Electron transfer flavoprotein domain II orientation monitored using double electron-electron resonance between an enzymatically reduced, native FAD cofactor and spin labels.

Department of Chemistry and Biochemistry, University of Denver, Denver, CO 80208.

Human electron transfer flavoprotein (ETF) is a soluble mitochondrial heterodimeric flavoprotein that links fatty acid ß-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 (aII) 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 P. denitrificans and human ETF. To characterize the orientations of the aII 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 aII 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 aI (A43C) were between 4.0 and 4.5±0.35 nm and for ßIII (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 aII 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).

Copyright © 2011 The Protein Society.

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