Probing the open state of cytochrome P450cam with ruthenium-linker substrates

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-Å deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates.

[1]  J Berendzen,et al.  The catalytic pathway of cytochrome p450cam at atomic resolution. , 2000, Science.

[2]  A. West,et al.  THEORETICAL STUDIES OF THE SUPRAMOLECULAR SYNTHON BENZENE · · · HEXAFLUOROBENZENE , 1997 .

[3]  R. Raag,et al.  Crystal structures of cytochrome P-450CAM complexed with camphane, thiocamphor, and adamantane: factors controlling P-450 substrate hydroxylation. , 1991, Biochemistry.

[4]  R. Ornstein,et al.  Dramatic differences in the motions of the mouth of open and closed cytochrome P450BM‐3 by molecular dynamics simulations , 1995, Proteins.

[5]  G. Lepesheva,et al.  Conformational dynamics and molecular interaction reactions of recombinant cytochrome p450scc (CYP11A1) detected by fluorescence energy transfer. , 1999, Biochimica et biophysica acta.

[6]  T. Poulos,et al.  Structure of cytochrome P450eryF involved in erythromycin biosynthesis , 1995, Nature Structural Biology.

[7]  T. Poulos,et al.  The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid , 1997, Nature Structural Biology.

[8]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[9]  S. Fischer,et al.  Binding of buried structural water increases the flexibility of proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Ema,et al.  Lipase-catalyzed transesterification of 2-hydroxy-2-(pentafluorophenyl)acetonitrile leading to (1R,2R)- and (1S,2S)-bis(pentafluorophenyl)ethane-1,2-diol , 2000, The Journal of organic chemistry.

[11]  Ortiz de Montellano,et al.  Cytochrome P-450: Structure, Mechanism, and Biochemistry , 1986 .

[12]  F. Guengerich,et al.  Cooperativity in oxidations catalyzed by cytochrome P450 3A4. , 1997, Biochemistry.

[13]  G. Moore,et al.  Influence of polyfluorination of the phenylalanine ring of angiotensin II on conformation and biological activity. , 1991, Biochimica et biophysica acta.

[14]  D. Nelson,et al.  Cytochrome P450 and the individuality of species. , 1999, Archives of biochemistry and biophysics.

[15]  H. Arakawa,et al.  Proton Delivery in NO Reduction by Fungal Nitric-oxide Reductase , 2000, The Journal of Biological Chemistry.

[16]  S. Prasad,et al.  Binding of camphor to Pseudomonas putida cytochrome P450cam: steady‐state and picosecond time‐resolved fluorescence studies , 2000, FEBS letters.

[17]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[18]  C. Di Primo,et al.  A critical role of protein‐bound water in the catalytic cycle of cytochrome P‐450 camphor , 1992, FEBS letters.

[19]  R. Wade,et al.  How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms. , 2000, Journal of molecular biology.

[20]  J Deisenhofer,et al.  Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. , 1993, Science.

[21]  Chu-Young Kim,et al.  Contribution of Fluorine to Protein−Ligand Affinity in the Binding of Fluoroaromatic Inhibitors to Carbonic Anhydrase II , 2000 .

[22]  R. Poorman,et al.  Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors. , 1999, Journal of medicinal chemistry.

[23]  W. Pryor.  Cytochrome P450: Structure, mechanism, and biochemistry , 1996 .

[24]  David J. Schuller,et al.  Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus * , 2000, The Journal of Biological Chemistry.

[25]  J Deisenhofer,et al.  Crystal structure and refinement of cytochrome P450terp at 2.3 A resolution. , 1994, Journal of molecular biology.

[26]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[27]  Slobodan Petar Rendic,et al.  Human cytochrome P450 enzymes: a status report summarizing their reactions, substrates, inducers, and inhibitors. , 1997, Drug metabolism reviews.

[28]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[29]  J. Sussman,et al.  Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica. , 2000, Journal of molecular biology.

[30]  I. Tanaka,et al.  Crystal structure of nitric oxide reductase from denitrifying fungus Fusarium oxysporum , 1997, Nature Structural Biology.

[31]  T. Poulos,et al.  Fatty acid metabolism, conformational change, and electron transfer in cytochrome P-450BM-3 , 1999 .

[32]  F. Guengerich,et al.  Cytochrome P-450 3A4: regulation and role in drug metabolism. , 1999, Annual review of pharmacology and toxicology.

[33]  B C Finzel,et al.  The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. , 1985, The Journal of biological chemistry.

[34]  D E McRee,et al.  Mammalian microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding and functional diversity. , 2000, Molecular cell.

[35]  C. R. Patrick,et al.  A Molecular Complex of Benzene and Hexafluorobenzene , 1960, Nature.

[36]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[37]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[38]  D. McRee,et al.  A visual protein crystallographic software system for X11/Xview , 1992 .

[39]  M. Sanner,et al.  Reduced surface: an efficient way to compute molecular surfaces. , 1996, Biopolymers.

[40]  C. Di Primo,et al.  Conformational dynamics of cytochrome P-450cam as monitored by photoacoustic calorimetry. , 1993, Biochemistry.

[41]  A. Fitch,et al.  Structure of the Lowest Temperature Phase of the Solid Benzene–Hexafluorobenzene Adduct† , 1992 .

[42]  C. Di Primo,et al.  Origin of the photoacoustic signal in cytochrome P-450cam: role of the Arg186-Asp251-Lys178 bifurcated salt bridge. , 1997, Biochemistry.

[43]  T. Poulos,et al.  Modeling protein-substrate interactions in the heme domain of cytochrome P450(BM-3). , 1994, Acta crystallographica. Section D, Biological crystallography.

[44]  T. Poulos,et al.  Crystal structure of cytochrome P450 14α-sterol demethylase (CYP51) from Mycobacterium tuberculosis in complex with azole inhibitors , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[45]  H. Gray,et al.  Optical detection of cytochrome P450 by sensitizer-linked substrates. , 1999, Proceedings of the National Academy of Sciences of the United States of America.