Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P- 450 BM3

ω-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the K(m) and decreasing the k(cat) for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (K(ic)= 0.9 μM, K(iu) = 5.7 μM), while 12- (imidazolyl)dodecanoic acid (K(ic)= 1.35 μM, K(iu) = 6.9 μM) was superior to 11-(imidazolyl)undecanoic acid (K(ic)= 7.5 μM, K(iu) = 16 μM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 μM (C12 azole) and 27 μM (C11 azole). The binding of 10(imidazolyl)decanoic acid was too tight for an absolute K(d) to be determined spectrophotometrically, but this value is