
D001665:Binding Sites; D002164:Camphor / Q000737:chemistry; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry; D008024:Ligands; D011487:Protein Conformation; D011489:Protein Denaturation; D013057:Spectrum Analysis; D013696:Temperature; D013816:Thermodynamics
D006420:Hemeproteins; D008024:Ligands; D002164:Camphor; D019475:Camphor 5-Monooxygenase