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© 2005 American Society of Plant Biologists
Moco Mojo: Crystal Structure Reveals Essential Features of Eukaryotic Assimilatory Nitrate Reductionneckardt{at}aspb.org The assimilatory nitrate reduction pathway is a vital biological process, as it is one of the principal routes by which inorganic nitrogen is incorporated into organic compounds in higher plants, algae, and fungi. After uptake into cells by nitrate transporters, the first and rate-limiting step in this pathway is the reduction of nitrate to nitrite catalyzed by NAD(P)H:nitrate reductase (NR), a molybdenum cofactor (Moco) enzyme that is highly regulated both at the transcriptional and posttranslational level.
An understanding of NR mechanism of action and regulation is of fundamental importance in plant biology and in addition has implications for the control of nitrogen pollution. Nitrate accumulation in surface and groundwater is of concern in many areas of the world because it poses human health threats and is a major contributor to eutrophication of lakes and streams. Basic research on eukaryotic NR may lead to the development of genetically engineered crops with increased nitrate use efficiency, leading to improved crop yields and reducing the need for application of nitrogen fertilizers (Campbell, 1999
There are three forms of eukaryotic NR: NADH-specific enzymes, found most commonly in plants and algae; NADPH-specific enzymes, found only in fungi; and bispecific NAD(P)H forms, found in all three groups but most commonly in fungi (Campbell and Kinghorn, 1990  ). The enzyme is active as a homodimer and reduces nitrate in an irreversible NAD(P)H-dependent reaction that requires the participation of b-type heme-Fe and flavin adenine nucleotide (FAD) cofactors in addition to the molybdenum binding cofactor molybdopterin (Campbell, 2001). The NR monomer is divided into three distinct regions (see figure): a C-terminal FAD binding fragment, which contains cytochrome b5 reductase activity and both FAD and NADH binding domains, the adjacent heme–Fe binding cytochrome b5 domain, and the N-terminal Moco fragment. Short linker sequences, known as hinge 1 and hinge 2, separate the Moco fragment from the cytochrome b5 domain and the cytochrome b5 domain from the C-terminal FAD fragment, respectively. The Moco fragment (required for NR activity) is further divided into a C-terminal dimerization domain and an N-terminal Moco binding domain. During catalysis, electrons are transferred from NADH (or NADPH) to FAD and then shuttled via reduction of the cytochrome b5 heme-Fe to Moco. The reduced Mo atom in the Moco domain subsequently transfers two electrons to nitrate, reducing it to nitrite and hydroxide/water.
Eukaryotic NRs belong to a larger family of cytochrome b5 and Moco-containing enzymes that includes sulfite oxidases, which catalyze the oxidation of sulfite to sulfate concomitant with transfer of electrons from the Moco to the heme cofactor. This reaction is thought to be essentially the reverse of nitrate reduction owing to the relative stabilities of the N=O and S=O bonds, which make nitrate an effective oxygen donor (electron acceptor), whereas sulfite is a more effective oxygen acceptor (electron donor). Previously, crystal structures of NR have included only the FAD fragment (Lu et al., 1994 , 1995) because of the recalcitrance of the holoenzyme and Moco fragment to crystallography. Crystal structures of Moco domains have been determined for sulfite oxidase from chicken (Kisker et al., 1997) and Arabidopsis (Schrader et al., 2003).
Despite the importance of nitrate reduction in plant biology, key elements of the reaction mechanism (the "mojo" of the enzyme alluded to in the title) and regulation of NR remain uncertain, in large part because of the lack of a high-resolution crystal structure of the Moco fragment containing the NR active site. Previous models of the NR holoenzyme (e.g., Campbell, 1999
The authors obtained two models of the NR Moco fragment (NR-Mo) from 2.6-Å (NR-Mo1) and higher-resolution 1.7-Å (NR-Mo2) data sets. The NR-Mo monomer showed a mixed  +ß structure divided into two distinct domains, similar to that of sulfite oxidases. The monomers formed tightly associated dimers along twofold crystallographic symmetry axes, mediated by hydrogen bonding and salt bridges formed mainly between the C-terminal dimerization domains. The higher resolution of the NR-Mo2 data set was attributed to a greater number of intersubunit bonds in NR-Mo2 relative to the NR-Mo1 crystal form. The authors concluded that dimerization is the native oligomeric state of the enzyme, which is consistent with previous studies.
Surrounding the nitrate binding active site there are several positively charged residues that create a cavity or substrate funnel, which is also common to sulfite oxidases and is consistent with binding of an anion substrate (e.g., nitrate or sulfite). However, NR contains a patch of negative charge that lies immediately above the entrance to the substrate funnel, a feature that has not been observed in sulfite oxidases. In addition, the NR substrate funnel forms a narrow slot, which provides enough space to allow entry of the planar nitrate molecule, but not the more bulky sulfate molecule. This observation may help to explain why sulfate does not inhibit NR, whereas nitrate is a potent inhibitor of sulfite oxidases (Kessler and Rajagopalan, 1974 NR-Mo2 crystal form contained sulfate ions bound near the active site, whereas NR-Mo1 did not, which allowed the authors to observe that binding of sulfate mediated conformational changes in the enzyme. They speculate this sulfate binding site and induced conformation changes could explain the phenomenon of phosphate activation of NR because sulfate is a structural mimic of phosphate. Unfortunately, experiments with phosphate in the buffer failed to produce crystals, so this hypothesis could not be tested directly. NR also failed to cocrystallize with nitrate or nitrite. However, the high-resolution data set of NR-Mo2 showed four ordered water molecules in the active site that appeared to mimic nitrate binding. The authors used these data to propose the orientation in which nitrate binds to the active site. Binding of nitrate places one of the nitrate oxygen atoms (Onitrate) in close proximity to the NR-Mo atom, whereupon it attacks the metal center and displaces a bound hydroxyl ligand to form a penta-coordinated reaction intermediate. Electrons in the Mo orbital then flip over to the Mo-Onitrate bond, causing oxidation from Mo(IV) to Mo(VI) and concomitant release of nitrite. The Mo center is subsequently rereduced by the electrons derived from NAD(P)H and transferred via FAD and the cytochrome b5 heme-Fe intramolecular electron transport chain. Importantly, many key amino acid residues were identified that are crucial for catalysis, paving the way for future investigations using site-directed mutagenesis.
An interesting feature unique to higher plant NRs is a Ser residue located in the hinge 1 region between the Moco fragment and the cytochrome b5 domain that mediates inhibition of NR activity through phosphorylation-dependent binding of a 14-3-3 protein (reviewed in MacKintosh and Meek, 2001 ). Higher plant NR activity is rapidly and reversibly modulated under conditions (such as light/dark transitions) that affect photosynthetic carbon assimilation (reviewed in Kaiser and Huber, 2001), and 14-3-3 regulation appears to be the major mechanism regulating this modulation. The structures of the Moco fragment of NR presented by Fischer et al. do not include the hinge 1 region but show the extreme C terminus of the Moco fragment adjacent to hinge 1 to be clearly separated from the dimerization domain. This observation suggests that the hinge 1 region is surface exposed, which is considered to be crucial for phosphorylation of the regulatory Ser and binding of inhibitory 14-3-3 proteins in plant NRs. A complete understanding of the regulation of plant NR awaits a crystal structure of a plant holoenzyme. Nonetheless, the work of Fischer et al. represents a landmark contribution to our understanding of this key eukaryotic enzyme that will undoubtedly spawn many further studies into the finer details of NR mechanism of action and regulation.
Campbell, W.H. (1999). Nitrate reductase structure, function, and regulation: Bridging the gap between biochemistry and physiology. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 277–303.[CrossRef][Web of Science][Medline] Campbell, W.H. (2001). Structure and function of eukaryotic NAD(P)H:nitrate reductase. Cell. Mol. Life Sci. 58, 194–204.[CrossRef][Web of Science][Medline] Campbell, W.H., and Kinghorn, K.R. (1990). Functional domains of assimilatory nitrate reductase and nitrite reductases. Trends Biochem. Sci. 15, 315–319.[CrossRef][Web of Science][Medline]
Fischer, K., Barbier, G.G., Hecht, H.-J., Mendel, R.R., Campbell, W.H., and Schwarz, G. (2005). Structural basis of eukaryotic nitrate reduction: Crystal structures of the nitrate reductase active site. Plant Cell 17, 1167–1179.
Kaiser, W.M., and Huber, S.C. (2001). Post-translational regulation of nitrate reductase: Mechanism, physiological relevance and environmental triggers. J. Exp. Bot. 52, 1981–1989. Kessler, D.L., and Rajagopalan, K.V. (1974). Hepatic sulfite oxidase. Effect of anions on interaction with cytochrome c. Biochim. Biophys. Acta 370, 389–398.[Medline] Kisker, C., Schindelin, H., Pachero, A., Wehbi, W.A., Garrett, R.M., Rajagopalan, K.V., Enemark, J.H., and Rees, D.C. (1997). Molecular basis of sulfite oxidase deficiency from the structure of sulfite oxidase. Cell 91, 973–983.[CrossRef][Web of Science][Medline] Lu, G., Campbell, W.H., Schneider, G., and Lindqvist, Y. (1994). Crystal structure of the FAD-containing fragment of corn nitrate reductase at 2.5 Å resolution: Relationship to other flavoprotein reductases. Structure 2, 809–821.[Medline] Lu, G., Lindqvist, Y., Schneider, G., Dwivedi, U., and Campbell, W. (1995). Structural studies on corn nitrate reductase: Refined structure of the cytochrome b reductase fragment at 2.5 Å, its ADP complex and an active-site mutant and modeling of the cytochrome b domain. J. Mol. Biol. 248, 931–948.[CrossRef][Web of Science][Medline] MacKintosh, C., and Meek, S.E.M. (2001). Regulation of plant NR activity by reversible phosphorylation, 14-3-3 proteins and proteolysis. Cell. Mol. Life Sci. 58, 205–214.[CrossRef][Web of Science][Medline] Patton, C.J., Fischer, A.E., Campbell, W.H., and Campbell, E.R. (2002). Corn leaf nitrate reductase: A nontoxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis. Environ. Sci. Technol. 36, 729–735.[Medline] Schrader, N., Fischer, K., Theis, K., Mendel, R.R., Schwarz, G., and Kisker, C. (2003). The crystal structure of plant sulfite oxidase provides insights into sulfite oxidation in plants and animals. Structure 11, 1251–1263.[Medline] Related articles in Plant Cell:
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