title: Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa.
abstract: Nitrate reductase (NR) is a complex molybdenum cofactor (Moco)-dependent homodimeric metalloenzyme that is vitally important for autotrophic organism as it catalyzes the first and rate-limiting step of nitrate assimilation. Beside Moco, eukaryotic NR also binds FAD and heme as additional redox active cofactors, and these are involved in electron transfer from NAD(P)H to the enzyme molybdenum center where reduction of nitrate to nitrite takes place. We report the first biochemical characterization of a Moco-free eukaryotic NR from the fungus Neurospora crassa, documenting that Moco is necessary and sufficient to induce dimer formation. The molybdenum center of NR reconstituted in vitro from apo-NR and Moco showed an EPR spectrum identical to holo-NR. Analysis of mutants unable to bind heme or FAD revealed that insertion of Moco into NR occurs independent from the insertion of any other NR redox cofactor. Furthermore, we showed that at least in vitro the active site formation of NR is an autonomous process.
authors.affiliation: Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
authors.forename: Phillip
authors.initials: P
authors.lastname: Ringel
authors.affiliation: Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Joern
authors.initials: J
authors.lastname: Krausze
authors.affiliation: Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
authors.forename: Joop
authors.initials: J
authors.lastname: van den Heuvel
authors.affiliation: Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.affiliation: Core Facility for Protein Spectroscopy, Institute of Cytobiology and Cytopathology, Phillips University, 35032 Marburg, Germany.
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Pierik
authors.affiliation: Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Herzog
authors.affiliation: Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany. Electronic address: r.mendel@tu-bs.de.
authors.forename: Ralf R
authors.initials: RR
authors.lastname: Mendel
authors.affiliation: Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kruse
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism*; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000378:metabolism; D008667:Metalloproteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D009249:NADP / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D050901:Nitrate Reductase / Q000378:metabolism; D009572:Nitrite Reductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011621:Pteridines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014461:Ultracentrifugation
chemical_list: D003067:Coenzymes; D006418:Heme; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009249:NADP; D050901:Nitrate Reductase; D009572:Nitrite Reductases; D011621:Pteridines; D011994:Recombinant Proteins; C022261:molybdopterin
keywords: Electron Paramagnetic Resonance (EPR); Heme; Molybdenum; Molybdenum Cofactor; Neurospora; Nitrate Reductase; Prosthetic Group Insertion; Ultracentrifugation
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