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 ofalpha-proteobacteria, such as Rhodobacter sphaeroides and Paracoccus pantotrophus, the gamma-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 mum 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 alpha-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 A, which is indicative of a water ligand. The potential range over which the Mo(6+) state is reduced to the Mo(5+) 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 Mo(6+/5+) > +350 mV), and the form of the Mo(5+) EPR signal is quite distinct. In E. coli NapA or NapAB, the Mo(5+) 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 Mo(5+) ion and where a stable des-oxo Mo(6+) species may participate.
Highlights
Bioinformatic analyses reveal that the periplasmic nitrate reductase is phylogenetically widespread in proteobacteria [2], MARCH 2, 2007 VOLUME 282 NUMBER 9
Bioinformatic analyses reveal that the periplasmic nitrate reductase is phylogenetically widespread in proteobacteria [2], 3 The abbreviations used are: Mo-bis-MGD, Mo-bis-molybdopterin guanine dinucleotide; MGD, molybdopterin guanine dinucleotide; Nap, periplasmic nitrate reductase; PDB, Protein Data Bank; Analytical ultracentrifugation (AUC), analytical ultracentrifugation
The catalytic subunit (NapA) from periplasmic nitrate reductases from the ␣-proteobacteria Paracoccus denitrificans and Rhodobacter sphaeroides forms a very tight complex with a di-c-heme subunit (NapB). This heterodimeric complex resists separation by a range of chromatographic methods that disrupt weak protein-protein interactions (10, 14 –15), and a KD of 0.5 nM has been experimentally determined for the reduced NapAB complex of the R. sphaeroides [10]
Summary
Growth Conditions, and Plasmids—The E. coli K12 strain LCB2048 is defective in the membrane-bound nitrate reductases A and Z [19] and was transformed with pJG460 (napDABCEFG). Cultures of strain LCB2048 (pJG460) were grown in glycerol and nitrate (GN) minimal salts medium containing 0.4% glycerol and 20 mM potassium nitrate [19] without aeration overnight at 37 °C after the addition of ampicillin (100 g1⁄7mlϪ1). The 250-ml GN cultures were transferred to 2-liter batches of fresh GN medium and grown overnight at 37 °C. Purification of NapA and NapB—Periplasmic proteins were extracted from the harvested cells as described previously [19] and applied to a DEAE-cellulose column equilibrated in 10 mM Tris-HCl, pH 8.0. The column was developed using a wash
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