Expression, purification and characterization of cytochrome P450 Biol: a novel P450 involved in biotin synthesis in Bacillus subtilis

The biol gene has been sub-cloned and over-expressed in Escherichia coli, a nd the protein purified to homogeneity. The protein is a cytochrome P450, a s indicated by its visible spectrum (low-spin haem iron Soret band at 419 n m) and by the characteristic carbon monoxide-induced shift of the Soret ban d to 448 nm in the reduced form. N-terminal amino acid sequencing and mass spectrometry indicate that the initiator methionine is removed from cytochr ome P450 BioI and that the relative molecular mass is 44,732 Da, consistent with that deduced from the gene sequence. SDS-PAGE indicates that the prot ein is homogeneous after column chromatography on DE-52 and hydroxyapatite, followed by FPLC on a quaternary ammonium ion-exchange column (Q-Sepharose ). The purified protein is of mixed spin-state by both electronic spectrosc opy and by electron paramagnetic resonance [g values=2.41, 2.24 and 1.97/1. 91 (low-spin) and 8.13, 5.92 and 3.47 (high-spin)]. Magnetic circular dichr oism and electron paramagnetic resonance studies indicate that P450 BioI ha s a cysteine-ligated b-type haem iron and the near-IR magnetic circular dic hroism band suggests strongly that the sixth ligand bound to the haem iron is water. Resonance Raman spectroscopy identifies vibrational signals typic al of cytochrome P450, notably the oxidation state marker nu (4) at 1373 cm (-1) (indicating ferric P450 haem) and the splitting of the spin-state mark er nu (3) into two components (1503 cm(-1) and 1488 cm(-1)), indicating cyt ochrome P450 BioI to be a mixture of high- and low-spin forms. Fatty acids were found to bind to cytochrome P450 BioI, with myristic acid (K-d=4.18 +/ -0.26 muM) and pentadecanoic acid (K-d=3.58 +/-0.54 muM) having highest aff inity. The fatty acid analogue inhibitor 12-imidazolyldodecanoic acid bound extremely tightly (K-d<1 M<mu>), again indicating strong affinity for fatt y acid chains in the P450 active site. Catalytic activity was demonstrated by reconstituting the P450 with either a soluble form of human cytochrome P 450 reductase, or a Bacillus subtilis ferredoxin and E. coli ferredoxin red uctase. Substrate hydroxylation at the omega -terminal position was demonst rated by turnover of the chromophoric fatty acid para-nitrophenoxydodecanoi c acid, and by separation of product from the reaction of P450 BioI with my ristic acid.