A model for effector activity in a highly specific biological electron transfer complex: the cytochrome P450(cam)-putidaredoxin couple.
The camphor hydroxylase cytochrome P450(cam) (CYP101) catalyzes the 5-exo hydroxylation of camphor in the first step of camphor catabolism by Pseudomonas putida. CYP101 forms a specific electron transfer complex with its physiological reductant, the Cys(4)Fe(2)S(2) ferredoxin putidaredoxin (Pdx). Pdx, along with other proteins and small molecules, has also been shown to be an effector for turnover by CYP101. Multidimensional nuclear magnetic resonance (NMR) techniques have been used to make extensive sequential (1)H, (15)N, and (13)C resonance assignments in CYP101 that permit a more complete characterization of the complex formed by CYP101 and Pdx. NMR-detected perturbations in CYP101 upon Pdx binding encompass regions of the CYP101 remote from the putative Pdx binding site, including in particular a region of the CYP101 molecule that has been implicated in substrate access to the active site via dynamical processes. A model for effector activity is proposed in which the primary role of the effector is to prevent uncoupling (formation of reduced oxo species without formation of hydroxycamphor) by enforcing conformations of CYP101 that prevent loss of substrate and/or intermediates prior to turnover. A secondary role could also be to enforce conformations that permit efficient proton transfer into the active site for coupled proton/electron transfer.
Department of Chemistry, Brandeis University, Waltham, Massachusetts 02454-9110, USA.
Susan Sondej
SS
Pochapsky
Thomas C
TC
Pochapsky
Julie W
JW
Wei
D000595:Amino Acid Sequence; D001665:Binding Sites; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D005288:Ferredoxins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C100137:putidaredoxin; D019475:Camphor 5-Monooxygenase