The nitric oxide reductase activity of cytochrome c nitrite reductase from Escherichia coli.
Cytochrome c nitrite reductase (NrfA) from Escherichia coli has a well established role in the respiratory reduction of nitrite to ammonium. More recently the observation that anaerobically grown E. coli nrf mutants were more sensitive to NO. than the parent strain led to the proposal that NrfA might also participate in NO. detoxification. Here we describe protein film voltammetry that presents a quantitative description of NrfA NO. reductase activity. NO. reduction is initiated at similar potentials to NrfA-catalyzed reduction of nitrite and hydroxylamine. All three activities are strongly inhibited by cyanide. Together these results suggest a common site for reduction of all three substrates as axial ligands to the lysine-coordinated NrfA heme rather than nonspecific NO. reduction at one of the four His-His coordinated hemes also present in each NrfA subunit. NO. reduction by NrfA is described by a K(m) of the order of 300 microm. The predicted turnover number of approximately 840 NO. s(-1) is much higher than that of the dedicated respiratory NO. reductases of denitrification and the flavorubredoxin and flavohemoglobin of E. coli that are also proposed to play roles in NO. detoxification. In considering the manner by which anaerobically growing E. coli might detoxify exogenously generated NO. encountered during invasion of a human host it appears that the periplasmically located NrfA should be effective in maintaining low NO. levels such that any NO. reaching the cytoplasm is efficiently removed by flavorubredoxin (K(m) approximately 0.4 microm).
van Wonderen
Jessica H
JH
Centre for Metalloprotein Spectroscopy and Biology, School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, United Kingdom.
Burlat
Bénédicte
B
Richardson
David J
DJ
Cheesman
Myles R
MR
Butt
Julea N
JN
D000693:Anaerobiosis / Q000502:physiology; D003574:Cytochrome c Group / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D004927:Escherichia coli Infections / Q000201:enzymology / Q000235:genetics; D006801:Humans; D019811:Hydroxylamine / Q000378:metabolism; D009154:Mutation; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*
D003574:Cytochrome c Group; D009573:Nitrites; D033903:Periplasmic Proteins; D019811:Hydroxylamine; D009569:Nitric Oxide; C022302:cytochrome C-552; D010088:Oxidoreductases; C034747:nitric-oxide reductase