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

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.

Nitrate reduction by Desulfovibrio desulfuricans: a periplasmic nitrate reductase system that lacks NapB, but includes a unique tetraheme c-type cytochrome, NapM.

Many sulphate reducing bacteria can also reduce nitrite, but relatively few isolates are known to reduce nitrate. Although nitrate reductase genes are absent from Desulfovibrio vulgaris strain Hildenborough, for which the complete genome sequence has been reported, a single subunit periplasmic nitrate reductase, NapA, was purified from Desulfovibrio desulfuricans strain 27774, and the structural gene was cloned and sequenced. Chromosome walking methods have now been used to determine the complete sequence of the nap gene cluster from this organism. The data confirm the absence of a napB homologue, but reveal a novel six-gene organisation, napC-napM-napA-napD-napG-napH. The NapC polypeptide is more similar to the NrfH subgroup of tetraheme cytochromes than to NapC from other bacteria. NapM is predicted to be a tetra-heme c-type cytochrome with similarity to the small tetraheme cytochromes from Shewanella oneidensis. The operon is located close to a gene encoding a lysyl-tRNA synthetase that is also found in D. vulgaris. We suggest that electrons might be transferred to NapA either from menaquinol via NapC, or from other electron donors such as formate or hydrogen via the small tetraheme cytochrome, NapM. We also suggest that, despite the absence of a twin-arginine targeting sequence, NapG might be located in the periplasm where it would provide an alternative direct electron donor to NapA.