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

omega-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 mu M, K-iu = 5.7 mu M), while 12-(imidazolyl)dodecanoic acid (K-ic = 1.35 mu M, K-iu = 6.9 mu M) was superior to 11-(imidazolyl)undecanoic acid (K-ic = 7.5 mu M, K-iu = 16 mu M). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 mu M (C12 azole) and 27 mu M (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