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Spectropotentiometric and structural analysis of the periplasmic nitrate reductase from Escherichia coli

Lookup NU author(s): Dr Clive Butler

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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 of α-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the γ-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 α-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 Mo 4+. We then propose a catalytic cycle for E. coli NapA in which nitrate binds to the Mo5+ ion and where a stable des-oxo Mo 6+ species may participate. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Publication metadata

Author(s): Jepson BJN, Mohan S, Clarke TA, Gates AJ, Cole JA, Butler CS, Butt JN, Hemmings AM, Richardson DJ

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2007

Volume: 282

Issue: 9

Pages: 6425-6437

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology, Inc.

URL: http://dx.doi.org/10.1074/jbc.M607353200

DOI: 10.1074/jbc.M607353200

PubMed id: 17130127

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