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Chemical Probes of Cytochrome P450 Structure

  • Chapter
Cytochrome P450

Part of the book series: Handbook of Experimental Pharmacology ((HEP,volume 105))

Abstract

Chemical modification of proteins is based on the fact that some amino acid side chains of proteins are accessible and, under specific conditions, are reactive enough to bind the probe (for detailed information see, e.g., Means and Feeney 1971). The main problem is of causing alterations of the protein structure when introducing the probe such as: (1) local effects (short-range steric and noncovalent binding interactions, including salt bridges, H-bonds, hydrophobic and van der Waals interactions as well as local conformation changes); (2) global effects (overall changes of conformation or solvation that may affect, or be propagated throughout, the molecule as a whole), and (3) specific long-range effects (these might include specific pathways whereby structural modifications at a site are transmitted to another site) (Ackers and Smith 1985). This is a problem common not only to methods introducing probes, but also to methods changing the primary structure of proteins (de novo and semisynthesis with amino acid exchanges, site-directed mutagenesis). That is why one has to be very careful in performing and interpreting modification studies on proteins. Nevertheless, chemical probes have been widely used to study the structure and function of cytochromes P450 as well as functionally linked protein-protein interactions in cytochrome P450 systems. The broad interest in using this method is due to the fact that the overwhelming number of cytochromes P450 so far isolated are membrane bound (Nebert et al. 1991) and therefore it is difficult to crystallize them so that direct structural information on these cytochromes P450 has so far not been available.

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References

  • Ackers GK, Smith FR (1985) Effects of site-specific amino acid modification on protein interactions and biological function. Annu Rev Biochem 54: 597–629

    Article  PubMed  CAS  Google Scholar 

  • Adamovich TB, Pikuleva IA, Chashchin VL, Usanov SA (1989) Selective chemical modification of cytochrome P-450scc lysine residues. Identification of lysines involved in the interaction with adrenodoxin. Biochim Biophys Acta 996: 247–253

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R, Gunsalus IC (1985) Heterologous reconstitution of cytochrome P-450LM2 activity with bacterial electron transfer systems. In: Vereczky L, Magyar K (eds) Cytochrome P-450, biochemistry, biophysics and induction. Akademia Kiado, Budapest, pp 159–162

    Google Scholar 

  • Bernhardt R, Ngoc Dao NT, Stiel H, Schwarze W, Friedrich J, Janig GR, Ruckpaul K (1983) Modification of cytochrome P-450 with fluorescein isothiocyanate. Biochim Biophys Acta 745: 140–148

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R, Makower A, Janig GR, Ruckpaul K (1984) Selective chemical modification of a functionally linked lysine in cytochrome P-450LM2. Biochim Biophys Acta 785: 186–190

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R, Pommerening K, Ruckpaul K (1987) Modification of carboxyl groups on NADPH-cytochrome P-450 reductase involved in binding of cytochromes c and P-450LM2. Biochem Int 14: 823–832

    PubMed  CAS  Google Scholar 

  • Bernhardt R, Kraft R, Otto A, Ruckpaul K (1988) Electrostatic interactions between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase. Biomed Biochim Acta 47: 581–592

    PubMed  CAS  Google Scholar 

  • Bernhardt R, Kraft R, Ruckpaul K (1989a) A simple determination of the sideness of the NH2-terminus in the membrane-bound cytochrome P-450LM2. Biochem Int 17: 1143–1150

    Google Scholar 

  • Bernhardt R, Stiel H, Ruckpaul K (1989b) Distance between lysine 384 and heme of cytochrome P-450LM2 (P-450IIB4) studied by fluorescence energy transfer measurements. Biochem Biophys Res Commun 163: 1282–1289

    Article  PubMed  CAS  Google Scholar 

  • Bernhardt R, Kraft R, Alterman M, Otto A, Schrauber H, Gunsalus IC, Ruckpaul K (1992a) Common mechanism of interaction between cytochrome P450 and electron donors in different monooxygenase systems. In: Archakow AI, Bachmanova GI (eds) Cytochrome P-450. Bichemistry and Biophysics. Proceedings of the 7th international meeting, Cytochrome P-450: structure and function, biotechnolocigal and ecological aspects. Moscow, 28 July–2 Aug 1991, INCO-TNC, Joint Stock Company, Moscow, Russia, pp 204–209

    Google Scholar 

  • Bernhardt R, Kraft R, Ruckpaul K, Gunsalus IC (1992b) Chemical modification of cytochromes P450 101/(cam) P450 111 (lin.) in preparation Black SD, Coon MJ (1985) Studies on the identity of the heme-binding cysteinyl residue in rabbit liver microsomal cytochrome P-450 isozyme 2. Biochem Biophys Res Commun 128: 82–89

    Google Scholar 

  • Chashchin VL, Turko IV, Akhrem AA, Usanov SA (1985) Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain. Biochim Biophys Acta 828: 313–324

    Article  PubMed  CAS  Google Scholar 

  • Chernogolov AA, Adamovich TB, Usanov SA (1992) Immunochemical mapping of ferredoxin-binding sites of cytochrome P-450scc. In: Archakov AI, Bachmanova GI (eds). Cytochrome P-450. Biochemistry and Biophysics. Proceedings of the 7th international meeting, cytochrome P-450: structure and function, biotechnological and ecological aspects, Moscow, 28 July–2 Aug 1991, INCOTNC, Joint Stock Company, Moscow, Russia, pp 57–59

    Google Scholar 

  • Coghlan VM, Vickery LE (1991) Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc. J Biol Chem 266: 18606–18612

    PubMed  CAS  Google Scholar 

  • DeLemos-Chiarandini C, Frey AB, Sabatini DD, Kreibich G (1987) Determination of the membrane topology of the phenobarbital-inducible rat liver cytochrome P-450 isoenzyme PB-4 using site-specific antibodies. J Cell Biol 104: 209–219

    Article  CAS  Google Scholar 

  • Geren LM, O’Brien P, Stonehuerner J, Millett F (1984) Identification of specific carboxylate groups on adrenodoxin that are involved in the interaction with adrenodoxin reductase. J Biol Chem 259: 2155–2160

    PubMed  CAS  Google Scholar 

  • Gibson G, Tamburini PP (1986) Chemical modification of the histidine residues of purified hepatic cytochrome P-450. Influence on substrate binding and the hemoprotein spin state. Chem Biol Interact 58: 185–198

    Article  PubMed  CAS  Google Scholar 

  • Gotoh O, Fujii-Kuriyama Y (1989) Evolution, structure, and gene regulation of cytochrome P-450. In: Ruckpaul K, Rein H (eds) Frontiers in biotrans-formation, vol 1. Akademie, Berlin, pp 195–243

    Google Scholar 

  • Gotoh O, Tagashira Y, Iizuka T, Fujii-Kuriyama Y (1983) Structural characterization of cytochrome P-450. Possible location of the heme binding cysteine in determined amino-acid sequence. J Biochem (Tokyo) 93: 807–817

    Article  CAS  Google Scholar 

  • Graham-Lorence S, Khalil MW, Lorence NC, Mendelson CR, Simpson ER (1991) Structure-function relationship of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis. J Biol Chem 266: 11939–11946

    PubMed  CAS  Google Scholar 

  • Gunsalus IC, Sligar SG (1978) Oxygen reduction by the P-450 monooxygenase systems. Adv Enzymol 47: 1–44

    PubMed  CAS  Google Scholar 

  • Hamamoto I, Ichikawa Y (1984) Modification of a lysine residue of adrenodoxin reductase, essential for complex formation with adrenodoxin. Biochim Biophys Acta 786: 32–41

    Article  PubMed  CAS  Google Scholar 

  • Haniu M, Yasunobu KT, Gunsalus IC (1983) Heme binding and substrate-protected cysteine residues in P-450cam. Biochem Biophys Res Commun 116: 30–38

    Article  PubMed  CAS  Google Scholar 

  • Hara T, Miyata T (1990) Structure-activity relationship of mitochondrial steroid hydroxylase covalent complexes. In: Ingelmann-Sundberg M, Gustafsson J-A, Orrenius S (eds) Abstract of the 8th international symposium on microsomes and drug oxidations, Stockholm, 25–29 July 1990, Karolinska Institute, p 129

    Google Scholar 

  • Imai M, Shimada H, Watanabe Y, Matsushima-Hibiya Y, Makino R, Koga H, Horiuchi T, Ishimura Y (1989) Uncoupling of the cytochrome P-450cam monooxygenase reaction by a single mutation, threonine 252 to alanine or valine. Proc Natl Acad Sci USA 86: 7823–7827

    Article  PubMed  CAS  Google Scholar 

  • Janig GR, Dettmer R, Usanov SA, Ruckpaul K (1983) Identification of the ligand trans to thiolate in cytochrome P-450LM2 by chemical modification. FEBS Lett 159: 58–62

    Article  PubMed  CAS  Google Scholar 

  • Janig GR, Makower A, Rabe H, Friedrich J, Ruckpaul K (1984) Chemical modification of tyrosine residues at the active centre of cytochrome P-450cam. Biomed Biochim Acta 43: K17–K24

    PubMed  CAS  Google Scholar 

  • Janig GR, Kraft R, Makower A, Rabe H, Ruckpaul K (1985) Assignment of tyrosine to the active centres of cytochrome P-450LM2 and cytochrome P450cam. In: Vereczky L, Magyar K (eds) Cytochrome P-450, biochemistry, biophysics and induction. Akademia Kiado, Budapest, pp 53–56

    Google Scholar 

  • Janig GR, Kraft R, Blanck J, Ristau O, Rabe H, Ruckpaul K (1987) Chemical modification of cytochrome P-450LM4. Identification of functionally linked tyrosine residues. Biochim Biophys Acta 916: 512–523

    Article  PubMed  CAS  Google Scholar 

  • Kalb VF, Loper JC (1988) Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity. Proc Natl Acad Sci USA 85: 7221–7225

    Article  PubMed  CAS  Google Scholar 

  • Kawalek JC, Levine W, Ryan D, Lu AYH (1977) Role of sulfhydryl groups in the hydroxylation of benzo[a]pyrene by purified rat and rabbit cytochrome P-448. Arch Biochem Biophys 183: 732–741

    Article  PubMed  CAS  Google Scholar 

  • Krainev AG, Weiner LM (1991) Localization of active centres of endoplasmic proteins in membranes. In: Ruckpaul K, Rein H (eds) Frontiers in biotransformation, vol 5. Akademie, Berlin, pp 138–183

    Google Scholar 

  • Kunz BC, Vergeres G, Winterhalter K, Richter C (1991) Chemical modification of rat liver microsomal cytochrome P-450: study of enzymic properties and membrane topology. Biochim Biophys Acta 1063: 226–234

    Article  PubMed  CAS  Google Scholar 

  • Lambeth JD (1990) Enzymology of mitochondrial side-chain cleavage by cytochrome P-450scc. In: Ruckpaul K, Rein H (eds) Frontiers in biotransformation, vol 3. Akademie, Berlin, pp 58–100

    Google Scholar 

  • Lambeth JD, Geren LM, Millett F (1984) Cytochrome P-450scc-adrenodoxin interactions. J Biol Chem 259: 10025–10029

    PubMed  CAS  Google Scholar 

  • Means GE, Feeney RE (1971) Chemical modification of proteins. Holden-Day, San Francisco

    Google Scholar 

  • Murray RI, Fisher MT, Debrunner PG, Sligar SG (1986) Structure and chemistry of cytochrome P-450. In: Ortiz de Montellano P (ed) Cytochrome P-450: structure, function, and biochemistry. Plenum, New York, pp 443–479

    Google Scholar 

  • Nadler SG, Strobel HW (1988) Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450. Arch Biochem Biophys 261: 418–429

    Article  PubMed  CAS  Google Scholar 

  • Nebert DW, Nelson DR, Coon MJ, Estabrook RW, Feyereisen R, Fujii-Kuriyama Y, Gonzalez FJ, Guengerich FP, Gunsalus IC, Johnson EF, Loper JC, Sato R, Waterman MR, Waxman D (1991) The P-450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Biol 10: 1–14

    Article  PubMed  CAS  Google Scholar 

  • Nelson DR, Strobel HW (1988) On the membrane topology of vertebrate cytochrome P-450 proteins. J Biol Chem 263: 6038–6050

    PubMed  CAS  Google Scholar 

  • Nelson DR, Strobel HW (1989) Secondary structure prediction of 52 membrane- bound cytochromes P-450 show a strong structural similarity to P-450cam. Biochemistry 28: 656–660

    Article  PubMed  CAS  Google Scholar 

  • Parkinson A, Ryan DE, Thomas PE, Jerina DM, Sayer JM, van Bladeren PJ, Haniu M, Shively JE, Levine W (1986a) Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4′-nitroacetophenone. J Biol Chem 261: 11478–11486

    PubMed  CAS  Google Scholar 

  • Parkinson A, Thomas PE, Ryan DE, Gorsky LD, Shively JE, Sayer JM, Jerina DM, Levine W (1986b) Mechanism of inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4′-nitroacetophenone. J Biol Chem 261: 11487–11495

    PubMed  CAS  Google Scholar 

  • Pikuleva IA, Lapko AG, Akhrem AA, Usanov SA, Chashchin VL (1987) Localization of tetranitromethane-modified tyrosine residues in the polypeptide chain of cholesterol-hydroxylating cytochrome P-450 (in Russian). Bioorg Khim 13: 739–747

    PubMed  CAS  Google Scholar 

  • Pikuleva IA, Usanov SA, Chashchin VL (1990) Chemical modification of adrenocortical cytochrome P-450scc with diethylpyrocarbonate (in Russian). Biokhimiya 55: 665–673

    CAS  Google Scholar 

  • Poulos TL, Finzel BC, Gunsalus IC, Wagner GC, Kraut J (1985) The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450. J Biol Chem 260: 16122–16130

    PubMed  CAS  Google Scholar 

  • Schwarze W, Bernhardt R, Janig GR, Ruckpaul K (1984) Fluorescent energy transfer measurements on fluorescein isothiocyanate modified cytochrome P-450LM2. Biochem Biophys Res Commun 113: 353–360

    Article  Google Scholar 

  • Schwarze W, Jaeger J, Jànig GR, Ruckpaul K (1988) Active site model of cytochrome P-450LM2. Biochem Biophys Res Commun 150: 996–1005

    Article  PubMed  CAS  Google Scholar 

  • Shimizu T, Tateishi T, Hatano M, Fujii-Kuriyama Y (1991) Probing the role of lysines and arginines in the catalytic function of cytochrome P-450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P-450 reductase. J Biol Chem 266: 3372–3375

    PubMed  CAS  Google Scholar 

  • Stayton PS, Poulos TL, Sligar SG (1989) Putidaredoxin competitively inhibits cytochrome b5-cytochrome P-450cam association: A proposed molecular model for a cytochrome P-450cam electron transfer complex. Biochemistry 28: 8201–8205

    Article  PubMed  CAS  Google Scholar 

  • Stayton PS, Sligar SG (1990) The cytochrome P-450cam binding surface as defined by site-directed mutagenesis and electrostatic modeling. Biochemistry 29: 7381–7386

    Article  PubMed  CAS  Google Scholar 

  • Tamburini PP, MacFarquhar S, Schenkman JB (1986) Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes. Biochem Biophys Res Commun 134: 519–526

    Article  PubMed  CAS  Google Scholar 

  • Tsubaki M, Tornita S, Tsuneoka Y, Ichikawa Y (1986) Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme- binding cysteine residue. Biochim Biophys Acta 870: 564–574

    Article  PubMed  CAS  Google Scholar 

  • Tsubaki M, Iwamoto Y, Hiwatashi A, Ichikawa Y (1989) Inhibition of electron transfer from adrenodoxin to cytochrome P-450scc by chemical modification with pyridoxal-5′-phosphate: identification of adrenodoxin-binding site of cytochrome P-450scc. Biochemistry 28: 6899–6907

    Article  PubMed  CAS  Google Scholar 

  • Tuls J, Geren L, Lambeth JD, Millett F (1987) The use of a specific fluorescence probe to study the interaction of adrenodoxin with adrenodoxin reductase and cytochrome P-450scc. J Biol Chem 262: 10020–10025

    PubMed  CAS  Google Scholar 

  • Tuls J, Geren L, Millett F (1989) Fluorescein isothiocyanate specifically modifies lysine 338 of cytochrome P-450scc and inhibits adrenodoxin binding. J Biol Chem 264: 16421–16425

    PubMed  CAS  Google Scholar 

  • Turko IV, Adamovich TB, Kirillova NM, Usanov SA, Chashchin VL (1989) Cross- linking studies of the cholesterol hydroxylation system from bovine adrenocortical mitochondria. Biochim Biophys Acta 996: 37–42

    Article  PubMed  CAS  Google Scholar 

  • Usanov SA, Pikuleva IA, Chashchin VL, Akhrem AA (1984) Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane. Biochim Biophys Acta 790: 259–267

    Article  PubMed  CAS  Google Scholar 

  • Usanov SA, Turko IV, Chashchin VL, Akhrem AA (1985) Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin. Biochim Biophys Acta 832 /3: 288–296

    Article  PubMed  CAS  Google Scholar 

  • Usanov SA, Pikuleva IA, Akhrem AA, Chashchin VL (1987) Chemical modification of cysteine residues in mitochondrial cytochrome P-450scc from adrenal cortex. Identification of the proximal ligand (in Russian). Bioorg Khim 13: 725–738

    PubMed  CAS  Google Scholar 

  • Usanov SA, Chernogolov AA, Chashchin VL (1989) Immunochemical analysis of adrenocortical cytochrome P-450scc. Topology of the hemoprotein polypeptide chain in the phospholipid membrane (in Russian). Biokhimiya 54: 916–925

    CAS  Google Scholar 

  • Usanov SA, Chashchin VL, Akhrem AA (1990) Cytochrome P-450 dependent pathways of the biosynthesis of steroid hormones. In: Ruckpaul K, Rein H (eds) Frontiers in biotransformation, vol 3. Akademie, Berlin, pp 1–57

    Google Scholar 

  • Vergeres G, Winterhalter K, Richter C (1991) Localization of the N-terminal methionine of rat liver cytochrome P-450 in the lumen of the endoplasmic reticulum. Biochim Biophys Acta 1063: 235–241

    Article  PubMed  CAS  Google Scholar 

  • Zvelebil MJJM, Wolf CR, Sternberg MJE (1991) A predicted three-dimensional structure of human cytochrome P-450: implications for substrate specificity. Protein Eng 4: 271–282

    Article  PubMed  CAS  Google Scholar 

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Authors
  1. R. Bernhardt
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Editors and Affiliations

  1. Department of Pharmacology, MC 1505, University of Connecticut Health Center, 263 Farmington Avenue, 06030, Farmington, CT, USA

    John B. Schenkman Ph.D.

  2. Institut für Toxikologie, Gesellschaft für Strahlen- und Umweltforschung mbH, Ingolstädter Landstr. 1, W-8042, Neuherberg, Fed. Rep. of Germany

    Helmut Greim M.D.

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© 1993 Springer-Verlag Berlin Heidelberg

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Bernhardt, R. (1993). Chemical Probes of Cytochrome P450 Structure. In: Schenkman, J.B., Greim, H. (eds) Cytochrome P450. Handbook of Experimental Pharmacology, vol 105. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-77763-9_35

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  • DOI: https://doi.org/10.1007/978-3-642-77763-9_35

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-642-77765-3

  • Online ISBN: 978-3-642-77763-9

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