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Originally published In Press as doi:10.1074/jbc.M601610200 on April 13, 2006

J. Biol. Chem., Vol. 281, Issue 24, 16340-16346, June 16, 2006
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A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase*Formula

Hein J. Wijma{ddagger}1, Lars J. C. Jeuken§, Martin P. Verbeet{ddagger}, Fraser A. Armstrong, and Gerard W. Canters{ddagger}2

From the {ddagger}Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, the §Institute of Molecular Biophysics, University of Leeds, Leeds LS2 9JT, United Kingdom, and the Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, United Kingdom

Received for publication, February 21, 2006 , and in revised form, April 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO–2] reduction of the type-2 site precedes nitrite binding, at high [NO–2] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an ~50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Copper-containing nitrite reductase (NiR)3 is one of the enzymes of the denitrification pathway (1). Denitrification globally recycles fixed nitrogen (Formula, Formula) to the atmosphere (N2) with NO and N2O being formed as by-products; they act as ozone scavenger and as greenhouse gas, respectively (2, 3). Copper-containing nitrite reductases are found in bacteria, Archaea, and fungi (1, 4, 5). In pathogens, copper-containing NiR is known to enhance the resistance against human sera in Neisseria gonorrhoeae (6) and allows Neisseria meningitidis to respire on nitrite under the microaerobic conditions encountered during disease in humans (7). Furthermore, there is an interest in applying NiR in amperometric biosensors for selectively monitoring toxic nitrite in natural waters and waste streams (8–10).

NiR is a homotrimer, in which each subunit contains a type-1 copper site that accepts electrons from a physiological electron donor and transfers them to a type-2 copper site that is located deeper inside the enzyme (11–13). The type-2 copper is coordinated by three histidines and forms the active site together with a water network, an aspartate, and a histidine. The latter two residues hydrogen bond to the nitrite and donate protons for the enzymatic reaction (14–18). In the absence of nitrite a water molecule occupies the fourth ligand position. Deprotonation of this water molecule occurs between pH 6 and 7 (18) and results in OH– as the fourth ligand (19). NiR catalyzes the reduction of nitrite bidirectionally (Formula) (20).

An important mechanistic question currently under debate (1) is whether nitrite binds to the oxidized type-2 copper first after which the electron is transferred to the type-2 site (5, 14, 21, 22) or whether the type-2 site is first reduced after which the nitrite binds to the copper (23–26). Also the possibility has been considered that the two routes operate in parallel (24, 26). Among key observations indicating that nitrite binds first to the oxidized copper are that the reduced type-2 site in NiR prefers a low occupancy of the fourth coordination position (14, 21, 22) and that the prereduced NiR only sluggishly reacts with nitrite (22, 27). On the other hand, in model complexes of the type-2 site, the copper is reduced prior to nitrite binding (28–30), and the nitrite dissociation constant of the oxidized type-2 site in NiR is more than 10-fold higher (25) than its Michaelis constant (Km).

Here we report on investigations of the catalytic mechanism of NiR by kinetic techniques for which solutions of NiR with three different electron donors at saturating concentrations have been employed. The experiments have been complemented by electrochemical experiments on NiR that was immobilized on a rotating disk graphite electrode. It appears that the results can be analyzed and fitted satisfactorily on the basis of a random sequential mechanism.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Material—Wild-type NiR (from Alcaligenes faecalis S-6) and NiR M150T were prepared as described (20).4 The copper content of native NiR and NiR M150T used for electrode experiments was 1.7 copper/monomer, as determined by the bicinchoninic acid method (32). For assays in solution, native NiR with a copper content of 2.0 per monomer was used.

Activity Assays in Solution—Unless reported otherwise, the buffer was 25 mM malate-MES-HEPES (MMH). The pH was adjusted with NaOH. Reported pH values are those of the assay buffer and were determined at the temperature of the assay. Nitrite reductase activity assays using saturating concentrations of reduced pseudoazurin (400 µM, which is well above Km) as the electron donor were carried out as described (20) at 25 °C. The activity was measured by monitoring the increase of the absorption band at 590 nm, typical of oxidized pseudoazurin ({epsilon}590 nm = 2900 M–1 cm–1 (33)). A linear increase of absorbance with time (providing real initial rates) was observed in all cases.

Activity assays that employed benzyl viologen as the electron donor at saturating concentrations were carried out at 25 °C in a sealed cuvette that had been flushed with argon to make it anaerobic. The benzyl viologen (200 µM) was reduced by dithionite to an initial absorbance at 600 nm of 1.2–1.6. Alternatively, reduced benzyl viologen was prepared electrochemically with a platinum working electrode and an auxiliary electrode (34). The catalytic activity was calculated from the rate of decrease in absorbance of reduced benzyl viologen ({epsilon}603 nm = 14,500 M–1 cm–1 (35)) after addition of NiR, corrected for baseline drift. The decrease in absorbance was linear with respect to time after addition of NiR and remained so until >95% of the reduced viologen had been oxidized.

The activity with benzyl viologen as an electron donor was higher than the activity determined from the rate of reduction of nitrite (results not shown). This discrepancy arises because the nitric oxide product is reduced further, non-enzymatically, by the viologen to give nitrous oxide or ammonia (1). Because the stoichiometry between nitrite conversion and viologen consumption depended on the precise reaction conditions and varied from case to case (not further investigated) the catalytic activity is reported in arbitrary units when benzyl viologen was used as a reductant (see for instance Figs. 1C and 3B, a.u.). In some assays methyl viologen was used instead of benzyl viologen. The activities with a viologen as the electron donor, as quantified by nitrite disappearance, are similar to the activities with pseudoazurin as the electron donor (for A. faecalis S-6 NiR at pH 7 the kcat of nitrite reduction is 338 s–1 with methyl viologen (36) and 392 s–1 with pseudoazurin (20) as the electron donor).

Activity Assays with a Rotating Disk Electrode—Activity assays employing a pyrolytic graphite edge electrode were carried out in an anaerobic cell (Metrohm) provided with an AUTOLAB rotating disk electrode (Eco Chemie), a platinum counter electrode, and an isothermal calomel electrode. During the experiments, the cell was kept anaerobic by flushing the head space with argon. The pyrolytic graphite edge electrode, constructed in the laboratory, had a surface area of 4 mm2. Pyrolytic graphite (GE Advanced Ceramics) was embedded in epoxy (Araldite CY1300:HY1300 mixed 3:1 w/w from Ciba/Robnor) so that the basal plane was perpendicular with respect to the surface that contacts the solution. For NiR immobilization, the pyrolytic graphite edge electrode was polished thoroughly using a polishing cloth (Buehler) covered with 6 micron diamond, sonicated in H2O, washed with buffer, then exposed to NiR (>100 µM) for ~30 s, rinsed with H2O, and inserted in the buffer. After sealing the cell, the electrode was cycled between 560 and –140 mV versus normal hydrogen electrode (NHE) to check that oxygen was absent. Observed currents were corrected for background currents measured in the absence of nitrite.

Experiments were initiated by holding the electrode at a potential of 560 mV versus NHE for 10 s, followed by a "jump" to the desired value. After the background current had stabilized, the dependence of activity on nitrite concentration was measured by stepwise increases in nitrite concentration. The current at each nitrite concentration was averaged over 10 s, and the current in the absence of nitrite was subtracted. The temperature was kept at 0 °C to improve the stability of the protein film on the electrode; from control experiments it was established that the loss of active coverage during the time of the experiment was <10%.

For reliable determination of enzymatic constants by PFV, the electrode rotation rate should be sufficiently fast to prevent transport of substrate to the electrode (or accumulation of product near the electrode) becoming rate-limiting (37). Varying the rate of rotation between 500 and 9000 rpm did not influence the catalytic current, not even at the lowest nitrite concentrations. Thus, the catalytically active coverage of NiR on the electrodes is sufficiently low to maintain steady-state levels of substrate close to the surface of the electrode during turnover. Also the low currents observed (~300 nA) were consistent with a low catalytically active coverage. The coverage was estimated by assuming that the turnover rate/active site (kt) is the same as measured in solution and is linearly related to the catalytic current (it) by Equation 1, it =–FA{Gamma} kt (Equation 1), in which A is the surface area of the electrode, {Gamma} is the enzymatic coverage (catalytically active subunits/unit of surface), F is the Faraday constant, and a negative sign signifies a reduction reaction. By combining the data from Figs. 1 and 4 the enzymatic coverage was estimated from Equation 1 to be 0.03 pmol cm–2. As a comparison, for fumarate reductase (38) no transport limitation is observed at the much higher coverage of 13 pmol cm–2. Also for hydrogenase (39), a far more active enzyme, no substrate depletion is observed provided the coverage is below 1 pmol cm–2. These observations justify the assumption that in our case no complications because of transport limitations affect the observations. The typical rotation speed during the experiments was 2000 rpm. The data were fitted using Igor Pro (WaveMetrics, Inc.).

Modeling—The experimental results (see above) were analyzed on the basis of Scheme 1. Depicted are the type-2 site and the various changes it may undergo during turnover. In the starting configuration the oxidized Cu(II) carries water as the fourth ligand. Replacement of the water (step 1, k1 and k–1) by nitrite and subsequent electron transfer from the type-1 site to the type-2 site (step 3, k3 and k–3) leads to the reduced nitrite-bound type-2 site. These two steps constitute the B route. The A route consists of step 2 (electron transfer from type-1 to type-2) and the subsequent step 4 (nitrite binding) and leads to the same nitrite-bound reduced type-2 site, which is subsequently returned to the initial state (step 6) by conversion of nitrite to NO, dissociation of the NO, and binding of a water molecule. Conversion of the H2O-bound reduced type-2 site to an inactive three-coordinate (14, 22) reduced form (step 5) is included in the scheme. The effect of this step (or equilibrium) on the reaction kinetics can be observed with freshly prepared enzyme in the initial phase of the reaction.5 Scheme 1 applies at low pH; at high pH the same scheme applies but with the water replaced by OH–. This also means that the kinetic constants in Scheme 1 are pH-dependent. In a random sequential mechanism as in Scheme 1, at low nitrite concentration electron transfer to the type-2 site will be faster than nitrite binding, and catalysis will mainly occur via the A route (step 2 and 4). At higher nitrite concentration, the nitrite binding will become faster than the electron transfer to the H2O/OH–-bound type-2 site, and catalysis will follow the B route (via steps 1 and 3).

When Scheme 1 applies the dependence of kt on the nitrite concentration ([S]) can be shown to obey Equation 2 (see supplemental materials for derivation and details), Formula (Equation 2). Equation 2 also applies when the isomerization (step 5) is omitted from Scheme 1. The terms Formula are primarily associated with the A and B routes, respectively. Thus, the Michaelis constants Formula and Formula are a measure of Formula and Formula, respectively. Michaelis constants approximate to dissociation constants when (a) the catalytic step is much faster than the electron-transfer steps, (b) the isomerization (step 5) is slow, and (c) the dissociation of nitrite is faster than the other steps (see Table S1 in supplemental material). Furthermore, when step 6 is not rate-limiting we find that (Table S1 in supplemental material) the ratio of Formula approximately equals the ratio of the electron transfer rates k3 and k2. In the case that NiR follows an ordered mechanism (only steps 2, 4, and 6 or only steps 1, 3, 5, and 6) standard Henri-Michaelis-Menten (HMM) behavior should be observed, as given by kt(S) = kcat[S]/(Km + [S]) (Equation 3).


Figure 1
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  		FIGURE 1.
Dependence of catalytic activity on pH and nitrite concentration. A, activity versus pH with pseudoazurin as the electron donor. The filled circles refer to data obtained in the presence of 5 mM nitrite, and the open circles correspond to 500 µM nitrite. The lines merely connect the points, they are not a fit. The buffer was 25 mM malate-MES-HEPES set at pH with NaOH; the temperature was 25 °C. B, activity versus nitrite concentration with pseudoazurin as the electron donor at pH 4.9. The solid line is a fit of the data to Equation 2 yielding Formula, Formula, Formula, Formula mM. C, activity versus nitrite concentration with benzyl viologen as the electron donor at pH 5.4. The solid line is a least-squares fit to Equation 2 resulting in Formula, Formula, Formula µM. a.u., arbitrary units.

 


Figure 2
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  		FIGURE 2.
Kinetic constants measured by protein film voltammetry. A, reductive current versus time upon subsequent nitrite additions. After the electrode was held at –70 mV versus NHE for 200 s, the background current was stable, and the nitrite concentration was increased stepwise. B, plot of the baseline-subtracted reductive current versus concentration of nitrite at pH 5.7. The solid line is a fit to Equation 2 and yielded Formula, Formula µM, Formula µM. The buffer was 25 mM malate-MES-HEPES at 0 °C. For experimental details see "Experimental Procedures."

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deviations from Henri-Michaelis-Menten Kinetics by NiR—The investigations were started by measuring the catalytic activity (kt) of nitrite reductase as a function of pH with pseudoazurin as the electron donor at saturating concentrations. With 500 µM nitrite maximum activity was observed around pH 5.6, whereas at 5 mM nitrite maximum activity occurred at pH 6.0 (Fig. 1A). Interestingly, the maximum activity was 25% higher with 500 µM nitrite than with 5 mM nitrite. To investigate this phenomenon further, we assayed the activity as a function of nitrite concentration. At pH 4.9, the activity with increasing nitrite concentration reached a maximum followed by a steady decrease at higher nitrite concentrations (Fig. 1B) that could be fitted to Equation 2 resulting in Formula.

To investigate the possible dependence of these kinetics on the nature of the electron donor, we used benzyl viologen instead of pseudoazurin, again at saturating concentrations. Benzyl viologen is a far stronger reductant (EM =–350 mV versus NHE) than pseudoazurin (EM = 295 mV at pH 6 (20)). Furthermore, benzyl viologen is expected to reduce NiR via a nonspecific collisional mechanism, whereas pseudoazurin forms a specific complex with NiR (40–42), so we might also ascertain the role of complex formation in the overall mechanism. Our results showed that with benzyl viologen as the electron donor, the catalytic rate also diminished at higher nitrite concentrations (Fig. 1C).


Figure 3
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  		FIGURE 3.
Deviations from Henri-Michaelis-Menten kinetics at high pH. A, activity versus nitrite concentration of two different batches of native NiR immobilized on a pyrolytic graphite edge electrode. Closed and open circles represent the different batches that are fitted to the Henri-Michaelis-Menten equation (Equation 3) by a continuous and a broken line, respectively. The two fits resulted in Km values of 47 ± 6 µM and 48 ± 5 µM (at pH 7.47). The inset shows the Eadie-Hofstee plot in which all points should lie on straight line were the Henri-Michaelis-Menten equation to be obeyed. The y axis in the Eadie-Hofstee plot is activity, the x axis is activity divided by substrate concentration. B, activity versus nitrite concentration at pH 7.14 with benzyl viologen as electron donor. Shown is a fit to the Henri-Michaelis-Menten Equation 3 (THIN solid line) and to Equation 2 (thick line, Formula µM, within the range of accessible nitrite concentrations (< 5 mM) no saturation of the rate occurred, meaning only lower estimates of Formula and Formula are possible).

 
Finally, in a separate experiment the catalytic activity of NiR immobilized on a rotating disk electrode was measured by observing the reductive current (Fig. 2A). The activity displayed the same dependence on nitrite concentration (Fig. 2B) as observed before in the bulk experiments, both at electrode potentials of –70 and +110 mV versus NHE. The latter observation shows that the measured activities do not depend on the kinetics of the electron transfer between electrode and type-1 site. If the experiment was started at high nitrite concentration, and the solution was diluted, an increase in catalytic current was observed (results not shown). In addition, the M150T mutant of NiR (in which intramolecular electron transfer from type-1 to type-2 is retarded), also displayed a decrease in icat at higher nitrite concentrations (Fig. S1 of supplemental materials). Thus, it was established that regardless of the nature of the electron donor the catalytic activity of NiR is optimized at a particular nitrite concentration and drops when this level is exceeded.

Equation 2 not only accounted for the kinetic data obtained below pH 6.5 but gave a better fit than the HMM Equation 3 at higher pH. At higher nitrite concentrations the best fits to Equation 3 deviate significantly from the experimental points (Fig. 3A, only fits to Equation 3 are shown). To check whether the observed extra activity at high nitrite concentration was not caused by a co-purified contaminant (such as proteolytically degraded enzyme or redox-active metal ions) two independent batches of NiR were used under otherwise identical conditions (Fig. 3A). Both batches gave the same deviations from HMM kinetics (Fig. 3A) and, when fitted to the HMM equation, produced the same Michaelis constants (47 ± 6 µM and 48 ± 5 µM). Thus, the deviation from HMM kinetics is characteristic of the intact NiR and is not caused by a contaminant.


Figure 4
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  		FIGURE 4.
Kinetic constants of electrode-immobilized NiR versus pH. Open circles denote the kinetic constants of wt NiR determined on a pyrolytic graphite edge electrode held at –70 mV versus NHE, closed circles denote those determined at +110 mV versus NHE, and gray-filled squares denote the data points of NiR M150T determined at –70 mV versus NHE (no data points are shown in B because Formula was difficult to determine because of the low amplitude). The thin line in D is a linear fit of log Formula versus pH of the data points at +110 mV, the dashed line is a fit to the data points at –70 mV for wt, and the thick line is a fit to the data points of M150T. In B and D no data points are shown above pH 7 because above this pH no saturation is observed and thus no data could be obtained. Formula versus pH; B, Formula versus pH; C, Formula versus pH; D, Formula versus pH.

 
With benzyl viologen as electron donor, identical deviations from HMM occurred reproducibly above pH 6 (Fig. 3B; the thin line is a fit according to HMM kinetics, Table 1). An Eadie-Hofstee plot, in which deviations from HMM kinetics appear as deviations from linearity, showed the same, non-linear, trends for assays on the electrode and in solution (Fig. 3, A and B, insets). It was possible, however, to fit the activity versus nitrite concentration to Equation 2 (Fig. 3B, thick line, and Table 1). Also for NiR M150T a substrate activation-like phenomenon occurred (results not shown). Thus, over the measured pH range the data are fit most satisfactorily assuming a random-sequential mechanism according to Equation 2. Because Formula below pH 6.5 and Formula above pH 6.5 (Table 1), it appears that electron transfer to the type-2 site is retarded if nitrite replaces a water molecule (below pH 6.5) but becomes faster above pH 6.5 where the nitrite replaces a hydroxyl ion.


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  		TABLE 1
Effect of pH on kinetic constants of NiR in solution assays

The reported errors are S.D. BV-SDT, benzyl viologen reduced in situ by sodium dithionite; BV, reduced benzyl viologen was prepared electrochemically. See "Experimental Procedures" for experimental details.

 
Kinetic Constants Versus pH—At the rotating disk electrode, a far lower background activity was observed than in solution assays, which made it possible to measure, accurately, Formula, Formula, and the ratio of Formula over a wide pH range (Fig. 4). The individual trends of Formula and Formula versus pH could not be determined accurately because the currents depended on the enzymatic coverage, which was only reproducible within 50% between different protein films (this is not apparent in Fig. 3A where results are reported on two different samples, which happen to exhibit similar surface coverages, but it can be clearly seen in Fig. 4A where, e.g. around pH 5.5, icat varies from 200 to 325 nA for wt NiR at –70 mV versus NHE). Even so, a plot of i Acat (Equation 1) versus pH determined for wt NiR at +110 mV versus NHE (Fig. 4A, closed circles) agrees with the activity versus pH at 500 µM nitrite when pseudoazurin is used as the electron donor (Fig. 1A). Although the data points at –70 mV exhibit a larger spread a similar trend is still observed in this case. Above pH 9 the catalytic currents for wt NiR were too low to be observed, whereas below pH 4 the higher background activity and the lower catalytic currents of NiR prevented measurements. For NiR M150T the observed currents were far lower (Fig. 4A), and as a result the observable range was limited from pH 4.5 to 7.5.

The Formula ratio was ~0.3 below pH 6.5 (Fig. 4B), whereas above pH 7 Formula was >1 (results not shown, exact values could not be obtained because Formula is larger than the experimentally accessible range of nitrite concentration). Below pH 6.0 in bulk solution (Table 1), and for M150T on the electrode, most data points for Formula were less accurate because of difficulties with subtracting the background activity at high nitrite concentration. For M150T around pH 6.5, the ratios were approximately the same as for the wt NiR (at pH 6.76 the ratio was 0.46, and at pH 6.57 it was 0.36).


Figure 5
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  		SCHEME 1.
Proposed catalytic mechanism of NiR. The catalytic and product release step k6 is in fact reversible (20), which is not modeled because in the current experiments the build-up of the product nitric oxide is prevented. The nitrite is depicted as deprotonated but it may also be protonated in the catalytic cycle.

 
For M150T the Formula was 2–10-fold lower than for wt NiR (Fig. 4C). A plot of log Formula versus pH (Fig. 4D) revealed a slope of 1 for both wt NiR and NiR M150T. For wt, there was no significant difference between the Formula values at –70 mV and at 110 mV. The values of M150T were two times lower than those of the wt NiR at the same electrode potential. The logarithmic plot of the solution data of Formula versus pH also displays a slope of ~1 (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Random-sequential Mechanism—The simplest explanation for our data invokes a random-sequential mechanism that accounts for nitrite binding and type-2 site reduction (Scheme 1). At low nitrite concentration, reduction of the type-2 site is faster than nitrite binding to the oxidized site (A route), whereas at high nitrite concentrations the binding is faster and the reaction is diverted through the B route. Below pH 6.5, we find that the B route is slower than the A route (indicated by Formula) because the rate-limiting step of electron transfer to the type-2 site is slower with nitrite-bound than with H2O-bound, in agreement with literature reports that electron transfer from the type-1 to the type-2 site is slower in the presence of nitrite (43–46) and that electron transfer is a rate-limiting step for turnover of the NiR (18, 44, 47). Above pH 6.5, the B route is faster than the A route (Formula), because the type-2 H2O ligand (which is easily substituted by nitrite) is replaced by/converted to OH– (18, 19), and electron transfer to the type-2 site is faster with nitrite bound than with OH– (Table 1, Fig. 3). Also, the absolute magnitude and pH dependence of Formula, which corresponds to the nitrite binding step in Scheme 1 (step 1), agrees with the Formula for nitrite (see above), consistent with the random-sequential mechanism. In the next three paragraphs we consider a number of alternatives to explain our data.

According to an ordered mechanism electron transfer from type-1 to type-2 occurs to either the H2O/OH–-bound or the nitrite-bound type-2 site. Such a sharp division is not observed in pulse-radiolysis experiments (43–46). Secondly, with assays employing a strong and fast electron donor, like benzyl viologen or methyl viologen, it is expected that at low nitrite concentrations a large proportion of the NiR will have both the type-1 and the type-2 site reduced. With viologens as the electron donor, the Formula (Table 1) and kcat values of NiR (see "Experimental Procedures") are similar to those found in the presence of the physiological electron donor, which is only able to reduce the type-1 site. This observation suggests that nitrite can bind to the reduced type-2 site and that the "nitrite-binds-prior-to-reduction" ordered mechanism does not apply. Assuming a random-sequential mechanism also obviates the need to postulate the existence of a sensor loop between type-1 and type-2 site (22, 48).

An alternative explanation for the observed substrate inhibition could be that a second nitrite may bind to the type-2 site (Scheme S1 in supplemental materials). We can rule out this possibility on the basis of the available crystallographic evidence. In nine crystal structures of oxidized NiR and five crystal structures of reduced NiR (16, 21, 49–53) only one nitrite is found at the type-2 site.

Against a "reduction-before-nitrite-binding" ordered mechanism (thereby supporting a nitrite-binding-prior-to-reduction ordered mechanism) it has been argued that reduction of type-2 site before nitrite binding may result in a three-coordinated inactive form (22). We have data5 indicating that after a reduction of the type-2 site a slow reversible isomerization to a reduced inactive form may occur (Scheme 1, step 5). However this step is slow compared with step 4 at nitrite concentrations above Formula, and the possible occurrence of this state is still compatible with a random-sequential mechanism.

The Effect of pH and Nitrite Concentration on Catalysis—Our data on the pH dependence of the catalytic activity of NiR (Fig. 1) are in agreement with literature data (54). Also our Formula value at pH 7 (79 ± 12 µM) with benzyl viologen as the electron donor agrees with the value of 74 µM determined by Kakutani et al. (54) with methyl viologen as the electron donor.

In this work, for the first time the effect of nitrite concentration on the activity at low pH is studied. An all determining factor is the logarithmic dependence of Formula on pH, i.e. the affinity constant for the B route. Our data show that below pH 6 the Formula becomes low enough that at typical assay conditions (~1 mM nitrite) catalysis via the B route begins to prevail, resulting in a drop in activity (indicated by Formula, Fig. 4B). Above pH 6, the Formula is so high that in typical activity assays most catalysis occurs via the A route. Thus, from pH 6 to pH 5 the loss of activity observed in plots of activity versus pH (17, 18, 25, 54, 55), can be assigned to the increasing prevalence of the slower B route. Below pH 5 the Formula and Formula values drop dramatically and at pH < 4 they fall below the detection limit (Fig. 4A). The simplest explanation for this is that the type-1 to type-2 electron transfer rate decreases below this pH, as it does in related nitrite reductases (44).

A key feature of Scheme 1, in agreement with literature data (43–46) is that the rate-limiting electron transfer rate between type-1 and type-2 site depends on the ligand that is bound to the type-2 site. The rates decrease in the order H2O > nitrite > OH–. The slower electron transfer to the type-2 site with nitrite/OH– bound may be because of a lower midpoint potential of the type-2 site and/or a higher reorganization energy (56) than with H2O as a ligand. In fact in pulse-radiolysis experiments a 100-mV decrease in type-2 site midpoint potential is found when the pH is raised from 6 to 8 (44).

The up to 10 times lower values of Formula for NiR M150T illustrate the dependence of this parameter on the electron-transfer kinetics. With slower electron transfer,5 Km will decrease because saturation will occur at lower nitrite concentration. It appears that the effect of a slower electron transfer rate is more pronounced for Formula (Fig. 4C) than for Formula (Fig. 4D), whereas Formula also follows a complicated behavior versus pH (for a simple protonation/deprotonation equilibrium slopes equal to 1, 0, or –1 are expected in a logarithmic plot). Apparently Formula is less sensitive than Formula to the various kinetic parameters in the system and thus provides a better probe of nitrite binding.

The linear dependence of log Formula on pH suggests that either a chemical group in NiR or nitrite itself needs to be protonated before catalysis and because no leveling off is observed that the pKa is < 4.5. The corresponding acid could be the nitrite (pKa = 3.25), the active site aspartate (usual pKa range in proteins (31) is 2–5.5) that is known to hydrogen bond to the bound nitrite (14, 16, 18), or the active site His-255 (usual range for histidine is 5–8). The linear dependence of log Formula on pH is similar to that found (25) for the Formula value of the type-2 site of Alcaligenes xylosoxidans NiR (in frozen samples < 30 µM at pH 5.2, 350 µM at pH 7.5). EPR experiments on A. faecalis S-6 NiR as a function of pH6 have shown that the Formula for the type-2 site ({approx}1 mM at pH 6, {approx}10 mM at pH 7 in frozen samples) is indeed approximately equal to the Formula (see Fig. 4D).

Conclusions—A random-sequential mechanism is in agreement with the steady-state kinetics observed for wt and M150T NiR with all tested electron donors. At low nitrite concentration, electron transfer occurs prior to nitrite binding, whereas at higher nitrite concentration nitrite binding occurs first. The velocity of the rate-limiting electron transfer step between the type-1 and the type-2 site depends on the ligand that is bound at the type-2 site and decreases in the order H2O > nitrite > OH–. The Formula and Formula values of NiR are similar and show a normal pH dependence with pKa < 4.5. The results are in agreement with NiR as a robust machinery that can effectively bind nitrite and catalyze its reduction over a wide range of nitrite concentration and pH.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Fig. S1, equations, and a supplemental scheme. Back

1 Present address: Duke University, Medical Center, Dept. of Biochemistry, Durham, NC 27710. Back

2 To whom correspondence should be addressed. E-mail: Canters{at}chem.leidenuniv.nl.

3 The abbreviations used are: NiR, nitrite reductase; MES, 4-morpholineethanesulfonic acid; NHE, normal hydrogen electrode; HMM, Henri-Michaelis-Menten; wt, wild type. Back

4 H. J. Wijma, I. MacPherson, M. Alexandre, R. E. M. Diederix, G. W. Canters, M. E. P. Murphy, and M. P. Verbeet, submitted for publication. Back

5 H. J. Wijma, L. J. C. Jeuken, M. P. Verbeet, F. A. Armstrong, and G. W. Canters, unpublished results. Back

6 M. Fittipaldi and H. J. Wÿma, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. H. A. Heering and Dr. A. W. J. W. Tepper for useful discussions, Dr. K. Vincent for help with some electrochemical experiments, and R. Zwier for manufacturing excellent equipment.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zumft, W. G. (1997) Microbiol. Mol. Biol. Rev. 61, 533–616[Abstract]
  2. Bange, H. W. (2000) Nature 408, 301–302[CrossRef][Medline] [Order article via Infotrieve]
  3. Robertson, G. P., Paul, E. A., and Harwood, R. R. (2000) Science 289, 1922–1925[Abstract/Free Full Text]
  4. Ichiki, H., Tanaka, Y., Mochizuki, K., Yoshimatsu, K., Sakurai, T., and Fujiwara, T. (2001) J. Bacteriol. 183, 4149–4156[Abstract/Free Full Text]
  5. Adman, E. T., and Murphy, M. E. P. (2001) in Handbook of Metalloproteins (Messerschmidt, A., Huber, R., Poulos, T., Wieghardt, K., eds), pp. 1381–1390, John Wiley & Sons, Ltd., Chicester
  6. Cardinale, J. A., and Clark, V. L. (2000) Infect. Immun. 68, 4368–4369[Abstract/Free Full Text]
  7. Rock, J. D., and Moir, J. W. (2005) Biochem. Soc. Trans. 33, 134–136[CrossRef][Medline] [Order article via Infotrieve]
  8. Sasaki, S., Karube, I., Hirota, N., Arikawa, Y., Nishiyama, M., Kukimoto, M., Horinouchi, S., and Beppu, T. (1998) Biosens. Bioelectron. 13, 1–5[CrossRef][Medline] [Order article via Infotrieve]
  9. Wu, Q., Storrier, G. D., Pariente, F., Wang, Y., Shapleigh, J. P., and Abruña, H. D. (1997) Anal. Chem. 69, 4856–4863[Medline] [Order article via Infotrieve]
  10. Astier, Y., Canters, G. W., Davis, J. J., Hill, H. A. O., Verbeet, M. P., and Wijma, H. J. (2005) Chem. Phys. Chem. 6, 1114–1120[CrossRef][Medline] [Order article via Infotrieve]
  11. Libby, E., and Averill, B. A. (1992) Biochem. Biophys. Res. Commun. 187, 1529–1535[CrossRef][Medline] [Order article via Infotrieve]
  12. Godden, J. W., Turley, S., Teller, D. C., Adman, E. T., Liu, M. Y., Payne, W. J., and LeGall, J. (1991) Science 253, 438–442[Abstract/Free Full Text]
  13. Kukimoto, M., Nishiyama, M., Murphy, M. E., Turley, S., Adman, E. T., Horinouchi, S., and Beppu, T. (1994) Biochemistry 33, 5246–5252[CrossRef][Medline] [Order article via Infotrieve]
  14. Murphy, M. E., Turley, S., and Adman, E. T. (1997) J. Biol. Chem. 272, 28455–28460[Abstract/Free Full Text]
  15. Boulanger, M. J., Kukimoto, M., Nishiyama, M., Horinouchi, S., and Murphy, M. E. (2000) J. Biol. Chem. 275, 23957–23964[Abstract/Free Full Text]
  16. Boulanger, M. J., and Murphy, M. E. (2001) Biochemistry 40, 9132–9141[CrossRef][Medline] [Order article via Infotrieve]
  17. Kataoka, K., Furusawa, H., Takagi, K., Yamaguchi, K., and Suzuki, S. (2000) J. Biochem. (Tokyo) 127, 345–350[Abstract/Free Full Text]
  18. Zhao, Y., Lukoyanov, D. A., Toropov, Y. V., Wu, K., Shapleigh, J. P., and Scholes, C. P. (2002) Biochemistry 41, 7464–7474[CrossRef][Medline] [Order article via Infotrieve]
  19. Ellis, M. J., Dodd, F. E., Strange, R. W., Prudencio, M., Sawers, G., Eady, R. R., and Hasnain, S. S. (2001) Acta. Crystallogr. Sect. D Biol. Crystallogr. 57, 1110–1118[CrossRef][Medline] [Order article via Infotrieve]
  20. Wijma, H. J., Canters, G. W., de Vries, S., and Verbeet, M. P. (2004) Biochemistry 43, 10467–10474[CrossRef][Medline] [Order article via Infotrieve]
  21. Adman, E. T., Godden, J. W., and Turley, S. (1995) J. Biol. Chem. 270, 27458–27474[Abstract/Free Full Text]
  22. Strange, R. W., Murphy, L. M., Dodd, F. E., Abraham, Z. H., Eady, R. R., Smith, B. E., and Hasnain, S. S. (1999) J. Mol. Biol. 287, 1001–1009[CrossRef][Medline] [Order article via Infotrieve]
  23. Hulse, C. L., and Averill, B. A. (1989) J. Am. Chem. Soc. 111, 2322–2323[CrossRef]
  24. Averill, B. A. (1996) Chem. Rev. 96, 2951–2964[CrossRef][Medline] [Order article via Infotrieve]
  25. Abraham, Z. H., Smith, B. E., Howes, B. D., Lowe, D. J., and Eady, R. R. (1997) Biochem. J. 324, 511–516
  26. Wasser, I. M., de Vries, S., Moenne-Loccoz, P., Schroder, I., and Karlin, K. D. (2002) Chem. Rev. 102, 1201–1234[CrossRef][Medline] [Order article via Infotrieve]
  27. Kashem, M. A., Dunford, H. B., Liu, M. Y., Payne, W. J., and LeGall, J. (1987) Biochem. Biophys. Res. Commun. 145, 563–568[CrossRef][Medline] [Order article via Infotrieve]
  28. Halfen, J. A., Mahapatra, S., Olmstead, M. M., and Tolman, W. B. (1994) J. Am. Chem. Soc. 116, 2173–2174[CrossRef]
  29. Casella, L., Carugo, O., Gulotti, M., Doldi, S., and Frassoni, M. (1996) Inorg. Chem. 35, 1101–1113[CrossRef][Medline] [Order article via Infotrieve]
  30. Monzani, E., Anthony, G. J., Koolhaas, A., Spandre, A., Leggieri, E., Casella, L., Gulotti, M., Nardin, G., Randaccio, L., Fontani, M., Zanello, P., and Reedijk, J. (2000) J. Biol. Inorg. Chem. 5, 251–261[CrossRef][Medline] [Order article via Infotrieve]
  31. Fersht, A. (1985) in Enzyme Structure and Mechanism, pp. 55, 133, 156, and 256, W. H. Freeman and Company, New York
  32. Brenner, A. J., and Harris, E. D. (1995) Anal. Biochem. 226, 80–84[CrossRef][Medline] [Order article via Infotrieve]
  33. Kakutani, T., Watanabe, H., Arima, K., and Beppu, T. (1981) J. Biochem. (Tokyo) 89, 463–472[Abstract/Free Full Text]
  34. Stankovich, M. T. (1980) Anal. Biochem. 109, 295–308[CrossRef][Medline] [Order article via Infotrieve]
  35. van Leeuwen, J. W., van Dijk, C., and Veeger, C. (1983) Eur. J. Biochem. 135, 601–607[Medline] [Order article via Infotrieve]
  36. Wijma, H. J., Boulanger, M. J., Molon, A., Fittipaldi, M., Huber, M., Murphy, M. E., Verbeet, M. P., and Canters, G. W. (2003) Biochemistry 42, 4075–4083[CrossRef][Medline] [Order article via Infotrieve]
  37. Heering, H. A., Hirst, J., and Armstrong, F. A. (1998) J. Phys. Chem. B. 102, 6889–6902[CrossRef]
  38. Léger, C., Heffron, K., Pershad, H. R., Maklashina, E., Luna-Chavez, C., Cecchini, G., Ackrell, B. A., and Armstrong, F. A. (2001) Biochemistry 40, 11234–11245[CrossRef][Medline] [Order article via Infotrieve]
  39. Jones, A. K., Lamle, S. E., Pershad, H. R., Vincent, K. A., Albracht, S. P. J., and Armstrong, F. A. (2003) J. Am. Chem. Soc. 125, 8505–8514[CrossRef][Medline] [Order article via Infotrieve]
  40. Kukimoto, M., Nishiyama, M., Ohnuki, T., Turley, S., Adman, E. T., Horinouchi, S., and Beppu, T. (1995) Protein Eng. 8, 153–158[Abstract/Free Full Text]
  41. Kukimoto, M., Nishiyama, M., Tanokura, M., Adman, E. T., and Horinouchi, S. (1996) J. Biol. Chem. 271, 13680–13683[Abstract/Free Full Text]
  42. Impagliazzo, A., and Ubbink, M. (2004) J. Am. Chem. Soc. 126, 5658–5659[CrossRef][Medline] [Order article via Infotrieve]
  43. Suzuki, S., Deligeer, Yamaguchi, K., Kataoka, K., Kobayashi, K., Tagawa, S., Kohzuma, T., Shidara, S., and Iwasaki, H. (1997) J. Biol. Inorg. Chem. 2, 265–274[CrossRef]
  44. Kobayashi, K., Tagawa, S., Deligeer, and Suzuki, S. (1999) J. Biochem. (Tokyo) 126, 408–412[Abstract/Free Full Text]
  45. Suzuki, S., Furusuwa, H., Kataoka, K., Yamaguchi, K., Kobayashi, K., and Tagawa, S. (2000) Inorg. React. Mechan. 2, 129–135
  46. Suzuki, S., kataoke, K, Yamaguchi, K, Inoue, T., and Kai, Y. (1999) Coord. Chem. Rev. 190–192, 245–265[CrossRef]
  47. Deligeer, Fukunaga, R., Kataoka, K., Yamaguchi, K., Kobayashi, K., Tagawa, S., and Suzuki, S. (2002) J. Inorg. Biochem. 91, 132–138[CrossRef][Medline] [Order article via Infotrieve]
  48. Hough, M. A., Ellis, M. J., Antonyuk, S., Strange, R. W., Sawers, G., Eady, R. R., and Hasnain, S. S. (2005) J. Mol. Biol. 350, 300–309[CrossRef][Medline] [Order article via Infotrieve]
  49. Dodd, F. E., Hasnain, S. S., Abraham, Z. H. L., Eady, R. R., and Smith, B. E. (1997) Acta Crystallogr. Sect. D 53, 406–418[CrossRef][Medline] [Order article via Infotrieve]
  50. Dodd, F. E., Van Beeumen, J., Eady, R. R., and Hasnain, S. S. (1998) J. Mol. Biol. 282, 369–382[CrossRef][Medline] [Order article via Infotrieve]
  51. Tocheva, E. I., Rosell, F. I., Mauk, A. G., and Murphy, M. E. P. (2004) Science 304, 867–870[Abstract/Free Full Text]
  52. Boulanger, M. J., and Murphy, M. E. P. (2003) Protein Sci. 12, 248–256[CrossRef][Medline] [Order article via Infotrieve]
  53. Murphy, M. E., Turley, S., Kukimoto, M., Nishiyama, M., Horinouchi, S., Sasaki, H., Tanokura, M., and Adman, E. T. (1995) Biochemistry 34, 12107–12117[CrossRef][Medline] [Order article via Infotrieve]
  54. Kakutani, T., Watanabe, H., Arima, K., and Beppu, T. (1981) J. Biochem. (Tokyo) 89, 453–461[Abstract/Free Full Text]
  55. Iwasaki, H., and Matsubara, T. (1972) J. Biochem. (Tokyo) 71, 645–652[Abstract/Free Full Text]
  56. Marcus, R. A., and Sutin, N. (1985) Biochim. Biophys. Acta 811, 265–322

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