Cytochrome P450 Biol (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that Biol 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 BioI-specifically 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 muM). Sigmoidal binding curves for titration of Biol with azole drugs suggests a cooperative process in this case. Biol 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 Biol = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound Biol = - 199 2 mV). Extraction of bound substrate-like molecule from purified Biol indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the Biol 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 Biol secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the Biol 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 Biol structure.