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. 1990 Jan;172(1):457–464. doi: 10.1128/jb.172.1.457-464.1990

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

B E Haigler 1, D T Gibson 1
PMCID: PMC208452  PMID: 2294092

Abstract

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.

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Selected References

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  1. Axcell B. C., Geary P. J. Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem J. 1975 Jan;146(1):173–183. doi: 10.1042/bj1460173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barnsley E. A. The induction of the enzymes of naphthalene metabolism in pseudomonads by salicylate and 2-aminobenzoate. J Gen Microbiol. 1975 May;88(1):193–196. doi: 10.1099/00221287-88-1-193. [DOI] [PubMed] [Google Scholar]
  3. Batie C. J., LaHaie E., Ballou D. P. Purification and characterization of phthalate oxygenase and phthalate oxygenase reductase from Pseudomonas cepacia. J Biol Chem. 1987 Feb 5;262(4):1510–1518. [PubMed] [Google Scholar]
  4. Beadle C. A., Smith A. R. The purification and properties of 2,4-dichlorophenol hydroxylase from a strain of Acinetobacter species. Eur J Biochem. 1982 Apr 1;123(2):323–332. doi: 10.1111/j.1432-1033.1982.tb19771.x. [DOI] [PubMed] [Google Scholar]
  5. Bernhardt F. H., Pachowsky H., Staudinger H. A 4-methoxybenzoate O-demethylase from Pseudomonas putida. A new type of monooxygenase system. Eur J Biochem. 1975 Sep 1;57(1):241–256. doi: 10.1111/j.1432-1033.1975.tb02296.x. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Bryan J. K. Molecular weights of protein multimers from polyacrylamide gel electrophoresis. Anal Biochem. 1977 Apr;78(2):513–519. doi: 10.1016/0003-2697(77)90111-7. [DOI] [PubMed] [Google Scholar]
  8. Chen J. S., Mortenson L. E. Inhibition of methylene blue formation during determination of the acid-labile sulfide of iron-sulfur protein samples containing dithionite. Anal Biochem. 1977 May 1;79(1-2):157–165. doi: 10.1016/0003-2697(77)90390-6. [DOI] [PubMed] [Google Scholar]
  9. ELLMAN G. L. Tissue sulfhydryl groups. Arch Biochem Biophys. 1959 May;82(1):70–77. doi: 10.1016/0003-9861(59)90090-6. [DOI] [PubMed] [Google Scholar]
  10. Ensley B. D., Gibson D. T., Laborde A. L. Oxidation of naphthalene by a multicomponent enzyme system from Pseudomonas sp. strain NCIB 9816. J Bacteriol. 1982 Mar;149(3):948–954. doi: 10.1128/jb.149.3.948-954.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ensley B. D., Gibson D. T. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J Bacteriol. 1983 Aug;155(2):505–511. doi: 10.1128/jb.155.2.505-511.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ensley B. D., Ratzkin B. J., Osslund T. D., Simon M. J., Wackett L. P., Gibson D. T. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science. 1983 Oct 14;222(4620):167–169. doi: 10.1126/science.6353574. [DOI] [PubMed] [Google Scholar]
  13. Enzymatic -oxidation. VI. Isolation of homogeneous reduced diphosphopyridine nucleotide-rubredoxin reductase. J Biol Chem. 1972 Apr 10;247(7):2109–2116. [PubMed] [Google Scholar]
  14. Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
  15. Foust G. P., Mayhew S. G., Massey V. Complex formation between ferredoxin triphosphopyridine nucleotide reductase and electron transfer proteins. J Biol Chem. 1969 Feb 10;244(3):964–970. [PubMed] [Google Scholar]
  16. Geary P. J., Saboowalla F., Patil D., Cammack R. An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy. Biochem J. 1984 Feb 1;217(3):667–673. doi: 10.1042/bj2170667. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gottschall D. W., Dietrich R. F., Benkovic S. J., Shiman R. Phenylalanine hydroxylase. Correlation of the iron content with activity and the preparation and reconstitution of the apoenzyme. J Biol Chem. 1982 Jan 25;257(2):845–849. [PubMed] [Google Scholar]
  18. Gunsalus I. C., Wagner G. C. Bacterial P-450cam methylene monooxygenase components: cytochrome m, putidaredoxin, and putidaredoxin reductase. Methods Enzymol. 1978;52:166–188. doi: 10.1016/s0076-6879(78)52019-3. [DOI] [PubMed] [Google Scholar]
  19. Haigler B. E., Gibson D. T. Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816. J Bacteriol. 1990 Jan;172(1):465–468. doi: 10.1128/jb.172.1.465-468.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Iyanagi T., Makino R., Anan F. K. Studies on the microsomal mixed-function oxidase system: mechanism of action of hepatic NADPH-cytochrome P-450 reductase. Biochemistry. 1981 Mar 31;20(7):1722–1730. doi: 10.1021/bi00510a004. [DOI] [PubMed] [Google Scholar]
  21. Katagiri M., Ganguli B. N., Gunsalus I. C. A soluble cytochrome P-450 functional in methylene hydroxylation. J Biol Chem. 1968 Jun 25;243(12):3543–3546. [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Lund J., Dalton H. Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH:acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath). Eur J Biochem. 1985 Mar 1;147(2):291–296. doi: 10.1111/j.1432-1033.1985.tb08749.x. [DOI] [PubMed] [Google Scholar]
  24. Peterson J. A., Basu D., Coon M. J. Enzymatic omega-oxidation. I. Electon carriers in fatty acid and hydrocarbon hydroxylation. J Biol Chem. 1966 Nov 10;241(21):5162–5164. [PubMed] [Google Scholar]
  25. Righetti P. G., Drysdale J. W. Isoelectric focusing in gels. J Chromatogr. 1974 Sep 25;98(2):271–321. doi: 10.1016/s0021-9673(00)92076-4. [DOI] [PubMed] [Google Scholar]
  26. Sauber K., Fröhner C., Rosenberg G., Eberspächer J., Lingens F. Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria. Eur J Biochem. 1977 Mar 15;74(1):89–97. doi: 10.1111/j.1432-1033.1977.tb11370.x. [DOI] [PubMed] [Google Scholar]
  27. Schell M. A. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonas putida. Proc Natl Acad Sci U S A. 1986 Jan;83(2):369–373. doi: 10.1073/pnas.83.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schocken M. J., Gibson D. T. Bacterial oxidation of the polycyclic aromatic hydrocarbons acenaphthene and acenaphthylene. Appl Environ Microbiol. 1984 Jul;48(1):10–16. doi: 10.1128/aem.48.1.10-16.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Subramanian V., Liu T. N., Yeh W. K., Gibson D. T. Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography. Biochem Biophys Res Commun. 1979 Dec 14;91(3):1131–1139. doi: 10.1016/0006-291x(79)91998-3. [DOI] [PubMed] [Google Scholar]
  30. Subramanian V., Liu T. N., Yeh W. K., Narro M., Gibson D. T. Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida. J Biol Chem. 1981 Mar 25;256(6):2723–2730. [PubMed] [Google Scholar]
  31. Subramanian V., Liu T. N., Yeh W. K., Serdar C. M., Wackett L. P., Gibson D. T. Purification and properties of ferredoxinTOL. A component of toluene dioxygenase from Pseudomonas putida F1. J Biol Chem. 1985 Feb 25;260(4):2355–2363. [PubMed] [Google Scholar]
  32. TAGAWA K., ARNON D. I. Ferredoxins as electron carriers in photosynthesis and in the biological production and consumption of hydrogen gas. Nature. 1962 Aug 11;195:537–543. doi: 10.1038/195537a0. [DOI] [PubMed] [Google Scholar]
  33. Ueda T., Coon M. J. Enzymatic oxidation. VII. Reduced diphosphopyridine nucleotide-rubredoxin reductase: properties and function as an electron carrier in hydroxylation. J Biol Chem. 1972 Aug 25;247(16):5010–5016. [PubMed] [Google Scholar]
  34. Vermilion J. L., Coon M. J. Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. J Biol Chem. 1978 Apr 25;253(8):2694–2704. [PubMed] [Google Scholar]
  35. Wackett L. P., Kwart L. D., Gibson D. T. Benzylic monooxygenation catalyzed by toluene dioxygenase from Pseudomonas putida. Biochemistry. 1988 Feb 23;27(4):1360–1367. doi: 10.1021/bi00404a041. [DOI] [PubMed] [Google Scholar]
  36. Yamaguchi M., Fujisawa H. Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla c-1. J Biol Chem. 1978 Dec 25;253(24):8848–8853. [PubMed] [Google Scholar]
  37. Yamaguchi M., Fujisawa H. Purification and characterization of an oxygenase component in benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1980 Jun 10;255(11):5058–5063. [PubMed] [Google Scholar]
  38. Yamaguchi M., Fujisawa H. Reconstitution of iron-sulfur cluster of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1981 Jul 10;256(13):6783–6787. [PubMed] [Google Scholar]
  39. Yen K. M., Gunsalus I. C. Plasmid gene organization: naphthalene/salicylate oxidation. Proc Natl Acad Sci U S A. 1982 Feb;79(3):874–878. doi: 10.1073/pnas.79.3.874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zabinski R., Münck E., Champion P. M., Wood J. M. Kinetic and Mössbauer studies on the mechanism of protocatechuic acid 4,5-oxygenase. Biochemistry. 1972 Aug 15;11(17):3212–3219. doi: 10.1021/bi00767a012. [DOI] [PubMed] [Google Scholar]

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