Abstract
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).
Highlights
Innate response of hosts to the infective invasion of pathogens
Detection of NO1⁄7 Reduction by NrfA Using protein film voltammetry (PFV)—Previous studies have established that electrocatalytically active NrfA films are formed when freshly polished pyrolytic graphite edge electrodes are exposed to ice-cold NrfA solutions [19, 21,22,23]
The work presented here has shown that E. coli NrfA is able to serve as a direct NO1⁄7 reductase
Summary
E. coli NrfA has been reported to reduce ϳ450 NO1⁄7 sϪ1 whereas the enzyme from Desulfovibrio desulfuricans reduces only 30 NO1⁄7 sϪ1 under comparable conditions [16]. It has been noted that D. desulfuricans NrfA exhibits similar rates of NO1⁄7 and nitrite reduction whereas the enzyme from Sulfurospirilum deleyianum reduces NO1⁄7 at one hundredth the rate of nitrite reduction [16, 17] Given these diverse observations and the proposed role for E. coli NrfA in NO1⁄7 detoxification, we have set about providing a more complete and quantitative description of the NO1⁄7 reductase activity of this enzyme. Enzyme activities were resolved across the electrochemical potential domain, revealing multiple modulations of the catalytic rate in response to the application of an increased driving force for the reaction being catalyzed. Because these modulations of activity were independent of the nature of the electrode surface, they were proposed to reflect intrinsic properties of NrfA nitrite and hydroxylamine reduction. To facilitate comparison with the other reductase activities of this enzyme we have chosen to define the NO1⁄7 reductase activity of E. coli NrfA by PFV
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