Oxidation of the FAD cofactor to the 8-formyl-derivative in human electron-transferring flavoprotein

J Biol Chem. 2018 Feb 23;293(8):2829-2840. doi: 10.1074/jbc.RA117.000846. Epub 2018 Jan 4.

Abstract

The heterodimeric human (h) electron-transferring flavoprotein (ETF) transfers electrons from at least 13 different flavin dehydrogenases to the mitochondrial respiratory chain through a non-covalently bound FAD cofactor. Here, we describe the discovery of an irreversible and pH-dependent oxidation of the 8α-methyl group to 8-formyl-FAD (8f-FAD), which represents a unique chemical modification of a flavin cofactor in the human flavoproteome. Furthermore, a set of hETF variants revealed that several conserved amino acid residues in the FAD-binding pocket of electron-transferring flavoproteins are required for the conversion to the formyl group. Two of the variants generated in our study, namely αR249C and αT266M, cause glutaric aciduria type II, a severe inherited disease. Both of the variants showed impaired formation of 8f-FAD shedding new light on the potential molecular cause of disease development. Interestingly, the conversion of FAD to 8f-FAD yields a very stable flavin semiquinone that exhibited slightly lower rates of electron transfer in an artificial assay system than hETF containing FAD. In contrast, the formation of 8f-FAD enhanced the affinity to human dimethylglycine dehydrogenase 5-fold, indicating that formation of 8f-FAD modulates the interaction of hETF with client enzymes in the mitochondrial matrix. Thus, we hypothesize that the FAD cofactor bound to hETF is subject to oxidation in the alkaline (pH 8) environment of the mitochondrial matrix, which may modulate electron transport between client dehydrogenases and the respiratory chain. This discovery challenges the current concepts of electron transfer processes in mitochondria.

Keywords: 8-formyl-FAD; dehydrogenase; electron transfer; flavin adenine dinucleotide (FAD); flavin semiquinone; mitochondria; respiratory chain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Biocatalysis
  • Catalytic Domain
  • Conserved Sequence
  • Electron Transport
  • Electron-Transferring Flavoproteins / chemistry
  • Electron-Transferring Flavoproteins / genetics
  • Electron-Transferring Flavoproteins / metabolism*
  • Flavin-Adenine Dinucleotide / analogs & derivatives*
  • Flavin-Adenine Dinucleotide / chemistry
  • Flavin-Adenine Dinucleotide / metabolism*
  • Humans
  • Hydrogen-Ion Concentration
  • Models, Molecular*
  • Multiple Acyl Coenzyme A Dehydrogenase Deficiency / enzymology
  • Multiple Acyl Coenzyme A Dehydrogenase Deficiency / genetics
  • Mutagenesis, Site-Directed
  • Mutation
  • Oxidation-Reduction
  • Protein Engineering
  • Protein Multimerization
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • ETFA protein, human
  • ETFB protein, human
  • Electron-Transferring Flavoproteins
  • Recombinant Proteins
  • Flavin-Adenine Dinucleotide

Supplementary concepts

  • Glutaric aciduria 2

Associated data

  • PDB/1EFV
  • PDB/2A1T
  • PDB/1O96
  • PDB/1EFP
  • PDB/4KPU
  • PDB/2A1U
  • PDB/3CLR
  • PDB/3CLU