Escherichia coli cytochrome c nitrite reductase is one of a large family of homologous enzymes that are particularly prevalent in pathogenic enterobacteria. The enzymes are periplasmic and in vivo may find themselves challenged by molecules that could enhance or compromise their performance. In the present study, we describe protein film voltammetry in which the activity of E. coli cytochrome c nitrite reductase is challenged by the presence of a number of small molecules. These results are discussed in light of the environment(s) that the enzyme may face before and after colonization of a human host.

Introduction

A number of Eubacteria, including many pathogenic enterobacteria, express a cytochrome c nitrite reductase to support anaerobic respiration on nitrite [1,2]. Given that these are periplasmic enzymes, they could be challenged by a range of molecules derived from their immediate environment, and for some of these molecules their concentrations approach millimolar levels. Thus there is plenty of opportunity for species present in the bacterial environment to have detrimental, or stimulatory, effects on the catalytic performance of the nitrite reductase.

In previous studies, we have shown that protein film voltammetry provides a sensitive route to resolve activities of Escherichia coli cytochrome c nitrite reductase [3,45]. A small sample of enzyme, typically less than 1 pmol, is adsorbed on a graphite electrode. Cyclic voltammetry is then performed. The electrode potential is swept back and forth between two limits and the consequent flow of current is recorded. In response to the potential applied to the electrode, electrons are ‘directly’ injected into, or withdrawn from, the haems of nitrite reductase. Application of sufficiently negative potentials in the presence of substrate stimulates a net transfer of electrons from the electrode to nitrite molecules in solution via the action of the enzyme. If, at the same time, rapid rotation of the electrode ensures continued delivery of substrate to the electrode surface, a continuous ‘catalytic’ current is recorded. The negative sign of this current signifies substrate reduction (as opposed to substrate oxidation that would give rise to a positive current). The magnitude of the catalytic current reflects the net rate of catalysis – the parameter traditionally measured by spectrophotometric enzyme assays. However, now the catalytic rate is defined as a function of electrode potential in addition to concentration, pH, temperature etc.

At pH 7, the catalytic current magnitude arising from films of E. coli cytochrome c nitrite reductase shows saturation kinetics and the Km for nitrite reduction (∼25 μM) is in good agreement with values from solution studies [3,4]. It is interesting that maximal enzyme activity occurs in a narrow potential window when nitrite concentrations are less than, or comparable with, Km (NO2). We suggested that this behaviour arises because reduction at the active site haem pair [Em,7 (reduction potential at pH 7)≈−107 mV] stimulates catalysis, whilst reduction of the low-potential haem or haems (Em,7≈−323 mV) has the opposite effect [3,6]. Protein film voltammetry also defines the contrasting inhibitory effects of cyanide and azide on enzyme activity [4,5]. Given the opportunity for E. coli cytochrome c nitrite reductase to be challenged by a wide range of chemical environments during colonization of a human host, we have now extended our studies to describe the influence, or otherwise, of a wider range of molecules on nitrite reductase activity.

Experimental

Purification and protein film voltammetry of E. coli cytochrome c nitrite reductase were performed as described previously and all potentials are reported relative to the standard hydrogen electrode [4]. To investigate the effect of a given chemical on nitrite reductase activity, the following procedure was adopted. An enzyme film was subject to continuous cyclic voltammetry in 20 μM nitrite at 30 mV/s with electrode rotation at 3000 rev./min. On every third scan, when the electrode potential was approx. 0.2 V, an aliquot (<10 μl) of a concentrated stock solution of the species of interest was added to the solution in the electrochemical cell (initially 3 ml). Control experiments showed that complete mixing of species in the resultant solutions occurred in less than 4 s. Subtracting baselines from the voltammograms recorded for a given composition showed only minor differences between current–potential profiles of the forward and reverse sweeps in a given potential cycle. Separate experiments quantified a first-order loss of signal magnitude, whose rate was independent of solution composition. Thus current magnitudes were adjusted to account for this effect prior to analysis and presentation in the Figures within this paper. Control experiments found no evidence that the chemical species studied here provided substrates for the enzyme.

Results

Catalytic current–potential profiles from films of E. coli cytochrome c nitrite reductase in 20 μM nitrite are illustrated in Figure 1. The consequences of introducing nitrate into the experiment are illustrated in Figure 1 (upper panel). The magnitude of the catalytic response is decreased by the presence of millimolar nitrate and demonstrates inhibition of enzyme activity by these elevated nitrate concentrations. Quantification of the catalytic current at −0.6 V as a function of nitrate concentration yields an IC50 of 50 mM (Figure 2).

Catalytic current–potential profiles for E. coli cytochrome c nitrite reductase in 20 μM nitrite and the indicated concentrations of nitrate (upper panel) and ammonium (lower panel)

Figure 1
Catalytic current–potential profiles for E. coli cytochrome c nitrite reductase in 20 μM nitrite and the indicated concentrations of nitrate (upper panel) and ammonium (lower panel)

Protein film voltammetry was recorded in 50 mM Hepes and 2 mM CaCl2 (pH 7) at 25°C. The scan rate was 30 mV/s with electrode rotation at 3000 rev./min. For clarity, only the current–potential profiles of the sweeps to increasingly negative potentials are displayed. SHE, standard hydrogen electrode.

Figure 1
Catalytic current–potential profiles for E. coli cytochrome c nitrite reductase in 20 μM nitrite and the indicated concentrations of nitrate (upper panel) and ammonium (lower panel)

Protein film voltammetry was recorded in 50 mM Hepes and 2 mM CaCl2 (pH 7) at 25°C. The scan rate was 30 mV/s with electrode rotation at 3000 rev./min. For clarity, only the current–potential profiles of the sweeps to increasingly negative potentials are displayed. SHE, standard hydrogen electrode.

Influence of inhibitors on the activity of E. coli cytochrome c nitrite reductase at pH 7

Figure 2
Influence of inhibitors on the activity of E. coli cytochrome c nitrite reductase at pH 7

Open circles, Na2SO4; open triangles, KCl; open squares, NH4+; closed squares, KOCN; closed circles, NaNO3; stars, NaN3; half-filled circles, KSCN. Experimental conditions were as in Figure 1.

Figure 2
Influence of inhibitors on the activity of E. coli cytochrome c nitrite reductase at pH 7

Open circles, Na2SO4; open triangles, KCl; open squares, NH4+; closed squares, KOCN; closed circles, NaNO3; stars, NaN3; half-filled circles, KSCN. Experimental conditions were as in Figure 1.

Ammonium is the product of nitrite reduction by cytochrome c nitrite reductase. The voltammograms in Figure 1 (lower panel) illustrate how enzyme activity is extremely resilient to product inhibition. Less than 20% inhibition is noted even in the presence of 100 mM ammonium (Figure 2). It is instructive that even in the presence of this great excess of product (the ammonium concentration is 5000 times greater than that of nitrite), maximal enzyme activity is still only displayed across a narrow window of potential. Clearly none of the features within the activity–potential profile of the enzyme arise from the consequences of product inhibition.

The results of similar experiments performed with a range of inhibitors are summarized in Figure 2. Sulphate and chloride, like ammonium, are ineffective inhibitors of cytochrome c nitrite reductase. Cyanate and nitrate have greater potencies and are comparable with azide in the extent of inhibition that they induce. Thiocyanate is the most effective of the inhibitors investigated here.

Further insight into the activity of certain of these inhibitors is afforded by resolution of activity across the potential domain by the protein film voltammetry. For example, the voltammograms recorded in the presence of nitrate at first glance appear to show a displacement of the catalytic wave to more negative potentials (Figure 1, upper panel). Closer inspection shows that this behaviour is confined to the potentials required to observe significant activity from the film on lowering the electrode potential. In contrast, the foot of the attenuation feature at lower potentials is insensitive to the concentration of nitrate. Similar behaviour was observed during inhibition by cyanate. The behaviour of the voltammograms is consistent with tighter binding of these inhibitors to the oxidized over reduced state of the active site as reported in our previous description of azide inhibition of this enzyme [4]. Dissociation constants for each of these binding events are afforded by the relationship between wave position and inhibitor concentration [4]. The results of such an analysis are presented in Table 1.

Table 1
Dissociation constants for anion binding to the active site of E. coli cytochrome c nitrite reductase as deduced from the effect of the ions on the catalytic protein film voltammetry
 Kd 
 Active site oxidized Active site reduced 
SCN 42±10 μM 2±0.5 mM 
NO3 73±10 μM 10±3 mM 
N3 39±10 μM 15±5 mM 
 Kd 
 Active site oxidized Active site reduced 
SCN 42±10 μM 2±0.5 mM 
NO3 73±10 μM 10±3 mM 
N3 39±10 μM 15±5 mM 

Discussion

The nitrite reductase activity of E. coli cytochrome c nitrite reductase is remarkably resilient to perturbation by the chemicals investigated here. While the present study does not determine the physiological determinants of such properties, their advantageous consequences are readily recognized when E. coli habitats are considered.

E. coli ingested by humans experience a range of anoxic and micro-oxic environments on transit from the mouth to the gut and beyond. Saliva contains nitrite concentrations of 0.05–0.3 mM and while some of this nitrite is derived directly from the diet, the majority is produced by the enzymatic reduction of nitrate [7]. Several litres of saliva are carried into the gut each day by swallowing. Thus E. coli has the opportunity to utilize respiratory nitrite reduction during and after ingestion. In addition to nitrite, saliva also contains nitrate (0.1–2 mM), derived predominantly from the diet, and thiocyanate (0.3–2 mM), derived from the detoxification of cyanide derived from tobacco smoke and foods such as peach and apricot [7,8,9,1011]. From the studies presented here, it is unlikely that even such high levels of nitrate or thiocyanate will compromise respiration at the level of nitrite reduction. For those strains of E. coli that invade the blood supply as a prelude to sepsis or meningitis, any respiratory nitrite reduction will be unaffected by the levels of plasma sulphate (0.2 mM) [12]. Similarly, any E. coli excreted in faeces may have their survival in municipal wastewater effluents aided by the resilience of nitrite respiration to the levels of chloride (1–14 mM) and ammonium (up to 1.5 mM) that may be found in these environments [13].

The 11th Nitrogen Cycle Meeting 2005: Independent Meeting held at Estación Experimental del Zaidín, Granada, Spain, 15–17 September 2005. Organized and Edited by E.J. Bedmar (Granada, Spain), M.J. Delgado (Granada, Spain) and C. Moreno-Vivián (Córdoba, Spain).

We thank Dr Christine Moore for enzyme purification, and the U.K. Biotechnology and Biological Sciences Research Council (grants 83/17233 and 83/13842) and a JIF (Joint Infrastructure Funding) award (062178) for financial support.

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