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. 1999 Feb;8(2):298–306. doi: 10.1110/ps.8.2.298

Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.

Q Zhao 1, S Modi 1, G Smith 1, M Paine 1, P D McDonagh 1, C R Wolf 1, D Tew 1, L Y Lian 1, G C Roberts 1, H P Driessen 1
PMCID: PMC2144264  PMID: 10048323

Abstract

The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c.

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Selected References

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  1. Bridges A., Gruenke L., Chang Y. T., Vakser I. A., Loew G., Waskell L. Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase. J Biol Chem. 1998 Jul 3;273(27):17036–17049. doi: 10.1074/jbc.273.27.17036. [DOI] [PubMed] [Google Scholar]
  2. Enoch H. G., Strittmatter P. Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase. J Biol Chem. 1979 Sep 25;254(18):8976–8981. [PubMed] [Google Scholar]
  3. Fukuyama K., Wakabayashi S., Matsubara H., Rogers L. J. Tertiary structure of oxidized flavodoxin from an eukaryotic red alga Chondrus crispus at 2.35-A resolution. Localization of charged residues and implication for interaction with electron transfer partners. J Biol Chem. 1990 Sep 15;265(26):15804–15812. [PubMed] [Google Scholar]
  4. Ilan Z., Ilan R., Cinti D. L. Evidence for a new physiological role of hepatic NADPH:ferricytochrome (P-450) oxidoreductase. Direct electron input to the microsomal fatty acid chain elongation system. J Biol Chem. 1981 Oct 10;256(19):10066–10072. [PubMed] [Google Scholar]
  5. Iyanagi T., Mason H. S. Some properties of hepatic reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase. Biochemistry. 1973 Jun 5;12(12):2297–2308. doi: 10.1021/bi00736a018. [DOI] [PubMed] [Google Scholar]
  6. Jenkins C. M., Genzor C. G., Fillat M. F., Waterman M. R., Gómez-Moreno C. Negatively charged anabaena flavodoxin residues (Asp144 and Glu145) are important for reconstitution of cytochrome P450 17alpha-hydroxylase activity. J Biol Chem. 1997 Sep 5;272(36):22509–22513. doi: 10.1074/jbc.272.36.22509. [DOI] [PubMed] [Google Scholar]
  7. Jones T. A., Zou J. Y., Cowan S. W., Kjeldgaard M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A. 1991 Mar 1;47(Pt 2):110–119. doi: 10.1107/s0108767390010224. [DOI] [PubMed] [Google Scholar]
  8. Karplus P. A., Daniels M. J., Herriott J. R. Atomic structure of ferredoxin-NADP+ reductase: prototype for a structurally novel flavoenzyme family. Science. 1991 Jan 4;251(4989):60–66. [PubMed] [Google Scholar]
  9. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Lu A. Y., Junk K. W., Coon M. J. Resolution of the cytochrome P-450-containing omega-hydroxylation system of liver microsomes into three components. J Biol Chem. 1969 Jul 10;244(13):3714–3721. [PubMed] [Google Scholar]
  11. Ludwig M. L., Pattridge K. A., Metzger A. L., Dixon M. M., Eren M., Feng Y., Swenson R. P. Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes. Biochemistry. 1997 Feb 11;36(6):1259–1280. doi: 10.1021/bi962180o. [DOI] [PubMed] [Google Scholar]
  12. Modi S., Gilham D. E., Sutcliffe M. J., Lian L. Y., Primrose W. U., Wolf C. R., Roberts G. C. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as a substrate of cytochrome P450 2D6: allosteric effects of NADPH-cytochrome P450 reductase. Biochemistry. 1997 Apr 15;36(15):4461–4470. doi: 10.1021/bi962633p. [DOI] [PubMed] [Google Scholar]
  13. Modi S., Paine M. J., Sutcliffe M. J., Lian L. Y., Primrose W. U., Wolf C. R., Roberts G. C. A model for human cytochrome P450 2D6 based on homology modeling and NMR studies of substrate binding. Biochemistry. 1996 Apr 9;35(14):4540–4550. doi: 10.1021/bi952742o. [DOI] [PubMed] [Google Scholar]
  14. Nadler S. G., Strobel H. W. Role of electrostatic interactions in the reaction of NADPH-cytochrome P-450 reductase with cytochromes P-450. Arch Biochem Biophys. 1988 Mar;261(2):418–429. doi: 10.1016/0003-9861(88)90358-x. [DOI] [PubMed] [Google Scholar]
  15. Nisimoto Y. Localization of cytochrome c-binding domain on NADPH-cytochrome P-450 reductase. J Biol Chem. 1986 Oct 25;261(30):14232–14239. [PubMed] [Google Scholar]
  16. PHILLIPS A. H., LANGDON R. G. Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem. 1962 Aug;237:2652–2660. [PubMed] [Google Scholar]
  17. Peterson J. A., Graham S. E. A close family resemblance: the importance of structure in understanding cytochromes P450. Structure. 1998 Sep 15;6(9):1079–1085. doi: 10.1016/s0969-2126(98)00109-9. [DOI] [PubMed] [Google Scholar]
  18. Porter T. D. An unusual yet strongly conserved flavoprotein reductase in bacteria and mammals. Trends Biochem Sci. 1991 Apr;16(4):154–158. doi: 10.1016/0968-0004(91)90059-5. [DOI] [PubMed] [Google Scholar]
  19. Porter T. D., Kasper C. B. NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins. Biochemistry. 1986 Apr 8;25(7):1682–1687. doi: 10.1021/bi00355a036. [DOI] [PubMed] [Google Scholar]
  20. Rao S. T., Shaffie F., Yu C., Satyshur K. A., Stockman B. J., Markley J. L., Sundarlingam M. Structure of the oxidized long-chain flavodoxin from Anabaena 7120 at 2 A resolution. Protein Sci. 1992 Nov;1(11):1413–1427. doi: 10.1002/pro.5560011103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ravichandran K. G., Boddupalli S. S., Hasermann C. A., Peterson J. A., Deisenhofer J. Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450's. Science. 1993 Aug 6;261(5122):731–736. doi: 10.1126/science.8342039. [DOI] [PubMed] [Google Scholar]
  22. Schacter B. A., Nelson E. B., Marver H. S., Masters B. S. Immunochemical evidence for an association of heme oxygenase with the microsomal electron transport system. J Biol Chem. 1972 Jun 10;247(11):3601–3607. [PubMed] [Google Scholar]
  23. Shen A. L., Kasper C. B. Role of acidic residues in the interaction of NADPH-cytochrome P450 oxidoreductase with cytochrome P450 and cytochrome c. J Biol Chem. 1995 Nov 17;270(46):27475–27480. doi: 10.1074/jbc.270.46.27475. [DOI] [PubMed] [Google Scholar]
  24. Shimizu T., Tateishi T., Hatano M., Fujii-Kuriyama Y. Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase. J Biol Chem. 1991 Feb 25;266(6):3372–3375. [PubMed] [Google Scholar]
  25. Smith G. C., Tew D. G., Wolf C. R. Dissection of NADPH-cytochrome P450 oxidoreductase into distinct functional domains. Proc Natl Acad Sci U S A. 1994 Aug 30;91(18):8710–8714. doi: 10.1073/pnas.91.18.8710. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Smith W. W., Pattridge K. A., Ludwig M. L., Petsko G. A., Tsernoglou D., Tanaka M., Yasunobu K. T. Structure of oxidized flavodoxin from Anacystis nidulans. J Mol Biol. 1983 Apr 25;165(4):737–753. doi: 10.1016/s0022-2836(83)80277-0. [DOI] [PubMed] [Google Scholar]
  27. Tamburini P. P., Schenkman J. B. Differences in the mechanism of functional interaction between NADPH-cytochrome P-450 reductase and its redox partners. Mol Pharmacol. 1986 Aug;30(2):178–185. [PubMed] [Google Scholar]
  28. WILLIAMS C. H., Jr, KAMIN H. Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver. J Biol Chem. 1962 Feb;237:587–595. [PubMed] [Google Scholar]
  29. Wang M., Roberts D. L., Paschke R., Shea T. M., Masters B. S., Kim J. J. Three-dimensional structure of NADPH-cytochrome P450 reductase: prototype for FMN- and FAD-containing enzymes. Proc Natl Acad Sci U S A. 1997 Aug 5;94(16):8411–8416. doi: 10.1073/pnas.94.16.8411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Watt W., Tulinsky A., Swenson R. P., Watenpaugh K. D. Comparison of the crystal structures of a flavodoxin in its three oxidation states at cryogenic temperatures. J Mol Biol. 1991 Mar 5;218(1):195–208. doi: 10.1016/0022-2836(91)90884-9. [DOI] [PubMed] [Google Scholar]
  31. Zhang Z., Huang L., Shulmeister V. M., Chi Y. I., Kim K. K., Hung L. W., Crofts A. R., Berry E. A., Kim S. H. Electron transfer by domain movement in cytochrome bc1. Nature. 1998 Apr 16;392(6677):677–684. doi: 10.1038/33612. [DOI] [PubMed] [Google Scholar]
  32. Zhao Q., Smith G., Modi S., Paine M., Wolf R. C., Tew D., Lian L. Y., Primrose W. U., Roberts G. C., Driessen H. P. Crystallization and preliminary X-ray diffraction studies of human cytochrome P450 reductase. J Struct Biol. 1996 Mar-Apr;116(2):320–325. doi: 10.1006/jsbi.1996.0048. [DOI] [PubMed] [Google Scholar]

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