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Updated: May 12, 2026

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1
Published on: November 26, 2014
Biochemical characterization of molybdenum cofactor-free nitrate reductase from Neurospora crassa
Phillip Ringel1, Joern Krausze2, Joop van den Heuvel2
1Department of Plant Biology, Braunschweig University of Technology, 38106 Braunschweig, Germany.
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.
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