Imidazolyl Carboxylic Acids As Mechanistic Probes of

Biochemistry 1998, 37, 15799-15807

15799

Imidazolyl Carboxylic Acids As Mechanistic Probes of Flavocytochrome P-450 BM3† M. A. Noble,§ L. Quaroni,‡ George D. Chumanov,‡ K. L. Turner,§ S. K. Chapman,§ R. P. Hanzlik,| and A. W. Munro*,§ Department of Chemistry, The UniVersity of Edinburgh, The King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K., Department of Chemistry, Iowa State UniVersity, Ames, Iowa 50011, and Department of Medicinal Chemistry, UniVersity of Kansas, Lawrence, Kansas 66045 ReceiVed February 26, 1998; ReVised Manuscript ReceiVed July 20, 1998 ABSTRACT: 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 Km and decreasing the kcat 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 (Kic ) 0.9 µM, Kiu ) 5.7 µM), while 12-(imidazolyl)dodecanoic acid (Kic ) 1.35 µM, Kiu ) 6.9 µM) was superior to 11-(imidazolyl)undecanoic acid (Kic ) 7.5 µM, Kiu ) 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 Kd to be determined spectrophotometrically, but this value is