Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1993 Oct;2(10):1675–1685. doi: 10.1002/pro.5560021013

A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain.

W R Taylor 1, D T Jones 1, A W Segal 1
PMCID: PMC2142254  PMID: 8251942

Abstract

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abo A., Boyhan A., West I., Thrasher A. J., Segal A. W. Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. J Biol Chem. 1992 Aug 25;267(24):16767–16770. [PubMed] [Google Scholar]
  2. Andrews S. C., Shipley D., Keen J. N., Findlay J. B., Harrison P. M., Guest J. R. The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases. FEBS Lett. 1992 May 18;302(3):247–252. doi: 10.1016/0014-5793(92)80452-m. [DOI] [PubMed] [Google Scholar]
  3. Babior B. M., Kipnes R. S. Superoxide-forming enzyme from human neutrophils: evidence for a flavin requirement. Blood. 1977 Sep;50(3):517–524. [PubMed] [Google Scholar]
  4. Claessens M., Van Cutsem E., Lasters I., Wodak S. Modelling the polypeptide backbone with 'spare parts' from known protein structures. Protein Eng. 1989 Jan;2(5):335–345. doi: 10.1093/protein/2.5.335. [DOI] [PubMed] [Google Scholar]
  5. Clark R. A., Volpp B. D., Leidal K. G., Nauseef W. M. Two cytosolic components of the human neutrophil respiratory burst oxidase translocate to the plasma membrane during cell activation. J Clin Invest. 1990 Mar;85(3):714–721. doi: 10.1172/JCI114496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dancis A., Roman D. G., Anderson G. J., Hinnebusch A. G., Klausner R. D. Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3869–3873. doi: 10.1073/pnas.89.9.3869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fillat M. F., Edmondson D. E., Gomez-Moreno C. Structural and chemical properties of a flavodoxin from Anabaena PCC 7119. Biochim Biophys Acta. 1990 Sep 3;1040(2):301–307. doi: 10.1016/0167-4838(90)90091-s. [DOI] [PubMed] [Google Scholar]
  8. Friemann A., Brinkmann K., Hachtel W. Sequence of a cDNA encoding the bi-specific NAD(P)H-nitrate reductase from the tree Betula pendula and identification of conserved protein regions. Mol Gen Genet. 1991 May;227(1):97–105. doi: 10.1007/BF00260713. [DOI] [PubMed] [Google Scholar]
  9. Garcia R. C., Segal A. W. Phosphorylation of the subunits of cytochrome b-245 upon triggering of the respiratory burst of human neutrophils and macrophages. Biochem J. 1988 Jun 15;252(3):901–904. doi: 10.1042/bj2520901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  11. Gowri G., Campbell W. H. cDNA Clones for Corn Leaf NADH:Nitrate Reductase and Chloroplast NAD(P):Glyceraldehyde-3-Phosphate Dehydrogenase : Characterization of the Clones and Analysis of the Expression of the Genes in Leaves as Influenced by Nitrate in the Light and Dark. Plant Physiol. 1989 Jul;90(3):792–798. doi: 10.1104/pp.90.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Harayama S., Rekik M., Bairoch A., Neidle E. L., Ornston L. N. Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases. J Bacteriol. 1991 Dec;173(23):7540–7548. doi: 10.1128/jb.173.23.7540-7548.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holm L., Sander C. Database algorithm for generating protein backbone and side-chain co-ordinates from a C alpha trace application to model building and detection of co-ordinate errors. J Mol Biol. 1991 Mar 5;218(1):183–194. doi: 10.1016/0022-2836(91)90883-8. [DOI] [PubMed] [Google Scholar]
  14. Hurst J. K., Loehr T. M., Curnutte J. T., Rosen H. Resonance Raman and electron paramagnetic resonance structural investigations of neutrophil cytochrome b558. J Biol Chem. 1991 Jan 25;266(3):1627–1634. [PubMed] [Google Scholar]
  15. Imajoh-Ohmi S., Tokita K., Ochiai H., Nakamura M., Kanegasaki S. Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis. J Biol Chem. 1992 Jan 5;267(1):180–184. [PubMed] [Google Scholar]
  16. Johnstone I. L., McCabe P. C., Greaves P., Gurr S. J., Cole G. E., Brow M. A., Unkles S. E., Clutterbuck A. J., Kinghorn J. R., Innis M. A. Isolation and characterisation of the crnA-niiA-niaD gene cluster for nitrate assimilation in Aspergillus nidulans. Gene. 1990 Jun 15;90(2):181–192. doi: 10.1016/0378-1119(90)90178-t. [DOI] [PubMed] [Google Scholar]
  17. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  18. Kakinuma K., Kaneda M., Chiba T., Ohnishi T. Electron spin resonance studies on a flavoprotein in neutrophil plasma membranes. Redox potentials of the flavin and its participation in NADPH oxidase. J Biol Chem. 1986 Jul 15;261(20):9426–9432. [PubMed] [Google Scholar]
  19. 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]
  20. Levitt M. Accurate modeling of protein conformation by automatic segment matching. J Mol Biol. 1992 Jul 20;226(2):507–533. doi: 10.1016/0022-2836(92)90964-l. [DOI] [PubMed] [Google Scholar]
  21. Levitt M. Conformational preferences of amino acids in globular proteins. Biochemistry. 1978 Oct 3;17(20):4277–4285. doi: 10.1021/bi00613a026. [DOI] [PubMed] [Google Scholar]
  22. Light D. R., Walsh C., O'Callaghan A. M., Goetzl E. J., Tauber A. I. Characteristics of the cofactor requirements for the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes. Biochemistry. 1981 Mar 17;20(6):1468–1476. doi: 10.1021/bi00509a010. [DOI] [PubMed] [Google Scholar]
  23. Morris A. L., MacArthur M. W., Hutchinson E. G., Thornton J. M. Stereochemical quality of protein structure coordinates. Proteins. 1992 Apr;12(4):345–364. doi: 10.1002/prot.340120407. [DOI] [PubMed] [Google Scholar]
  24. Nugent J. H., Gratzer W., Segal A. W. Identification of the haem-binding subunit of cytochrome b-245. Biochem J. 1989 Dec 15;264(3):921–924. doi: 10.1042/bj2640921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Okamoto P. M., Fu Y. H., Marzluf G. A. Nit-3, the structural gene of nitrate reductase in Neurospora crassa: nucleotide sequence and regulation of mRNA synthesis and turnover. Mol Gen Genet. 1991 Jun;227(2):213–223. doi: 10.1007/BF00259673. [DOI] [PubMed] [Google Scholar]
  26. Parkos C. A., Allen R. A., Cochrane C. G., Jesaitis A. J. Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with relative molecular weights of 91,000 and 22,000. J Clin Invest. 1987 Sep;80(3):732–742. doi: 10.1172/JCI113128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Parkos C. A., Dinauer M. C., Walker L. E., Allen R. A., Jesaitis A. J., Orkin S. H. Primary structure and unique expression of the 22-kilodalton light chain of human neutrophil cytochrome b. Proc Natl Acad Sci U S A. 1988 May;85(10):3319–3323. doi: 10.1073/pnas.85.10.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ponder J. W., Richards F. M. Tertiary templates for proteins. Use of packing criteria in the enumeration of allowed sequences for different structural classes. J Mol Biol. 1987 Feb 20;193(4):775–791. doi: 10.1016/0022-2836(87)90358-5. [DOI] [PubMed] [Google Scholar]
  29. Prosser I. M., Lazarus C. M. Nucleotide sequence of a spinach nitrate reductase cDNA. Plant Mol Biol. 1990 Jul;15(1):187–190. doi: 10.1007/BF00017743. [DOI] [PubMed] [Google Scholar]
  30. Roos D., De Boer M., De Klein A., Bolscher B. G., Weening R. S. Chronic granulomatous disease: mutations in cytochrome b558. Immunodeficiency. 1993;4(1-4):289–301. [PubMed] [Google Scholar]
  31. Royer-Pokora B., Kunkel L. M., Monaco A. P., Goff S. C., Newburger P. E., Baehner R. L., Cole F. S., Curnutte J. T., Orkin S. H. Cloning the gene for an inherited human disorder--chronic granulomatous disease--on the basis of its chromosomal location. Nature. 1986 Jul 3;322(6074):32–38. doi: 10.1038/322032a0. [DOI] [PubMed] [Google Scholar]
  32. Schluchter W. M., Bryant D. A. Molecular characterization of ferredoxin-NADP+ oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the gene product. Biochemistry. 1992 Mar 31;31(12):3092–3102. doi: 10.1021/bi00127a009. [DOI] [PubMed] [Google Scholar]
  33. Segal A. W., Jones O. T. Novel cytochrome b system in phagocytic vacuoles of human granulocytes. Nature. 1978 Nov 30;276(5687):515–517. doi: 10.1038/276515a0. [DOI] [PubMed] [Google Scholar]
  34. Segal A. W. The electron transport chain of the microbicidal oxidase of phagocytic cells and its involvement in the molecular pathology of chronic granulomatous disease. J Clin Invest. 1989 Jun;83(6):1785–1793. doi: 10.1172/JCI114083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shaw J. P., Harayama S. Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2. Eur J Biochem. 1992 Oct 1;209(1):51–61. doi: 10.1111/j.1432-1033.1992.tb17260.x. [DOI] [PubMed] [Google Scholar]
  36. Stainthorpe A. C., Lees V., Salmond G. P., Dalton H., Murrell J. C. The methane monooxygenase gene cluster of Methylococcus capsulatus (Bath). Gene. 1990 Jul 2;91(1):27–34. doi: 10.1016/0378-1119(90)90158-n. [DOI] [PubMed] [Google Scholar]
  37. Taylor W. R. A flexible method to align large numbers of biological sequences. J Mol Evol. 1988 Dec;28(1-2):161–169. doi: 10.1007/BF02143508. [DOI] [PubMed] [Google Scholar]
  38. Taylor W. R. Towards protein tertiary fold prediction using distance and motif constraints. Protein Eng. 1991 Dec;4(8):853–870. doi: 10.1093/protein/4.8.853. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES