Resolution of two substrate-binding sites in an engineered cytochrome P450eryF bearing a fluorescent probe.
To elucidate the mechanisms of cooperativity of cytochrome P450eryF an SH-reactive fluorescent probe was introduced close to the substrate-binding site. Cys-154, the only accessible cysteine, was eliminated by site-directed mutagenesis, and a novel cysteine was substituted for Ser-93 in the B'/C loop. S93C, C154A, C154S, S93C/C154A, and S93C/S154C were characterized in terms of affinity for 1-pyrenebutanol (1-PB), cooperativity, and ionic-strength dependence of the 1-PB-induced spin shift. S93C/C154S retains the key functional properties of the wild-type, and modification by three different SH-reactive probes had little effect on the characteristics of the enzyme. The labeled proteins exhibited fluorescence resonance energy transfer from 1-PB to the label, which allowed us to resolve two substrate-binding events, and to determine the corresponding KD values (KD1 = 1.2 +/- 0.2 microM, KD2 = 9.4 +/- 0.8 microM). Using these values for analysis of the substrate-induced spin transition, we demonstrate that the interactions of P450eryF with 1-PB are consistent with a sequential binding mechanism, where substrate interactions at a higher-affinity site cause a conformational transition crucial for the binding of the second substrate molecule and subsequent spin shift. This transition is apparently associated with an important rearrangement of the system of salt links in the proximity of Cys-154.
Davydov
Dmitri R
DR
Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, Texas 77555-1031, USA. d.davydov@utmb.edu
Botchkareva
Alexandra E
AE
Davydova
Nadezhda E
NE
Halpert
James R
JR
D000495:Allosteric Site; D001426:Bacterial Proteins; D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000494:pharmacology*; D007477:Ions; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D011721:Pyrenes / Q000737:chemistry; D016930:Saccharopolyspora / Q000201:enzymology*; D012492:Salts / Q000494:pharmacology; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013379:Substrate Specificity
C463480:1-pyrenebutanol; D001426:Bacterial Proteins; D017931:DNA Primers; D005456:Fluorescent Dyes; D007477:Ions; D011721:Pyrenes; D012492:Salts; D012694:Serine; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C054444:eryF protein, Saccharopolyspora erythraea; D003545:Cysteine