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Originally published In Press as doi:10.1074/jbc.M409694200 on May 16, 2005

J. Biol. Chem., Vol. 280, Issue 28, 26049-26054, July 15, 2005
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Viscosity Effects on Eukaryotic Nitrate Reductase Activity*

Guillaume G. Barbier{ddagger} and Wilbur H. Campbell§

From the The Nitrate Elimination Company, Inc., Lake Linden, Michigan 49945 and Department of Biological Sciences, Michigan Technological University, Houghton, Michigan, 49931

Received for publication, August 23, 2004 , and in revised form, May 13, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1 [EC] –3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Nitrate reductase (NaR,1 EC 1.7.1.1 [EC] –3) is a homodimer with each monomer consisting of a ~100-kDa polypeptide and cofactors FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT) with a stoichiometry of 1:1:1 (1, 2). Mixing crude extracts of different homozygous Nicotiana nia mutants with no NaR activity restored enzyme activity, making the case for intragenic complementation and modular structure of the enzyme (3, 4). Subsequently, detailed biochemical analysis demonstrated that NaR indeed has a modular structure with independently folded regions of the large polypeptide (1). Nitrate to nitrite reduction is catalyzed by NaR with NAD(P)H as the reductant and is accompanied by a very large free energy release. During catalysis, electrons and protons are transferred from NAD(P)H to nitrate via electroactive redox cofactors, which act like an internal electron/proton transfer (IET) system (Fig. 1). NaR is a member of the sulfite oxidase (SOX) structure family of Mo-MPT-containing enzymes with which it shares high sequence similarity for the Mo-containing module (5). The Cyt b reductase fragment (CbR) of NaR for which the structure has been determined (6, 7) is related to the ferredoxin NADP+ reductase structural family (8). The Cyt b of NaR is related to mammalian Cyt b5 and SOX Cyt b with which it shares a high sequence homology (1, 9). Flexible hinge regions of NaR join the major functional modules: 1) Hinge 1 between the Mo-containing/nitrate-reducing module and the Cyt b domain and 2) Hinge 2 between the Cyt b domain and the electron-donating CbR module (Fig. 1). Hinge 1 contains the regulatory Ser phosphorylation and 14-3-3-binding sites in plant NaR (10). This important functionality clearly played a role in the evolution of this region of plant NaR but not in fungal and yeast NaR forms that lack the key Ser and 14-3-3-binding residues. Hinge 2 has no defined physiological or regulatory role (1). However, NaR loses activity if the hinges are cleaved by proteases and depends on the integrity of these regions for full functionality.

In previous kinetic studies of NaR, each subunit was shown to operate independently in catalysis with a kcat of ~200 s–1 for reaction of NADH with nitrate (1, 2, 7, 11, 12). NADH transfers two electrons and a proton to FAD with a rate constant ranging from 250 to 400 s–1 (11). Reduced FAD transfers electrons one by one to the Cyt b domain and a proton to solution. The average rate constant of these reactions is 300 s–1. Cyt b transfers one electron at a time to convert the MoVI of the Mo-MPT cofactor in two 1-electron steps to MoIV with an average rate constant of 260 s–1. A proton is also transferred to an oxy-ligand of the Mo in this process, and it is probably donated by a side chain of a nearby residue but ultimately comes from solution. When the Mo center is fully reduced and nitrate is bound in the active site, two electrons are transferred to reduce nitrate to nitrite and hydroxide. The rate constant of nitrate reduction to nitrite by the Mo-MPT cofactor is ~400 s–1 (11). Therefore, according to this study, Cyt b might be involved in rate-limiting events of catalysis but no individual step in catalysis was greatly slower than the others (11). On the other hand, NAD(P)H-dependent ferricyanide reduction is catalyzed by NaR with a kcat of ~1000 s–1 and reduced methyl viologen (MV) nitrate reduction is catalyzed by NaR with a kcat ~800 s–1 (1, 11). Thus, IET is likely the rate-limiting event in NaR catalysis but no study has been able to directly demonstrate this possibility.

Recent studies of mammalian SOX kinetics showed that the IET rate was impacted by solution viscosity (13, 14), confirming the hypothesis that the Cyt b domain was undergoing a large conformational change during catalysis relative to the Mo-MPT-containing sulfite-oxidizing module (9). NaR has a similar structure to SOX in that the Cyt b domain is tethered to the Mo-MPT-containing nitrate-reducing module via a flexible "hinge" sequence. However, NaR is more complex than SOX because the Cyt b domain is also tethered to the FAD-containing CbR. Thus, by analogy, if IET is the rate-limiting event in NaR catalysis and involves large conformational movement of Cyt b domain relative to the other larger modules of the enzyme to which it is tethered via Hinges 1 and 2 (Fig. 1), solution viscosity may have a significant impact on NaR-catalytic rates and help to demonstrate the rate-limiting nature of IET in NaR catalysis.

Monomeric polyhydroxylated molecules such as glycerol and sucrose, also called microviscosogens (15, 16), have been used in many studies of protein conformational changes (17–21). The kcat observed in the viscosity studies was shown to follow a linear dependence on the negative power of the viscosity and was described by Kramers equation (Equation 1) (14, 17–21),

(Eq. 1)
where {eta} = solution viscosity.

To further investigate the predicted rate-limiting processes in NaR catalysis, we have studied the effects of two viscosogens (glycerol and sucrose) on NAD(P)H:NaR activity of three forms of holoenzyme (recombinant yeast NaR, recombinant Arabidopsis NaR2, and natural corn leaf NaR) and NADH-ferricyanide reductase activity of the holo-NaR forms and the recombinant NaR fragments known as CbR and molybdenum reductase (MoR) (Fig. 1). We also assayed the MV:NaR activity of one form of holo-NaR. We observed a slight impact of the viscosity on the NADH:ferricyanide reductase activity of CbR and MV:NaR activity of the holoenzyme, confirming that during catalysis no large conformational change occurred in the FAD- and Mo-containing fragments. A large negative impact of viscosity was observed in the other assays performed, leading to the conclusion that a large conformational change involving the gating of Cyt b is most likely to be the limiting step in catalysis by NaR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 
Enzymes and NaR Fragments—Zea mays NaR (ZmNaR) and Arabidopsis thaliana NaR (AtNR2) were prepared as described previously (11, 22, 23). Yeast NaR (YNaR1) was obtained by cloning the YNaR1 gene from Pichia angusta (24) with N-terminal His tag into an appropriate expression vector and production in Pichia pastoris (results not shown). YNaR1 was purified by metal-chelate affinity chromatography, as described for S-NaR1, its recombinant nitrate-reducing fragment (25). The methods used to obtain Z. mays Cyt b reductase fragment (ZmCbR), spinach molybdenum reductase (SoMoR), and corn molybdenum reductase (ZmMoR) were previously described (12, 26–28). Enzymes and fragments were buffer exchanged into 25 mM MOPS, pH 7.0, and stored at –80 °C.

Enzyme Assays—All of the chemicals were from Sigma. NAD(P)H: NaR activity assays were done by monitoring change in A340 at 22 °C in 25 mM MOPS (ultra pure grade), pH 7.0, 10 mM KNO3, and 0.1 mM NAD(P)H, all made up in viscosogen. Nitrite appearance was monitored by the standard NaR assay at 30 °C and pH 7.5 (11, 12), which demonstrated that the effects of the viscosogens were the same in both assays. NAD(P)H:ferricyanide reductase activity assays were done in the same manner as the NaR assays with 1 mM potassium ferricyanide substituted for nitrate. Ferricyanide reduction {Delta}A340 rates were converted to µmol/min using an extinction coefficient of 6.9 mM–1 cm–1 (28), which corrects for the change in A340 due to reduction of ferricyanide. Each assay was replicated five times, and the mean ± S.E. was calculated. Each experiment was done twice, and representative data are shown. MV:NaR and Cyt c reductase activity assays were done with YNaR1 as previously described (12, 25) at 0 and 50% glycerol.

Concentrations of NaR forms containing heme-Fe were determined by taking their absorbances at 413 nm and calculated with an extinction coefficient of 120 mM–1 cm–1 (2, 11). For CbR containing only the FAD cofactor, the absorbance at 460 nm was measured and an extinction coefficient of 10 mM–1 cm–1 was used (6, 11).

Viscosity of Assay Buffers—Viscosogen buffers were prepared as weight percent solutions of 0 (buffer only), 10, 20, 30, 40, 50, and of MoBio grade glycerol and sucrose. The viscosity ({eta}) of buffer and each viscosogen solution was determined with glass viscometers at 22 °C compared with deionized water, which was shown to have virtually the same viscosity as the buffer with no added viscosogen. Each viscosity was determined five times, and the results were averaged. The viscosities of the 0, 10, 20, 30, 40, and 50% glycerol solutions were, respectively, 1.046, 1.329, 1.672, 2.265, 3.283, and 5.544 centiPoise. The viscosities of the 0, 10, 20, 30, 40, and 50% sucrose solutions were, respectively, 1.058, 1.373, 1.879, 2.847, 5.396, and 13.268 centiPoise. The impact of substrates and enzymes were ignored, because these have only a very small effect on viscosity. The viscosity of the assay buffers for the nitrate reductase assays of holo-NaR was slightly different from that shown above.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
CONCLUSION
 REFERENCES
 
Properties of Enzyme Forms—Characteristics of the holo-NaR forms and recombinant NaR fragments studied are summarized in Table I. Although CbR has two domains, one for binding NADH and one for binding FAD, the linker joining them is tightly associated with the NADH domain and is not considered a flexible hinge (1, 6, 7). MoR adds the heme-Fe or Cyt b domain to CbR via the flexible Hinge 2 of holo-NaR (Fig. 1 and Table I). CbR and MoR are monomeric enzyme fragments. Holo-NaR is a homodimer, and its monomeric unit is most complex with five domains and two flexible hinge regions (Fig. 1 and Table I). The Mo-MPT and dimer interface domains of the nitrate-reducing module have recently been shown (29) to be closely associated as in mammalian and plant SOX (9, 30). Hinge 1 joins the Cyt b domain to the nitrate-reducing module. The combined nitrate-reducing/Hinge 1/Cyt b fragment of NaR is equivalent to mammalian SOX in domain composition, and SOX is also a homodimer (9).


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  		TABLE I
Characteristics of eukaryotic nitrate reductase forms used in this study

 



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  		FIG. 1.
Functional schematic for eukaryotic NAD(P)H:NaR, which was adapted from a previous model (1). Ferricyanide is shown as only accepting electrons from the heme-iron center in the Cyt b domain based on the results presented here. P, Ser phosphorylation site for regulatory control of NaR activity in plants, which is also a 14-3-3-binding site (10).

 
In each set of assays with a particular enzyme form, an appropriate dilution was determined for the enzyme stock solution and it was prepared in the viscogen solution just prior to assay. The kcat values were calculated based on the Mo-MPT content when available or normalized to known values (1, 2, 11, 12). No Km determinations were done, because it has been shown that this parameter was independent of the viscosity for some enzymes (14, 17) and it was assumed that all of the forms would be impacted to the same degree.

Effect of Viscosity on Nitrate Reductase Activity—When MV: NaR activity of YNaR1 was assayed at 0 and 50% glycerol, ~93% activity was retained in presence of viscosogen. For comparison, only ~32% NADPH:NaR activity of YNaR1 was retained in 50% glycerol. Thus, it appeared that the nitrate-reducing step in NaR catalysis, represented by MV:NaR activity, was not greatly impacted by increased solution viscosity. In contrast, when nitrate reduction involved IET, represented by NADPH:NaR activity, solution viscosity had a major impact.

NAD(P)H:NaR activity of ZmNaR1, AtNR2, and YNaR1 were all strongly negatively impacted by increases in solution viscosity (Fig. 2 and Table II). Glycerol and sucrose had approximately the same effect. The loss of activity due to increased solution viscosity is approximately the same for all of the NaR forms despite significant differences in overall size of the subunit (Table I) and length of the hinge regions (Table II).


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  		TABLE II
Impact of solution viscosity on NAD(P)H:NaR activity of eukaryotic NaR forms

 
For holo-NaR with two flexible hinge regions (Fig. 1 and Table I), the interpretation of the results is clearly more difficult than for enzymes with a single flexible hinge region such as mammalian SOX and yeast flavocytochrome b2 (13, 14, 31). However, it is clear that IET in NaR is gated by large conformational changes of the Cyt b domain relative to the electron-donating and nitrate-reducing modules of the enzyme, which is illustrated in Scheme 1, where the gating effects of the two hinges are grouped together.

Several approaches could be taken to evaluate the relative effects of Hinge 1 versus Hinge 2. As is described below, we have taken advantage of the collection of recombinant NaR fragments already available to characterize Hinge 2 effects in relation to viscosity. Laser flash photolysis has been used to directly study IET in mammalian SOX where the Cyt b heme-Fe was rapidly reduced and the effect of viscosity on the equilibration of the electron with the Mo-MPT center was observed (14). Although laser flash photolysis would be useful to show a direct effect of viscosity on NaR IET, there would be complexity to the results because NaR has more redox centers than SOX. Thus, recombinant fragments of NaR with two redox centers such as MoR (12) and the recently constructed S-NaR2, which is the N-terminal fragment of YNaR1 including the Cyt b domain, Hinge 1, and S-NaR12 will be useful in these future studies.



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  		FIG. 2.
Plots of log kcat versus log viscosity for NAD(P)H:NaR activity of holo-NaR. The data were fitted with linear regression analysis for each set of enzyme assays.

 
Effect of Viscosity on Reductase Activities of NaR—The reductase portion of NaR comprised of CbR, Hinge 2, and Cyt b domain, which we call MoR (Table I), catalyzes NAD(P)H-dependent reduction of mammalian Cyt c, ferricyanide, and a number of other electron acceptors (1, 12). Cyt c reductase activity of YNaR1 assayed in 50% glycerol was inhibited by 79%, whereas ferricyanide reductase activity was inhibited by 74%. Because both activities were impacted to a significant extent by increased solution viscosity, we used the ferricyanide reductase activity in the current study.

Corn NaR (ZmNaR1) and its fragments (ZmCbR and ZmMoR) were used as models to study the impact of solution viscosity on ferricyanide reductase activity. Solution viscosity had little impact on ferricyanide reductase activity of ZmCbR, which had a very low slope close to 0 up to 30% solutions (Fig. 3 and Table III). The results (Fig. 3 and Table III) showed that there was a substantial impact of solution viscosity on the ferricyanide reductase activity of ZmNaR1 and ZmMoR and that, basically, sucrose and glycerol acted the same. Similar results were found for all of the NaR forms studied that contain Hinge 2 (Table III). Hinge 2 joins the CbR fragment to the Cyt b domain in all of these NaR forms with the exception of CbR, which has no hinge (Fig. 1 and Table I). Because the ferricyanide reductase activity of ZmCbR, which lacks the heme-Fe Cyt b domain (Table I), was only slightly impacted by solution viscosity (Table III and Fig. 3), ferricyanide reductase activity involves the heme-Fe when it is present in holo-NaR and MoR. Thus, NAD(P)H reduction of ferricyanide catalyzed by the whole enzyme and its larger reductase fragment appears to involve gating of IET, as indicated by the large impact solution viscosity on the activity. Therefore, the effect observed on ZmCbR probably represents the effect of viscosity on diffusion of substrates since this reaction was known to be near the diffusion limit (1, 12). Thus, the diffusion effect of viscogens on the substrates was negligible.


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  		TABLE III
Impact of Solution Viscosity on Ferricyanide Reductase Activity

 



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  		SCHEME 1
 
Because the negative impact of solution viscosity on the kcat and therefore the IET process is large, it can be suggested to involve large conformational change of the position of the Cyt b domain relative to the FAD domain of CbR during IET. Furthermore, it is clear from the impact of solution viscosity on ferricyanide reductase activity that IET is the rate-limiting process in this very rapid catalytic activity of NaR. These results are similar to those found for other redox enzymes with IET from or to a Cyt b domain, such as flavocytochrome b2 and SOX (13, 14, 31). Thus, the movement of Cyt b domain relative to CbR is gating the electron and proton transfer processes in MoR and holo-NaR (Scheme 2). In this simplified kinetic model for the reductase activity of NaR, "Hinge 2 gating" represents the state of the enzyme where Cyt b has moved from its resting position and optimized its position for one electron reduction by reduced flavin in the FAD domain. This step must occur twice to fully oxidize NAD(P)H. Because the actual transfer of the electron is extremely rapid (5), the time required for the conformational change is the slow step in catalysis of ferricyanide reduction by MoR and holo-NaR.

Ferricyanide reductase activity of ZmNaR1 was lower, and solution viscosity had a greater negative impact on it than ZmMoR. Similar results were found when comparing SoMoR and AtNR2 (Fig. 3 and Table III). Concentrating on corn NaR forms to avoid species differences, the greater structural complexity of holo-NaR must account for the slower conformational change of Cyt b in holo-NaR compared with MoR. MoR is a monomer and clearly less encumbered than the homodimeric holo-NaR with the nitrate-reducing module attached to Cyt b via Hinge 1 (Fig. 1 and Table I). Thus, it is expected that greater drag of the extra mass (Hinge 1 plus the nitrate-reducing module) will retard the movement of Cyt b relative to the FAD domain of CbR and account for both the lower kcat and greater viscosity effect on holo-NaR. There probably is also an effect of the second subunit of holo-NaR, but it is more difficult to discern its impact and we have generally treated NaR subunits as independent to simplify analysis (1, 11).

Nevertheless, a contradictory result was observed when matching the ferricyanide reductase activity of YNaR1 with the NaR activity. The ferricyanide reductase activity appeared to be more impacted by the viscosity than the NaR activity (Tables II and III). This means that the drag forces applied on Cyt b during IET were greater in the case of ferricyanide reductase activity. This observation is probably due to the shorter length of Hinge 2 in YNaR1, which might make IET from reduced flavin to Cyt b more efficient when electrons are transferred to Mo center than to an artificial electron acceptor in solution-like ferricyanide. However, more detailed kinetic studies, especially with transient time analysis of early events in catalysis, are needed to completely understand the differences among the eukaryotic families of NaR.

Evaluation of Hinge Length and Sequence in Relation to Viscosity Effects—For flavocytochrome b2, it was shown that the heme domain was involved in a large conformational change occurring during catalysis, and when the hinge linking the two distinct domains of flavocytochrome b2 was shortened, a 5-fold decrease of heme reduction rate was observed (31). Hinges 1 and 2 have natural differences among the NaRs we studied, and because varying hinge length by design has not yet been done, we made an initial analysis of the impact of hinge length on the effect of viscosity on NaR activities.

Fungal and yeast NaR forms have the longest Hinge 1 and shortest Hinge 2 sequences of all of the NaR sequences in GenBankTM. Plant NaR forms have significantly longer Hinge 2 sequences with those from dicots (AtNR2 and SoMoR) longer than those from monocots (ZmNaR1) (Tables II and Table III). In Fig. 4A, the four NaR forms studied here were compared for Hinge 2 sequence with its extent defined by the secondary structure of the bounding domains (1, 6, 7). In plant NaR forms, Hinge 2 sequences appear to have conserved residues, which suggests that secondary and/or tertiary structure may play a role in optimizing the longer Hinge 2 as compared with yeast and fungal NaR forms where few conserved residues were found.

NAD(P)H:ferricyanide reductase activities of holo-NaR show that a longer Hinge 2, similarly found in plant NaR, seems to be more optimal for IET between the FAD domain of CbR and the Cyt b domain. For YNaR1 with the shortest Hinge 2, the kcat for ferricyanide reductase activity is lower and the negative impact slope for viscosity shown in Table III is greater than either the monocot or dicot plant NaR forms. Thus, a trend for NaR similar to that found for flavocytochrome b2 was observed, which leads to the hypothesis that shortening the length of Hinge 2 involved in gating the first step of IET might result in lower activity.

In contrast, Hinge 1 of yeast and fungal NaR forms is longer than the corresponding region in plant NaR forms (Table II and Fig. 4B). The important regulatory Ser residue and 14-3-3-binding site in Hinge 1 of plant NaR forms (10) is clearly a highly conserved feature of most plant NaR forms and absent from yeast and fungal NaR forms (Fig. 4B). NAD(P)H:NaR activities of holo-NaR show that plant and yeast NaR forms have approximately the same kcat, and solution viscosity has approximately the same negative impact on NaR activity (Fig. 2 and Table II). Thus, in evolving with the different driving forces impacting Hinge 1, all of the NaR forms have arrived in the present with similar high efficiency for catalyzing nitrate reduction. NaR activity is obviously not dependent only on Hinge 1 features and so the overall catalytic efficiency of the enzyme probably reflects optimization of both hinges over evolutionary time. More experimental study such as substitution of Hinge 2 from YNaR1 for Hinge 2 in a plant NaR form would be needed to delve more deeply into the relative importance of Hinge 1 versus Hinge 2 in determining NaR activity.



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  		FIG. 3.
Plots of the log kcat versus log viscosity for NAD(P)H:ferricyanide reductase activity of holo-NaR and its fragments. The data were fitted with linear regression analysis.

 



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  		SCHEME 2
 

   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
REFERENCES
 
NaR catalyzes several partial activities in addition to the complete NAD(P)H:NaR activity, which is clearly linked to the internal electron transport chain of the enzyme (Fig. 1). Employing the partial activities as "probes" permitted us to isolate the impact of solution viscosity on catalytic activities that involved IET. First, it was shown that MV:NaR activity was only slightly effected by solution viscosity, implying that no large conformational changes occurred during the catalysis of nitrate reduction by the Mo-MPT. NADH:ferricyanide reductase activity of CbR showed a similar result, leading to the conclusion that no large conformational change occurred when the flavin of the enzyme was reduced and transferred electrons directly to an acceptor in solution. Neither of these catalytic reactions involves IET, and the absence of a viscosity effect is consistent with the concept established in studies of the impact of viscosity on IET in SOX (13, 14). NaR, similar to SOX, utilizes IET in catalysis of the NAD(P)H:NaR reaction and partial reactions such as Cyt c reduction. For both of these reactions, solution viscosity had a major negative impact on activity. Because Cyt c reduction and apparently ferricyanide reduction (Table III and Fig. 3) involve only the first IET step, it can be suggested that this electron transfer involves large conformation changes that limit the reaction as illustrated in Scheme 2. NAD(P)H: NaR activities require both IET steps in catalysis, and the large impact of solution viscosity (Table II and Fig. 2) was taken to mean that large conformational changes of the Cyt b domain of NaR were involved in IET. Applying the mass center rule to the three functional modules of holo-NaR, only Cyt b is predicted to move because it is much smaller than the two other modules (nitrate-reducing module at the N terminus with ~500 residues and CbR module at the C terminus with ~270 residues). Consequently, the NaR kcat is proportional to the reciprocal of solution viscosity due to the drag force of the solution retarding the movement of Cyt b relative to its C-terminal electron-donating module (CbR) and the N-terminal nitrate-reducing module. The magnitude of the Cyt b motion must increase the drag force in the NaR activities, because Cyt b has to go from FAD to Mo-MPT to transfer an electron, increasing the viscosity impact. More detailed studies will be required to determine the relative impact of Hinge 1 versus Hinge 2 on IET steps in NaR catalysis. However, it is clear that IET in NaR requires large conformational changes that can be studied using solution viscosity as a tool.



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  		FIG. 4.
Comparison of Hinge sequences for AtNR2, SoNR1, ZmNaR1, and YNaR1. Inclusive residue numbers are shown for the sequence fragment from each enzyme form. Conserved amino acid residues, in at least three of these sequences, are shown in boldface. In each alignment, the longest hinge sequence is numbered. For the Hinge 2 set, AtNR2 has 38 residues (1), and for the Hinge 1 set, YNaR1 has 37 residues. A, Hinge 2 sequence alignment. To define the extent of Hinge 2, secondary structure assignments for ZmNaR1 predicted for the model of the MoR fragment were used (6, 7). B, Hinge 1 sequence alignment. The N terminus of Hinge 1 is defined by secondary structure assignments for the structure of the dimer interface domain of the nitrate-reducing fragment of YNaR1 (29), as shown above the alignment. The C terminus of Hinge 1 is defined by predicted secondary structure of the Cyt b domain of AtNR2 (1). Ser, which is phosphorylated in plant NaR forms, is also identified (1). It should be noted that the phospho-Ser is located in a 14-3-3-binding site (10). Yeast NaR has neither a corresponding Ser nor a 14-3-3-binding site.

 

   FOOTNOTES
 
* This work was supported in part by a SBIR grant (Contract R44GM56598) (to NECi). 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

{ddagger} Present address: Dept. of Plant Biology, Michigan State University, East Lansing, MI 48824. Back

§ To whom correspondence should be addressed. Tel.: 906-296-1000; Fax: 906-296-8003; E-mail: bill{at}nitrate.com.

1 The abbreviations used are: NaR, eukaryotic NAD(P)H:nitrate reductase; At, A. thaliana; Cyt b, cytochrome b domain of NaR; CbR, cytochrome b reductase fragment of NaR; IET, internal electron/proton transfer; Mo-MPT, molybdenum-molybdopterin; MOPS, 3-(N-morpholino)propanesulfonic acid; MoR, Mo-MPT reductase fragment of NaR; MV, methyl viologen; S-NaR1, simplified nitrate reductase; SOX, mammalian sulfite oxidase; YNaR1, yeast NAD(P)H:NaR; ZmNaR, Z. mays NaR; AtNR, A. thaliana NaR; ZmCbR, Z. mays Cyt b reductase fragment; ZmMoR, corn molybdenum reductase; SoMoR, Spinacea oleracea molybdenum reductase. Back

2 G. G. Barbier and W. H. Campbell, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank David J. Lowe from the John Innes Center for suggestions and Marc Zimmermann, Andreas Weber, Lars Voll, and Andrea Braeutigam from Michigan State University for comments on the manuscript. We also thank Michael J. Campbell and Troy Kinnunen-Skidmore from the Nitrate Elimination Company, Inc. (NECi) for technical and experimental assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 CONCLUSION
 REFERENCES
 

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