Thermodynamic and Biophysical Characterization of Cytochrome P450 BioI from Bacillus subtilis ² 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, 3 and Andrew W. Munro* ,‡ Department of Biochemistry, UniVersity of Leicester, The Adrian Building, UniVersity Road, Leicester LE1 7RH, U.K., School of Chemical Sciences, UniVersity of East Anglia, Norwich, NR4 7TJ, U.K., Department of Pure & Applied Chemistry, UniVersity of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, U.K., Centre for Chemical Biology, Department of Chemistry, UniVersity of Sheffield, Dainton Building, Brook Hill, Sheffield, S3 7HF, U.K., and Biotechnology Research and DeVelopment, DSM Nutritional Products, P.O. Box 3255, Building 203/20A, CH-4002, Basel, Switzerland ReceiVed April 29, 2004; ReVised Manuscript ReceiVed July 23, 2004 ABSTRACT: Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an 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 binding features of P450 BioIsspecifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K d values in the range 0.1-160 µM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C 3 position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI )-330 ( 1 mV; for BioI as purified from E. coli (mixed-spin) ) 228 ( 2 mV; for palmitoleic acid-bound BioI )-199 ( 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure. The cytochromes P450 are a superfamily of heme b- containing monooxygenase enzymes found in all major life forms (1, 2). Their activity is central to synthesis and interconversions of numerous lipids, steroids and polyketides, and mammalian hepatic microsomal P450s are the front line of defense in the body against drugs and other xenobiotics (3). Eukaryotic P450s are, almost without exception, integral membrane proteins, and this has been a major factor hampering the determination of atomic structures for these proteins. However, genetic manipulation of microsomal CYP2C5 allowed production of a soluble monomeric form of the enzyme and enabled its crystallization and structural elucidation (4). Structures of other human P450s have been determined recently by industrial and academic consortia using similar strategies. By contrast, bacterial P450s are generally soluble enzymes, simplifying their expression and purification. The structures of several bacterial P450s have been solved, including forms from pathogenic bacteria (5) and P450s involved in catabolism of environmental pollutants (6, 7) and synthesis of antibiotics (8). In Bacillus subtilis, the genome sequence revealed eight distinct P450 systems (Table 1) (9). Two of these (CYP102A2 and CYP102A3) are homologues of the well-characterized B. megaterium enzyme flavocytochrome P450 BM3 (10, 11). P450 BM3 is a fatty acid hydroxylase in which the P450 is fused to its redox partner, a flavin adenine dinucleotide (FAD)- and flavin mononucleotide (FMN)-containing cyto- ² These studies were funded by the Biotechnology and Biological Sciences Research Council (BBSRC, U.K.) and through a BBSRC studentship award to R.J.L. cofunded by DSM Nutritional Products. * Author for correspondence: e-mail awm9@le.ac.uk; phone 0044 116 252 3464; fax 0044 116 252 3369. ‡ University of Leicester. § University of East Anglia. ⊥ University of Strathclyde. | University of Sheffield. 3 DSM Nutritional Products. 12410 Biochemistry 2004, 43, 12410-12426 10.1021/bi049132l CCC: $27.50 © 2004 American Chemical Society Published on Web 09/04/2004