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.
Jepson
Brian J N
BJ
Centre for Metalloprotein Spectroscopy and Biology, University of East Anglia, Norwich NR4 7TJ, United Kingdom.
Mohan
Sudesh
S
Clarke
Thomas A
TA
Gates
Andrew J
AJ
Cole
Jeffrey A
JA
Butler
Clive S
CS
Butt
Julea N
JN
Hemmings
Andrew M
AM
Richardson
David J
DJ
D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008982:Molybdenum / Q000737:chemistry*; D050901:Nitrate Reductase / Q000737:chemistry*; D010084:Oxidation-Reduction; D011485:Protein Binding; D055672:Static Electricity
D029968:Escherichia coli Proteins; D008982:Molybdenum; D050901:Nitrate Reductase; C494404:napA protein, E coli