Disruption of the gene encoding the NADH-binding subunit of NADH: ubiquinone oxidoreductase in Neurospora crassa. Formation of a partially assembled enzyme without FMN and the iron-sulphur cluster N-3

Eur J Biochem. 1994 Mar 1;220(2):551-8. doi: 10.1111/j.1432-1033.1994.tb18655.x.

Abstract

In this study, the gene of the 51-kDa NADH-binding subunit of the mitochondrial NADH:ubiquinone oxidoreductase (complex I) in Neurospora crassa was inactivated by homologous replacement with a defective gene copy. The resulting mutant, nuo51, lacks the 51-kDa subunit and shows no complex I activity but still grows at one third of the wild-type growth rate. The enzyme activity of the alternative NADH:ubiquinone oxidoreductase(s) is increased twofold while the activities of the other mitochondrial respiratory enzymes are normal. Complex I is almost completely assembled except for the NADH-binding subunit and still possesses three out of the four EPR-detectable iron-sulphur clusters. Since the deleted subunit contains the sequence motif for one tetranuclear iron-sulphur cluster, the missing cluster N-3 is considered to be bound to this subunit.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Binding Sites
  • Centrifugation, Density Gradient
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Cloning, Molecular
  • Electron Spin Resonance Spectroscopy
  • Escherichia coli
  • Flavin Mononucleotide / metabolism
  • Genes, Fungal*
  • Genetic Vectors
  • Iron-Sulfur Proteins / metabolism
  • Macromolecular Substances
  • Mitochondria / enzymology
  • Molecular Weight
  • Mutagenesis
  • NAD / metabolism
  • NAD(P)H Dehydrogenase (Quinone) / biosynthesis*
  • NAD(P)H Dehydrogenase (Quinone) / genetics*
  • NAD(P)H Dehydrogenase (Quinone) / isolation & purification
  • Neurospora crassa / enzymology*
  • Neurospora crassa / genetics*
  • Oxidation-Reduction
  • Protein Conformation
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification

Substances

  • Iron-Sulfur Proteins
  • Macromolecular Substances
  • Recombinant Proteins
  • NAD
  • Flavin Mononucleotide
  • NAD(P)H Dehydrogenase (Quinone)