Thermodynamic and Biophysical Characterization of Cytochrome P450 BioI from Bacillus subtilis
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- 作者：Rachel J. Lawson ; David Leys ; Michael J. Sutcliffe ; Carol A. Kemp ; Myles R. Cheesman ; Susan J. Smith ; John Clarkson ; W. Ewen Smith ; Ihtshamul Haq ; John B. Perkins ; and Andrew W. Munro
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：October 5, 2004
- 年：2004
- 卷：43
- 期：39
- 页码：12410 - 12426
- 全文大小：279K
- 年卷期：v.43,no.39(October 5, 2004)
- ISSN：1520-4995
文摘
Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages ofbiotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also performan acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G.,Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok,J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel bindingfeatures of P450 BioI-specifically that it binds steroids (including testosterone and progesterone) andpolycyclic azole drugs with similar affinity to that for fatty acids (Kd values in the range 0.1-160 
M).Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in thiscase. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH ofthe buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content).Steroids containing a carbonyl group at the C3 position induce a shift in heme iron spin-state equilibriumtoward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electronparamagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from opticaltitrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reductionpotential becomes progressively more positive as the proportion of high-spin heme iron increases (potentialfor low-spin BioI = -330 ± 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 ± 2 mV; forpalmitoleic acid-bound BioI = -199 ± 2 mV). Extraction of bound substrate-like molecule from purifiedBioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioIprotein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonanceRaman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotropeguanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is presentbetween Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered byaddition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.
M).Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in thiscase. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH ofthe buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content).Steroids containing a carbonyl group at the C3 position induce a shift in heme iron spin-state equilibriumtoward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electronparamagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from opticaltitrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reductionpotential becomes progressively more positive as the proportion of high-spin heme iron increases (potentialfor low-spin BioI = -330 ± 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 ± 2 mV; forpalmitoleic acid-bound BioI = -199 ± 2 mV). Extraction of bound substrate-like molecule from purifiedBioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioIprotein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonanceRaman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotropeguanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is presentbetween Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered byaddition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.