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Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A

Nature Structural & Molecular Biology volume 10, pages 681–687 (2003)Cite this article

Abstract

The facultative anaerobe Escherichia coli is able to assemble specific respiratory chains by synthesis of appropriate dehydrogenases and reductases in response to the availability of specific substrates. Under anaerobic conditions in the presence of nitrate, E. coli synthesizes the cytoplasmic membrane-bound quinol-nitrate oxidoreductase (nitrate reductase A; NarGHI), which reduces nitrate to nitrite and forms part of a redox loop generating a proton-motive force. We present here the crystal structure of NarGHI at a resolution of 1.9 Å. The NarGHI structure identifies the number, coordination scheme and environment of the redox-active prosthetic groups, a unique coordination of the molybdenum atom, the first structural evidence for the role of an open bicyclic form of the molybdo-bis(molybdopterin guanine dinucleotide) (Mo-bisMGD) cofactor in the catalytic mechanism and a novel fold of the membrane anchor subunit. Our findings provide fundamental molecular details for understanding the mechanism of proton-motive force generation by a redox loop.

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Figure 1: Proposed mechanism for the proton-motive force generating redox loop by NarGHI and FdnGHI.
Figure 2: Overall structure of E. coli NarGHI.
Figure 3: Overall structure of NarG and the catalytic site.
Figure 4: The Mo-bisMGD cofactor.
Figure 5: Structure of the integral membrane NarI subunit.

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References

  1. Blasco, F. et al. The coordination and function of the redox centres of the membrane-bound nitrate reductases. Cell. Mol. Life Sci. 58, 179–193 (2001).

    Article  CAS  Google Scholar 

  2. Rothery, R.A., Blasco, F., Magalon, A. & Weiner, J.H. The diheme cytochrome b subunit (Narl) of Escherichia coli nitrate reductase A (NarGHI): structure, function, and interaction with quinols. J. Mol. Microbiol. Biotechnol. 3, 273–283 (2001).

    CAS  PubMed  Google Scholar 

  3. Mitchell, P. Possible molecular mechanisms of the protonmotive function of cytochrome systems. J. Theor. Biol. 62, 327–367 (1976).

    Article  CAS  Google Scholar 

  4. Jormakka, M., Tornroth, S., Byrne, B. & Iwata, S. Molecular basis of proton motive force generation: structure of formate dehydrogenase-N. Science 295, 1863–1868 (2002).

    Article  Google Scholar 

  5. von Heijne, G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J. Mol. Biol. 225, 487–494 (1992).

    Article  CAS  Google Scholar 

  6. Forget, P. The bacterial nitrate reductases. Solubilization, purification and properties of the enzyme A of Escherichia coli K12. Eur. J. Biochem. 42, 325–332 (1974).

    Article  CAS  Google Scholar 

  7. Jones, R.W. & Garland, P.B. Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane. Biochem. J. 164, 199–211 (1977).

    Article  CAS  Google Scholar 

  8. Page, C.C., Moser, C.C., Chen, X. & Dutton, P.L. Natural engineering principles of electron tunnelling in biological oxidation-reduction. Nature 402, 47–52 (1999).

    Article  CAS  Google Scholar 

  9. Lancaster, C.R., Kroger, A., Auer, M. & Michel, H. Structure of fumarate reductase from Wolinella succinogenes at 2.2 Å resolution. Nature 402, 377–385 (1999).

    Article  CAS  Google Scholar 

  10. Xia, D. et al. Crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277, 60–66 (1997).

    Article  CAS  Google Scholar 

  11. Berks, B.C., Ferguson, S.J., Moir, J.W. & Richardson, D.J. Enzymes and associated electron transport systems that catalyse the respiratory reduction of nitrogen oxides and oxyanions. Biochim. Biophys. Acta 1232, 97–173 (1995).

    Article  Google Scholar 

  12. Schindelin, H., Kisker, C., Hilton, J., Rajagopalan, K.V. & Rees, D.C. Crystal structure of DMSO reductase: redox-linked changes in molybdopterin coordination. Science 272, 1615–1621 (1996).

    Article  CAS  Google Scholar 

  13. Schneider, F. et al. Crystal structure of dimethyl sulfoxide reductase from Rhodobacter capsulatus at 1.88 Å resolution. J. Mol. Biol. 263, 53–69 (1996).

    Article  CAS  Google Scholar 

  14. Boyington, J.C., Gladyshev, V.N., Khangulov, S.V., Stadtman, T.C. & Sun, P.D. Crystal structure of formate dehydrogenase H: catalysis involving Mo, molybdopterin, selenocysteine, and an Fe4-S4 cluster. Science 275, 1305–1308 (1997).

    Article  CAS  Google Scholar 

  15. Czjzek, M. et al. Crystal structure of oxidized trimethylamine N-oxide reductase from Shewanella massilia at 2.5 Å resolution. J. Mol. Biol. 284, 435–447 (1998).

    Article  CAS  Google Scholar 

  16. Dias, J.M. et al. Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods. Struct. Fold. Des. 7, 65–79 (1999).

    Article  CAS  Google Scholar 

  17. Blasco, F. et al. NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli. Mol. Microbiol. 28, 435–447 (1998).

    Article  CAS  Google Scholar 

  18. Volbeda, A. et al. Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373, 580–587 (1995).

    Article  CAS  Google Scholar 

  19. Peters, J.W., Lanzilotta, W.N., Lemon, B.J. & Seefeldt, L.C. X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 Å resolution. Science 282, 1853–1858 (1998).

    Article  CAS  Google Scholar 

  20. Magalon, A. et al. Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved His-50 residue in the NarG subunit. Biochemistry 37, 7363–7370 (1998).

    Article  CAS  Google Scholar 

  21. Rabenstein, D., Greenberg, M. & Saetre, R. Potentiometric and polarimetric studies of complexation of molybdenum(VI) and tungsten(VI) by aspartic acid and glutamic acid. Inorg. Chem. 16, 1241–1243 (1977).

    Article  CAS  Google Scholar 

  22. Enemark, J. & Garner, C. The coordination chemistry and function of the molybdenum centres of the oxomolybdoenzymes. J. Biol. Inorg. Chem. 2, 817–822 (1997).

    Article  CAS  Google Scholar 

  23. Berks, B.C. et al. Sequence analysis of subunits of the membrane-bound nitrate reductase from a denitrifying bacterium: the integral membrane subunit provides a prototype for the dihaem electron-carrying arm of a redox loop. Mol. Microbiol. 15, 319–331 (1995).

    Article  CAS  Google Scholar 

  24. Sieker, L.C., Adman, E. & Jensen, L.H. Structure of the Fe-S complex in a bacterial ferredoxin. Nature 235, 40–42 (1972).

    Article  CAS  Google Scholar 

  25. Stephens, P.J., Jollie, D.R. & Warshel, A. Protein control of redox potentials of iron-sulfur proteins. Chem. Rev. 96, 2491–2514 (1996).

    Article  CAS  Google Scholar 

  26. Yankovskaya, V. et al. Architecture of succinate dehydrogenase and reactive oxygen species generation. Science 299, 700–704 (2003).

    Article  CAS  Google Scholar 

  27. Zhao, Z., Rothery, R.A. & Weiner, J.H. Transient kinetic studies of heme reduction in Escherichia coli nitrate reductase A (NarGHI) by menaquinol. Biochemistry 42, 5403–5413 (2003).

    Article  CAS  Google Scholar 

  28. Blasco, F. et al. Formation of active heterologous nitrate reductases between nitrate reductases A and Z of Escherichia coli. Mol. Microbiol. 6, 209–219 (1992).

    Article  CAS  Google Scholar 

  29. Guigliarelli, B. et al. Complete coordination of the four Fe-S centers of the β subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S] clusters. Biochemistry 35, 4828–4836 (1996).

    Article  CAS  Google Scholar 

  30. Otwinowski, Z.M. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  31. Terwilliger, T. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999).

    Article  CAS  Google Scholar 

  32. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998).

    Article  Google Scholar 

  33. De La Fortelle, E.B., Gerard. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997).

    Article  CAS  Google Scholar 

  34. McRee, D.E. XtalView/Xfit—a versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999).

    Article  CAS  Google Scholar 

  35. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  36. Bacon, D. & Anderson, W. A fast algorithm for rendering space-filling molecule pictures. J. Mol. Graph. 6, 219–220 (1988).

    Article  Google Scholar 

  37. Honig, B. & Nicholls, A. Classical electrostatics in biology and chemistry. Science 268, 1144–1149 (1995).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the US Department of Energy for access to data collection facilities at the SSRL and NSLS synchrotrons, and also S. Szigety and D. Mroczko for technical assistance. This work was supported by a Human Frontiers Science Program grant to N.C.J.S., J.H.W. and F.B. N.C.J.S. also acknowledges the Canadian Institutes of Health Research and the Howard Hughes Medical Institute International Scholar program for financial support.

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Authors and Affiliations

  1. Department of Biochemistry, University of British Columbia, 2146 Health Sciences Mall, Vancouver, V6T 1Z3, British Columbia, Canada

    Michela G Bertero, Cynthia Hou, Daniel Lim & Natalie C J Strynadka

  2. Department of Biochemistry, CIHR Membrane Protein Research Group, University of Alberta, 4-25 Medical Sciences Building, Edmonton, T6G 2H7, Alberta, Canada

    Richard A Rothery, Monica Palak & Joel H Weiner

  3. Laboratoire de Chimie Bactérienne, CNRS, 31 chemin Joseph Aiguier, Marseille, 13402, Cedex 9, France

    Francis Blasco

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  1. Michela G Bertero
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Correspondence to Natalie C J Strynadka.

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Bertero, M., Rothery, R., Palak, M. et al. Insights into the respiratory electron transfer pathway from the structure of nitrate reductase A. Nat Struct Mol Biol 10, 681–687 (2003). https://doi.org/10.1038/nsb969

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