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J. Biol. Chem., Vol. 276, Issue 29, 26995-27002, July 20, 2001

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Pre-steady-state Kinetic Analysis of Recombinant Arabidopsis NADH:Nitrate Reductase

RATE-LIMITING PROCESSES IN CATALYSIS^*

Lawrie Skipper, Wilbur H. Campbell^§¶, Jeffrey A. Mertens^§, and David J. Lowe

From the  Biological Chemistry Department, John Innes Centre, Norwich NR4 7UH, United Kingdom and the ^§ Department of Biological Sciences, Michigan Technological University, Houghton, Michigan 49931

Received for publication, January 16, 2001, and in revised form, April 24, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

Recombinant Arabidopsis NADH:nitrate reductase was expressed in Pichia pastoris using fermentation. Large enzyme quantities were purified for pre-steady-state kinetic analysis, which had not been done before with any eukaryotic nitrate reductase. Basic biochemical properties of recombinant nitrate reductase were similar to natural enzyme forms. Molybdenum content was lower than expected, which was compensated for by activity calculation on molybdenum basis. Stopped-flow rapid-scan spectrophotometry showed that the enzyme FAD and heme were rapidly reduced by NADH with and without nitrate present. NADPH reduced FAD at less than one-tenth of NADH rate. Reaction of NADH-reduced enzyme with nitrate yielded rapid initial oxidation of heme with slower oxidation of flavin. Rapid-reaction freeze-quench EPR spectra revealed molybdenum was maintained in a partially reduced state during turnover. Rapid-reaction chemical quench for quantifying nitrite production showed that the rate of nitrate reduction was initially greater than the steady-state rate, but rapidly decreased to near steady-state turnover rate. However, rates of internal electron transfer and nitrate reduction were similar in magnitude with no one step in the catalytic process appearing to be much slower than the others. This leads to the conclusion that the catalytic rate is determined by a combination of rates with no overall rate-limiting individual process.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

Nitrate reductase (NR,¹ EC 1.6.6.1-3) is a molybdenum-containing enzyme, which catalyzes the pyridine nucleotide-dependent reduction of nitrate to nitrite in plants, fungi, and algae (1, 2). NR contains an internal electron transfer system consisting of an FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). The internal redox cofactors, which have a stoichiometry of 1:1:1 for FAD:heme:Mo-MPT, are bound to independently folding domains of the ~100-kDa polypeptide, which forms homodimers and -tetramers in the active enzyme (3). NR has two active sites: one for NAD(P)H to donate electrons to the FAD, which is connected via a cyt b domain to the Mo-MPT domain that possesses the nitrate reducing activity. The structure of the recombinant cyt b reductase fragment of corn NADH:NR, representing the C-terminal 270 residues containing the FAD and NADH binding sites, has been determined (4, 5). The structure of the cyt b reductase fragment demonstrates that this portion of the enzyme is related to the ferredoxin NADP⁺ reductase family of enzymes (6). The N-terminal region of NR, containing the Mo-MPT binding site and nitrate-reducing active site, is related by sequence similarity to sulfite oxidase, another Mo-MPT-containing enzyme, for which a structure was recently determined (7). The bridge between these parts of NR is the cyt b domain, which is related by sequence similarity to mammalian cyt b₅ and to sulfite oxidase's cyt b domain (1, 7). Thus, a model for holo-NR has been generated by combining atom replacement models of the N-terminal Mo-MPT and cyt b domains with the cyt b reductase structure (1, 5). The holo-NR model is dimeric with the dimer interface domain modeled after sulfite oxidase (1, 7). However, in the sulfite oxidase structure, the heme in the cyt b domain is 32 Å from the Mo-MPT center in the sulfite-oxidizing domain. It has been suggested these prosthetic groups in sulfite oxidase must move closer during catalysis for efficient electron transfer (8), since the rate of electron transfer is close to 1,000 s¹ between the centers in sulfite oxidase and the rate predicted by current models is <100 s¹ for a 32-Å separation (9). However, it is conceivable that electron transfer between centers in different subunits is more efficient than the intrasubunit process. Evidence for a shift of the coordinating ligands to molybdenum in NR, when going from resting to turnover forms, was recently obtained by x-ray absorption spectroscopy (10). Indeed, domain-domain movement to an enzyme conformation required for efficient electron transfer may be part of the catalytic mechanism of NR.

Although structural aspects of NR biochemistry remain unresolved, steady-state kinetic studies have contributed significantly to our understanding of enzyme catalysis (1, 2). The two-site ping-pong steady-state kinetic mechanism best describes the interactions of the substrates, NAD(P)H and nitrate, with the enzyme's two active sites (1). The catalytic efficiency (k_cat/K_m) of the pyridine nucleotide electron donation active site is about 2 orders of magnitude greater than that of the nitrate-reducing active site. In contrast to many other redox enzymes, NR activity is not limited by its rate of reduction and it has a high k_cat of ~200 s¹ (1). The high rate of reduction of the enzyme's FAD by NADH has been demonstrated in kinetic studies of the recombinant cyt b and cyt c reductase fragments of NR, which had FAD reduction rates of 474 and 560 s¹ at 10 °C, respectively (11, 12). This suggested that NR catalysis is limited either by internal electron transfer to molybdenum, or the rate of nitrate reduction at the molybdenum center, or the rate of release of nitrite. Furthermore, based on the reduced dye (methyl viologen and bromphenol blue) nitrate-reducing activity of NR, which exceeds NADH:NR activity, it has been suggested that internal electron transfer from heme to molybdenum is rate-limiting in NR (1, 2, 13, 14).

We report here the first determination of rates of internal electron transfer within holo-NR, the reduction status of redox centers during steady-state turnover, and the catalytic rate for nitrate reduction by fully reduced enzyme. These experiments were made possible by recombinant expression of Arabidopsis NIA2 (AtNR2) in Pichia pastoris (15). Using a fermenter to produce the enzyme permitted us to purify the large quantities of NR needed for pre-steady-state kinetic analysis involving all three redox centers, including stopped-flow, rapid-scan spectrophotometry of the FAD and heme centers, and rapid-reaction, freeze-quench EPR of the molybdenum center, FAD semiquinone and heme-iron. Rapid reaction of NADH-reduced NR with nitrate, which was quenched by zinc acetate and followed by quantification of the nitrite formed, resulted in a direct evaluation of the rate of formation of nitrite. We find that molybdenum does not remain fully reduced in steady-state turnover, when approached from the fully reduced state, indicating that internal electron transfer to molybdenum is at least a partially rate-limiting step under these conditions. However, when fully reduced enzyme reacts with nitrate, and nitrite production determined over the first 100 ms, the initial rate of nitrite formation is faster than the steady-state rate. This suggests that there is a rough equality between the electron transfer and nitrate reduction rates with neither process independently limiting the catalytic activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

Recombinant AtNR2 was expressed in P. pastoris as described previously (15), except that a Bio-Flo3000 fermenter (New Brunswick Scientific Co., Inc., Edison, NJ) was used to grow the cells to high density at 30 °C. The cells were lysed at 2-4 °C with a continuous-flow Dyno Mill model KDL (Glen Mills, Clifton, NJ) with 0.6-liter stainless steel grinding container filled with glass beads using a flow rate of 10 liters h¹ and two passes, as described previously (16). After centrifugation to remove cell debris, AtNR2 was purified by the method described previously (10, 15). The enzyme was shown to be homogeneous by denaturing polyacrylamide gel electrophoresis (3, 16). NADH:NR activity was determined by the rate of nitrite formation (3) and also by monitoring the rate of oxidation of NADH in a spectrophotometer. The molar concentration of AtNR2 was determined at 413 nm using  = 120 mM¹ cm¹ (3). The molybdenum content of AtNR2 was determined by inductively coupled plasma mass spectrometry (University of Georgia, Chemical Analysis Laboratory, Athens, GA) on four different enzyme preparations with the results averaged after correction for background, using buffer as the background matrix. The MPT content of AtNR2 was determined as described previously (17). Anaerobic redox titrations were done as previously described (16), except for EPR analysis of molybdenum, which was done in an anaerobic glove box by poising AtNR2 at various potentials versus a calomel electrode using sodium dithionite and potassium ferricyanide, with a mixture of redox dyes to allow efficient equilibration (18) and freezing the samples in EPR tubes using liquid nitrogen. The midpoint redox potentials are expressed relative to the standard hydrogen electrode. For EPR, anaerobic electron titration was done in a glove box by titrating NR with aliquots of NADH calculated to add 0.5 electron eq/heme and the series of aliquots frozen in liquid nitrogen in EPR tubes. For UV-visible spectral analysis, anaerobic electron titration was done in a cuvette with a septum-containing screw cap (Starna Cells, Inc., Atascadero, CA), where anaerobic NR was titrated with anaerobic NADH, added in 0.5-electron eq steps with a gas-tight syringe. Before the first addition and after each addition and brief mixing, the solution was scanned using an HP8452A UV-visible diode array spectrophotometer at 22 °C.

Stopped-flow rapid-scan spectrophotometry was done on a Hi-Tech KinetAsyst double mixing stopped-flow system (Hi-Tech Scientific, Wiltshire, United Kingdom) within an anaerobic glove box, using the KinetaScan detector, as described previously (16). The mixing manifold and glove box environment were thermostatically controlled to provide temperatures from 5 °C to 25 °C. Data were analyzed using Hi-Tech KinetAsyst 2.0 and SPECFIT 3.0.12 (Spectrum Software Associates, Chapel Hill, NC) programs. Molar concentrations of NADH, NADPH, and nitrate were determined by spectrophotometry using appropriate extinction coefficients. The buffer in all experiments was 25 mM MOPS, pH 7.0. NADH, NADPH, and MOPS were from Sigma, and all other chemicals were of the purest grade available. In most experiments, the Hi-Tech KinetaScan diode array detector was used in the multiwavelength mode and spectra were recorded from 350 to 700 nm (Fig. 1), although rates at particular wavelengths were checked in the single wavelength mode to ensure photolysis was not occurring. The stopped flow apparatus of this system has a dead time of ~1 ms and to observe the most rapid changes, 100 spectra were taken over the first 200 ms of the reactions. To observe changes in the oxidation state of FAD, the A₄₆₀ was analyzed, whereas for the heme-iron, A₅₅₇ was analyzed since the Sorét peaks at A₄₁₃ and A₄₂₄ overlap.

Rapid-freeze and rapid-quench reactions were carried out as described previously (19). The sample syringes, mixing chamber, and aging tubing were held in a water bath at various temperatures as described under "Results and Discussion." Differing reaction times are achieved by varying the length of the aging tubing without altering the flow rate of reactants. For freeze-quench rapid reactions, the contents of the aging tubing were expelled into a vessel containing isopentane at 140 °C, which is joined to an EPR tube. The frozen reaction mixture "snow" is tamped into the EPR tube with a rod having a Teflon^® head with vents for the isopentane. EPR spectroscopy was carried out on an updated Bruker ER 200 SH spectrometer with samples maintained at 15 and 80 K using an ER 900 liquid helium boil-off system (Oxford Instruments). The EPR spectrometer was set up with a modulation frequency of 100 kHz, modulation amplitude of 0.2607 millitesla, microwave frequency of 9.4 ± 0.1 GHz, and microwave power of 5.02 milliwatts. EPR spectra were analyzed with the Bruker Win-EPR 2.11 program. For rapid reactions to measure nitrite formation, the reaction mixtures were expelled into microcentrifuge tubes containing 0.2 ml of 0.5 M zinc acetate. After centrifugation and reaction with the nitrite determination reagents (3), the absorbance at 540 nm was taken and quantity of nitrite determined using a standard curve prepared with nitrite standards. The progress of the reaction was fitted to the function [nitrite] = A(1  e^kt) + Bt, with A and k varied to give a least squares fit to the data using the "Solver" function of Excel, and B set to the steady-state activity of the enzyme of 20 s¹.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

Basic Biochemical Properties of Recombinant AtNR2-- Recombinant AtNR2 was produced in P. pastoris using fermenter cultures with a gene construct described previously (15). Up to 20 mg of AtNR2 was produced per liter of culture in the fermenter. The enzyme was purified to homogeneity and gave specific activities ranging from 20 to 40 µmol of nitrite produced min¹ mg¹ using AMP-Sepharose and blue Sepharose affinity chromatographies, and enzyme was eluted by NADH from both media (10, 15). Yields were ~2 mg of NR/100 g of wet P. pastoris cells. In initial studies, electrophoretically homogeneous enzyme was used and in subsequent experiments, less pure enzyme prepared by just AMP-Sepharose was used to make larger quantities available. Analysis of AtNR2 gave a molybdenum content of 0.20 ± 0.02 mol/mol of heme, which agreed with a low molybdenum content found by x-ray absorption spectroscopy (10). The MPT content was similar to the molybdenum content at 0.18 mol/mol of heme. Analysis by native gradient gel electrophoresis and gel filtration indicated recombinant AtNR2 was mostly tetrameric with a small amount of dimer (Ref. 16; data not shown). These results indicate the majority AtNR2 enzyme molecule is a tetramer with one subunit containing FAD, heme-iron, and molybdenum and the other three subunits containing only FAD and heme-iron, although minority species containing different proportions are likely to be present statistically.

Under anaerobic conditions, the visible spectra of oxidized and NADH-reduced AtNR2 were very similar to those reported previously for eukaryotic NR forms (Fig. 1). The extinction coefficients for the wavelengths of interest in this study were determined (Table I). The values were based on the previously reported extinction coefficient for the Sorét peak of oxidized squash NADH:NR at 413 nm (3), and the values for reduced AtNR2 agree well with those for reduced squash NR. The extinction coefficients for NADPH-reduced enzyme were virtually identical. The data indicate that the FAD and heme were spectrally identical in all the subunits of the holo-enzyme despite the absence of the Mo-MPT cofactor from some of its subunits. EPR spectra of oxidized and fully reduced AtNR2 were devoid of signals at 80 K, whereas the oxidized heme-iron of NR's internal cyt b, observed at 15 K by EPR, had a typical low spin ferric heme spectrum. When AtNR2 was reduced with 1 eq of NADH/enzyme heme under anaerobic conditions, a strong Mo^V signal was observed at 80 K and a diminished heme-iron signal at 15 K. These EPR spectra were similar to those reported previously for spinach and Chlorella NR (20-22). Anaerobic redox titrations of AtNR2 showed the FAD had a similar mid point potential at 270 mV to those found for NR and its FAD-containing fragments, whereas the heme was less negative at 50 mV than previously reported for holo-NR (1, 2, 11, 16, 23). Anaerobic redox titration of the molybdenum observed by EPR at 80 K yielded the Mo^VI to Mo^V couple at 40 mV and the Mo^V to Mo^IV couple at 30 mV, which gives an average for the Mo^VI to Mo^IV couple at 5 mV. This value is similar to spinach and Chlorella NR at 25 °C, but significantly more positive than found for Chlorella NR at 173 K (24-26). Thus, it appears that the molybdenum center in AtNR2 is similar in redox properties to other NR forms.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Visible spectra of oxidized and NADH-reduced AtNR2. Spectra were recorded with the Hi-Tech stopped-flow rapid-scan System with enzyme (13 µM) mixed with 25 mM MOPS, pH 7.0, under anaerobic conditions at 5 °C, for the oxidized. Reduced spectrum was generated by reducing enzyme with 120 µM NADH. The 1- and 200-ms spectra were recorded during the reaction of NADH-reduced AtNR2 (13 µM) with 75 µM nitrate. All spectra were converted from absorbance to extinction coefficient by assuming mM = 120 mM1 cm1 (3).

Steady-state Kinetic Constants and Electron Titration of AtNR2-- Using an NADH oxidation progress curve with the stline curve fitting method (27), the K_m for NADH was determined to be 0.7 ± 0.3 µM at 30 °C, which is slightly lower than previously reported values (1). The nitrate K_m was 90 ± 20 µM, which is similar to Chlorella NR but higher than some other NR forms (1, 13, 26). The steady-state k_cat is 210 ± 30 s¹ when based on the molybdenum content. This compares well with the "intrinsic" k_cat values for other NADH:NR forms with known molybdenum content (Table II; Refs. 3 and 28). The k_cat/K_m values for NADH and nitrate are 300 and 2.3 µM¹ s¹, respectively, which are similar to previously reported values (1). Overall, AtNR2 appears to be similar in steady-state kinetic properties to other NR forms despite its low Mo-MPT content, which suggests that the fully functional portion of the enzyme is not perturbed into an unusual state by the absence of molybdenum from some of its subunits or by being folded differently in the recombinant environment.

Anaerobic electron titration of AtNR2 using NADH was carried out in two separate experiments (Fig. 2). The first experiment was analyzed by EPR and showed that the Mo^V signal peaked at an integrated intensity of 0.20 ± 0.04 electrons/molybdenum after addition of 2 electrons/enzyme heme, whereas the cyt b signal decreased in a more or less linear fashion with each addition of NADH until the enzyme reached capacity at ~4 electrons/subunit, based on heme content. The other experiment was done using visible spectral analysis, and the cyt b was reduced by NADH under anaerobic conditions to the same extent as in the corresponding EPR experiment (Fig. 2). FAD remained oxidized until 2 electrons/subunit had been added and was fully reduced by ~4 electrons/heme. These results are in reasonable agreement with the AtNR2 model presented above with 1 subunit fully loaded with cofactors, which would be fully reduced with 5 electrons, and 3 subunits with only FAD and heme-iron, which would be fully reduced with 3 electrons each. Thus, fully reduced AtNR2 in our preparations has capacity for a total of 14 electrons or 3.5 electrons/heme.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Electron titration of AtNR2 with NADH under anaerobic conditions. EPR spectra were taken at 15 and 80 K of enzyme (20 µM) before and after addition of aliquots of NADH representing 0.5 electron eq. Visible spectra were recorded with an HP8452A spectrophotomer with enzyme (2 µM) in an anaerobic cuvette, closed with a septum, before and after addition of 0.5 electron eq of NADH from an anaerobic solution. Both reaction sets were carried out in 25 mM MOPS, pH 7.0, at 22 °C.

The Reductive Half-reaction Measured by Stopped-flow Rapid-scan Visible Spectrophotometry-- When oxidized AtNR2 reacts with NADH or NADPH in the absence of an electron acceptor, biphasic kinetics are observed for reduction of both the FAD and cyt b of the enzyme (Table III). Time courses for AtNR2 reduction by NADH have been published previously (29). For NADH, the rates of reduction of the FAD are greater than those for cyt b during the first phase at lower temperatures. For NADPH, which has a steady-state turnover rate of ~7% of that with NADH, the reduction rates of FAD and cyt b are similar, which is consistent with the rate-limiting event being the initial reduction of the enzyme by this substrate. It is clear from these results that the enzyme's FAD is reduced very rapidly by NADH, but that the electrons are also rapidly transferred along the internal electron pathway to the heme and possibly molybdenum centers. However, it is not possible to observe the reduction of the molybdenum by this method since the Mo-MPT does not have a sufficiently strong visible absorbance (12). It is not possible reliably to deconvolute changes due to NADH oxidation since overlapping changes occur in the absorption of the enzyme. It should also be noted that semiquinone forms of the FAD are not observed in spectra taken during the reductive reaction.

Enzymatic Turnover Measured by Stopped-flow Rapid-scan Visible Spectrophotometry-- In order to determine how AtNR2 responds kinetically to turnover initiated on fully oxidized and reduced enzyme, two principal sets of pre-steady-state experiments were done under anaerobic conditions at temperatures from 5 °C to 25 °C. In one set, oxidized enzyme was mixed with NADH and nitrate (referred to as "turnover 1" conditions), and in the second set, pre-NADH reduced enzyme was mixed with nitrate (referred to as "turnover 2" conditions).

When oxidized AtNR2 reacts with a mixture of NADH and nitrate, the FAD and cyt b, which are 27 and 15% reduced when first observed 1 ms after initiating the reaction, become progressively more reduced during the following 200 ms (Fig. 3). Under turnover 1 conditions, the rates of cofactor reduction are similar to those observed in the reduction reaction with NADH (Table IV). Rates of cofactor reduction are slower with NADPH as electron donor, with the FAD reduction rate being about 10% of the NADH rate; this presumably explains why NADPH supports a slower steady-state rate of nitrate reduction. Under turnover 1 conditions, these reactions are also biphasic at both low and high concentrations of the substrates. In this case, FAD reduction rates using both NADH and NADPH are more rapid than the cyt b reduction rates. Although the results for turnover 1 tend to suggest that nitrate is not able to oxidize reduced enzyme rapidly enough to keep up with the input of electrons via the reduced flavin, an equally plausible explanation is that internal electron transfer from FADH₂ via heme to molybdenum is too slow to maintain a maximum level of nitrate reduction, resulting in an accumulation of electrons in the flavin and heme of the enzyme. The observations are complicated by the low molybdenum content of the enzyme, since electrons will be accumulating in the AtNR subunits lacking Mo-MPT.

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Stopped-flow rapid-scan kinetic time courses for the reaction of AtNR2 with a mixture of NADH and nitrate at 15 °C. Final concentrations were: enzyme = 4 µM; NADH = 13 µM; and nitrate = 15 µM. Reaction buffer was 25 mM MOPS, pH 7.0, and anaerobic conditions were used. The A557 and A460 were extracted from two full data sets of ~1-nm data points for 350 to 700 nm, averaged, and fitted with an equation using two exponentials with the fit shown as a solid line and the averaged data points superimposed. The rate constants for the biphasic reactions were: k1 = 232 ± 8 and k2 = 19.5 ± 0.3, for the reduction of the enzyme's heme at 557 nm; and k1 = 280 ± 10 and k2 = 15.7 ± 0.5, for the reduction of the enzyme's FAD at 460 nm.

When fully reduced enzyme is mixed with nitrate, the oxidation of the cofactors is observed. Under turnover 2 conditions with NADH, the rates of oxidation of FADH₂ and cyt b are equal to or greater than rates of reduction observed under turnover 1 conditions (Table IV). Time courses for cofactor oxidation under turnover 2 conditions at 15 °C are biphasic as steady-state conditions are approached (Fig. 4, left panel). NADH oxidation is linear up to 1.5 s of the reaction when it is ~93% depleted, based on change in A₃₅₀. The k_cat is ~100 s¹ during linear NADH oxidation, which is about one-half the steady-state k_cat at 30 °C. A longer time course, up to 10 s, shows that as NADH is exhausted, the cofactors become progressively more oxidized as the enzyme returns to its oxidized form (Fig. 4, right panel). During this slow approach to the fully oxidized state, the oxidation rates for the cofactors are about 1% of the rates in the approach to steady state and probably reflect rates of intersubunit electron transfer under these conditions, as electrons are transferred slowly to the subunit containing the Mo-MPT and nitrate reducing site within the largely oxidized enzyme.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Stopped-flow rapid-scan kinetic time courses for the reaction of NADH-reduced AtNR2 with nitrate at 15 °C. Final concentrations were: enzyme = 2 µM; NADH = 40 µM; and nitrate = 90 µM. Reaction conditions were the same as in Fig. 3, and data averaged from two data sets were handled in the same manner. Left panel, the rate constants for the biphasic reactions in the 1-s time courses were: k1 = 260 ± 10 and k2 = 6.0 ± 0.2, for the oxidation of the enzyme's heme at 557 nm; and k1 = 270 ± 30 and k2 = 8.3 ± 0.4, for the oxidation of the enzyme's FAD at 460 nm. Insets in left panels were for the first 200 ms of the reaction. Right panel, the rate constants for the biphasic reactions in the 9-s time courses were: k1 = 3.2 ± 0.6 and k2 = 0.13 ± 0.1, for the oxidation of the enzyme's heme at 557 nm; and k1 = 0.52 ± 0.01, for the oxidation of the enzyme's FAD at 460 nm, which was best fitted with a single exponential.

Rapid-reaction Kinetic Analysis of the Reaction of Fully Reduced AtNR2 with Nitrate-- To observe the state of the molybdenum during the turnover of AtNR2 under conditions where the fully reduced enzyme is reacted with nitrate, a rapid-reaction freeze-quench experiment was carried out. The rapid-reactions, at 15 °C, were at discrete times of 5, 48, and 400 ms. The EPR spectra at 80 K show that a large Mo^V signal, with integrated intensity 95 ± 20% total molybdenum, is detected at 5 ms, which changes form as the reaction proceeds (Fig. 5). Also present in the 5- and 48-ms spectra is a signal from FAD semiquinone to low field, which integrates to 5 ± 2% of total flavin during the steady-state phase and disappears by 400 ms. The line width for the flavin semiquinone is ~1.3 millitesla, which indicates it is of the red anionic type. This is in agreement with earlier observations of the FAD semiquinone in NR and recombinant cyt c reductase fragment under turnover conditions (12, 20, 26). The cyt b EPR signal was very weak at 5 and 48 ms and slightly stronger at 400 ms, indicating the heme is largely reduced during turnover. A parallel stopped-flow rapid-scan spectral kinetic analysis was run with a lower concentration of NR but similar ratios of NADH and nitrate at 15 °C. During the time courses from 1 ms to 1.9 s, the FAD was 97%, 94%, and 34% reduced at 5, ~50, and 400 ms, whereas the cyt b was 83%, 82%, and 39% reduced at the same time points. The EPR and UV-visible results are consistent and indicate that the initial fast electron transfer is over rapidly and followed by steady-state turnover for more than 100 ms, whereas the reaction reaches the NADH depleted state by 400 ms. These results are similar to those shown in Fig. 4, except that the turnover phase is shorter. Thus, during the time course of turnover (5-125 ms), NR has a highly reduced FAD, a small amount of FAD semiquinone, a less reduced cyt b, and a strong, but lower Mo^V concentration with an integrated intensity of 45 ± 10% total molybdenum at 48 ms. Thus, molybdenum is about 25% reduced during steady-state turnover. In this reaction, where NR begins fully reduced, it appears to remain highly reduced during turnover, which agrees with the results shown in Fig. 4. After NADH depletion, only the flavin semiquinone disappears, but the Mo^V signal returns to 105 ± 20% total molybdenum, and the flavin and cyt b become substantially oxidized. These results are complicated by the low molybdenum content of the enzyme, which would mean that less of the reduced FAD and cyt b would be directly coupled to the Mo-MPT nitrate reducing site. However, it is clear that about 50% of the molybdenum is at the Mo^V reduction level during the steady-state phase, even when the nitrate concentration is close to its K_m; this is consistent with internal electron transfer being rate-limiting and not capable of reducing molybdenum as fast as nitrate oxidizes it.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   EPR spectra of NADH-reduced AtNR2 reacted with nitrate using rapid-reaction, freeze-quench method. Aliquots of enzyme (22 µM) reduced with 110 µM NADH were reacted with 125 µM nitrate in 25 mM MOPS, pH 7.0, at 15 °C, and frozen in isopentane at liquid nitrogen temperatures. EPR spectra were recorded at 80 K. A, reaction time is 5 ms. B, reaction times is 48 ms. C, reaction time is 400 ms.

Rapid-reaction Kinetic Analysis of Nitrite Production by Fully Reduced AtNR2-- To complement these experiments, we devised a rapid-reaction chemical quench method for quantifying the nitrite formed during the type 2 turnover experiment. The NR was pre-reduced with 5 mM NADH and reacted with 5 mM nitrate for 0, 8, 18, 25, 56, and 110 ms, at 21 °C (Fig. 6). Two additional experiments at lower NADH concentration yielded a similar nitrite formation pattern. The data are fit using the function [nitrite] = A(1  e^kt) + Bt, with B set to 20 s¹, the measured steady-state rate under these conditions achieved at the end of the pre-steady-state phase. The best fit values for A and k are 3.1 moles of nitrite (mole of heme)¹ and 78 s¹, respectively. This indicates that initial rate of nitrate reduction is ~400 s¹ on the basis of molybdenum content, which exceeds the steady state of 100 s¹ found under these conditions as shown in Fig. 4. Thus, it is clear that the rate of nitrate reduction does not limit the steady-state catalytic activity of AtNR2.

View larger version (10K): [in this window] [in a new window]   Fig. 6.   Nitrite production in the reaction of NADH-reduced AtNR2 with nitrate using rapid-reaction, zinc acetate-quench method. Enzyme (5 µM) reduced with 5 mM NADH for 30 min was reacted with 5 mM nitrate in 25 mM MOPS, pH 7.0, at 21 °C, at 0, 8, 18, 25, 56, and 110 ms, and the nitrite produced was quantified as described under "Experimental Procedures." Each aliquot of reaction mixture contained 0.5 nmol of NR (based on heme), which was used to calculate the nanomoles of nitrite produced/nmol of heme (right-hand scale). The data points were fitted to Equation 1, which is shown as the solid line.

Electron Distributions in AtNR2 Turnover Forms-- In the two approaches used here to study NR pre-steady-state kinetics and steady-state turnover, the enzyme appears to behave differently in both the transient and steady-state phases of catalysis. For turnover 1, where oxidized AtNR2 was reacted with NADH and nitrate, the functional subunit with molybdenum can become reduced with 4 electrons by two additions of NADH before reduction of nitrate. The non-functional subunits without molybdenum can become reduced with only 2 electrons by addition of 1 NADH, if they are isolated kinetically from the functional subunit and cannot transfer electrons to the functional subunit rapidly enough to contribute to the steady-state rate. Thus, after each subunit reacts with 1 NADH, the enzyme has 8 electrons, or 2/heme, which we write as NR² with the superscript giving the average number of electrons per heme. Addition of another NADH to the functional molybdenum subunit results in 10 electrons or NR^2.5. From the experimental data, when steady-state is reached, NR is 53% reduced or the average state of reduction of each subunit is NR^1.9; this was calculated using reduction states of FAD and cyt b from Fig. 3 and assuming molybdenum is 25% reduced. Electron distribution during turnover is roughly three subunits with 2 electrons and one with 1 to 2 electron, which gives a total of 7-8 electrons/tetramer. Overall, it appears the molybdenum subunit is operating catalytically between NR² and NR⁴ during turnover with the other subunits largely isolated and transferring electrons to the molybdenum subunit at a rate slower than turnover.

For turnover 2, where fully NADH-reduced AtNR2 was reacted with nitrate, the functional subunit with molybdenum begins with 5 electrons, by two additions of NADH and equilibration of 1 electron from another subunit or another AtNR2 molecule, and reacts with nitrate as the first catalytic step. The non-functional subunits begin fully reduced with 3 electrons by 1 NADH addition and equilibration of electrons with other subunits to allow additional NADH reduction until the enzyme reaches 14 electrons or NR^3.5. From the experimental data, when steady state is reached, NR is 63% reduced using reduction state of FAD and cyt b from Fig. 4, and assuming molybdenum is 25% reduced; the average state of reduction of each subunit is NR^2.2. The electron distribution is therefore roughly three subunits with 2 electrons and one with 3 electrons for a total of 9 electrons/tetramer or 64% reduction. There are, however, no data to support a particular distribution of electrons among the subunits. Clearly, since fully reduced subunits without Mo-MPT would contain 3 electrons, rapid intersubunit electron transfer has taken place in the approach to steady-state. In contrast to turnover 1, the molybdenum-containing subunit in turnover 2 appears to cycle between NR³ and NR⁵. However, the non-molybdenum-containing subunits do not appear to actively transfer electrons to the molybdenum subunit in steady state, which is similar to turnover 1. Not only does NR as a whole remain more reduced in turnover 2 versus turnover 1 conditions, but so does the flavin; this suggests the two steady states are not exactly kinetically equivalent, although they are very similar in character.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

Various studies of NR catalysis have suggested that the limiting process is internal electron transfer (1, 2, 12-14), but no one had measured the actual reaction rates, probably due to the lack of sufficient enzyme from plants and other natural sources. Recombinant expression of AtNR2 provided us with the amounts of protein needed to study the rates of internal electron transfer in NR under pre-steady-state and turnover conditions (Tables III and IV, Figs. 3 and 4). Rapid reaction freeze-quench EPR was used to quantify the reduction state of molybdenum during the turnover reaction of fully reduced AtNR2 with nitrate, which demonstrated that the molybdenum center was about 25% reduced during turnover when nitrate was at a limiting concentration (Fig. 5). We also devised an experiment to measure directly the initial rate of nitrite formation by fully reduced enzyme, which exceeded the steady-state rate by a factor of about 4 (Fig. 6).

Analysis of the reductive half-reaction of AtNR2 with NADH showed the FAD and heme of the enzyme were rapidly reduced with rates of 300-400 and 250-350 s¹, respectively (Table III). When oxidized AtNR2 was reacted with a mixture of NADH and nitrate, the FAD and heme were also rapidly reduced at similar rates to those observed in the absence of nitrate (Table IV). Thus, it appeared that nitrate could not react fast enough to keep up with electron input. However, it was equally plausible that steps in internal electron transfer were slower than turnover at the nitrate-reducing active site. When fully reduced AtNR2 was reacted with nitrate, the reduction levels of the FAD and heme, which were initially very high, decreased to intermediate levels with the FAD 84% reduced and heme 42% reduced in the steady state (Fig. 4). Evaluation of the reduction state of molybdenum during this reaction showed that it was always partially reduced (Fig. 5). This is consistent with electron transfer from the flavin via heme to molybdenum being at least partially rate-limiting during steady-state turnover. Our results indicate that more than one NADH initially reacts with the catalytically active subunit per nitrate reduced; however, when the reaction is started with fully reduced enzyme, NADH reduction does not keep the enzyme fully reduced during steady-state turnover, so that electron transfer from flavin to heme must also be partially rate-limiting. The chemical quench experiment (Fig. 6) demonstrates that nitrate reduction is initially higher than the steady-state rate, which is approached at longer times of reaction of fully reduced enzyme with nitrate. These results substantiate that nitrate reduction is not rate-limiting in the approach to steady state.

To explain the rate-limiting events in nitrate reductase catalysis, a reaction mechanism for the oxidized enzyme reacting with NADH and nitrate was prepared (Scheme 1; each intermediate microstate of the enzyme is numbered from 1 to 15 and the cofactors are abbreviated as follows: oxidized, reduced and semiquinone flavin are FAD, FADH2, and FADH, respectively; oxidized and reduced cyt b are box and bred; and the 3 redox states of molybdenum are shown as MoVI, MoV, and MoIV for oxidized, 1 electron reduced, and 2 electron reduced metallocenter, respectively). In this reaction mechanism (Scheme 1), which applies only to the AtNR2 subunit with molybdenum, multiple NADH reaction steps are shown since the flavin becomes fully oxidized via electron transfer to the molybdenum, which permits addition of more electrons to the enzyme during turnover. However, this complication does not influence the catalytic rate since reduction of the FAD appears to be the fastest step. From the data collected in this study, rate constants for the individual steps in the mechanism can be assigned as follows (referring to Scheme 1): k₃ = 400 to 700 s¹, k₄ + k₆ = ~300 s¹, k₅ + k₇ = ~260 s¹, and k₁₀ = 400 s¹. All the rate constants are of similar magnitude, and the overall k_cat of NR is determined by a combination of these similar rate constants. This can be shown by the following equation.

(Eq. 1)

Applying the rate constants shown above yields a k_cat = 80-90 s¹, which is very close to the measured steady-state rate of 100 s¹ for the reactions at 15-20 °C. To prove that the reaction mechanism is valid, an integrated rate equation was generated and simulations were carried out. Although the general shape of the simulated results are like those in Fig. 3 for oxidized enzyme reacting with NADH and nitrate, the presence of non-molybdenum subunits complicates these results such that the simulation based on the reaction mechanism was not adequate. Thus, the full validation of the proposed reaction mechanism for NR must await studies of enzyme more fully loaded with molybdenum.

View larger version (18K): [in this window] [in a new window]   Scheme 1.

Although our results are complicated by the low molybdenum content of the enzyme, the biochemical characteristics of AtNR2 indicate that a fully functional subunit is present and representative of natural enzyme with a higher molybdenum content. X-ray absorption spectroscopy of AtNR2 has provided detailed definition of the molybdenum center and its coordination sphere in oxidized and reduced enzyme, which shows it is similar to previous studies of NR and sulfite oxidase (10). Our results suggest that, in the steady state, NR subunits are catalytically independent, although rapid intersubunit electron transfer cannot be ruled out by data collected so far, especially when the enzyme is fully reduced prior to reaction with nitrate. This work is a steppingstone toward a full understanding of the catalytic mechanism of NR and the processes limiting the activity of the enzyme.

	ACKNOWLEDGEMENTS

We thank Prof. Roger N. F. Thorneley (John Innes Centre, Norwich, United Kingdom) for use of the stopped-flow rapid-scanning system, Gillian Ashby for technician assistance, Dr. Robert R. Eady (John Innes Centre, Norwich, United Kingdom) for critical reading of the manuscript and helpful suggestions, and Prof. Ralf Mendel and Dr. Gunter Schwarz (Technical University of Braunschweig, Braunschweig, Germany) for MPT analysis of AtNR2.

	FOOTNOTES

^* This work was supported by a Biotechnology and Biological Sciences Research Council core strategic grant to the John Innes Centre, by National Science Foundation Grant MCB-9727982 (to W. H. C.), and by an Underwood fellowship from the Biotechnology and Biological Sciences Research Council of the United Kingdom (to W. H. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biological Sciences, Michigan Technological University, Houghton, MI 49931. Tel.: 906-487-2214; Fax: 906-487-3167; E-mail: wcampbel@mtu.edu.

Published, JBC Papers in Press, May 16, 2001, DOI 10.1074/jbc.M100356200

	ABBREVIATIONS

The abbreviations used are: NR, nitrate reductase; AtNR2, A. thaliana NIA2 GenBank^TM accession no. J03240; MPT, molybdopterin; Mo-MPT, molybdenum-molybdopterin; cyt, cytochrome; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

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