title: Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor, and spin labels.
abstract: Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid β-oxidation to the main respiratory chain. The crystal structure of human ETF bound to medium chain acyl-CoA dehydrogenase indicates that the flavin adenine dinucleotide (FAD) domain (αII) is mobile, which permits more rapid electron transfer with donors and acceptors by providing closer access to the flavin and allows ETF to accept electrons from at least 10 different flavoprotein dehydrogenases. Sequence homology is high and low-angle X-ray scattering is identical for Paracoccus denitrificans (P. denitrificans) and human ETF. To characterize the orientations of the αII domain of P. denitrificans ETF, distances between enzymatically reduced FAD and spin labels in the three structural domains were measured by double electron-electron resonance (DEER) at X- and Q-bands. An FAD to spin label distance of 2.8 ± 0.15 nm for the label in the FAD-containing αII domain (A210C) agreed with estimates from the crystal structure (3.0 nm), molecular dynamics simulations (2.7 nm), and rotamer library analysis (2.8 nm). Distances between the reduced FAD and labels in αI (A43C) were between 4.0 and 4.5 ± 0.35 nm and for βIII (A111C) the distance was 4.3 ± 0.15 nm. These values were intermediate between estimates from the crystal structure of P. denitrificans ETF and a homology model based on substrate-bound human ETF. These distances suggest that the αII domain adopts orientations in solution that are intermediate between those which are observed in the crystal structures of free ETF (closed) and ETF bound to a dehydrogenase (open).
authors.affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Swanson
authors.forename: Velavan
authors.initials: V
authors.lastname: Kathirvelu
authors.forename: Tomas
authors.initials: T
authors.lastname: Majtan
authors.forename: Frank E
authors.initials: FE
authors.lastname: Frerman
authors.forename: Gareth R
authors.initials: GR
authors.lastname: Eaton
authors.forename: Sandra S
authors.initials: SS
authors.lastname: Eaton
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D044927:Electron-Transferring Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D050770:Glutaryl-CoA Dehydrogenase / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry; D017434:Protein Structure, Tertiary; D013113:Spin Labels
chemical_list: D001426:Bacterial Proteins; D044927:Electron-Transferring Flavoproteins; D013113:Spin Labels; D005182:Flavin-Adenine Dinucleotide; D050770:Glutaryl-CoA Dehydrogenase