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Jepson, Brian J. N., Mohan, Sudesh, Clarke, Thomas A. 
ORCID: https://orcid.org/0000-0002-6234-1914, Gates, Andrew J. ORCID: https://orcid.org/0000-0002-4594-5038, Cole, Jeffrey A., Butler, Clive S., Butt, Julea N. ORCID: https://orcid.org/0000-0002-9624-5226, Hemmings, Andrew M. ORCID: https://orcid.org/0000-0003-3053-3134 and Richardson, David J. ORCID: https://orcid.org/0000-0002-6847-1832
(2007)
Spectropotentiometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli.
Journal of Biological Chemistry, 282 (9).
pp. 6425-6437.
ISSN 1083-351X
Abstract
The Escherichia coli NapA (periplasmic nitrate reductase) contains a [4Fe-4S] cluster and a Mo-bis-molybdopterin guanine dinucleotide cofactor. The NapA holoenzyme associates with a di-heme c-type cytochrome redox partner (NapB). These proteins have been purified and studied by spectropotentiometry, and the structure of NapA has been determined. In contrast to the well characterized heterodimeric NapAB systems ofa-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the ?-proteobacterial E. coli NapA and NapB proteins purify independently and not as a tight heterodimeric complex. This relatively weak interaction is reflected in dissociation constants of 15 and 32 µm determined for oxidized and reduced NapAB complexes, respectively. The surface electrostatic potential of E. coli NapA in the apparent NapB binding region is markedly less polar and anionic than that of the a-proteobacterial NapA, which may underlie the weaker binding of NapB. The molybdenum ion coordination sphere of E. coli NapA includes two molybdopterin guanine dinucleotide dithiolenes, a protein-derived cysteinyl ligand and an oxygen atom. The Mo–O bond length is 2.6 Å, which is indicative of a water ligand. The potential range over which the Mo6+ state is reduced to the Mo5+ state in either NapA (between +100 and -100 mV) or the NapAB complex (-150 to -350 mV) is much lower than that reported for R. sphaeroides NapA (midpoint potential Mo6+/5+ > +350 mV), and the form of the Mo5+ EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo5+ state could not be further reduced to Mo4+. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo6+ species may participate.
| Item Type: | Article |
|---|---|
| Faculty \ School: | Faculty of Science > School of Biological Sciences Faculty of Science > School of Chemistry (former - to 2024) |
| UEA Research Groups: | Faculty of Science > Research Groups > Molecular Microbiology Faculty of Science > Research Centres > Centre for Molecular and Structural Biochemistry Faculty of Science > Research Groups > Energy Materials Laboratory Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017) Faculty of Science > Research Groups > Chemistry of Light and Energy Faculty of Science > Research Groups > Chemistry of Life Processes Faculty of Science > Research Groups > Plant Sciences Faculty of Science > Research Groups > Organisms and the Environment |
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| Depositing User: | EPrints Services |
| Date Deposited: | 01 Oct 2010 13:38 |
| Last Modified: | 24 Sep 2024 09:38 |
| URI: | https://ueaeprints.uea.ac.uk/id/eprint/1410 |
| DOI: | 10.1074/jbc.M607353200 |
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