Stabilization of Non-productive Conformations Underpins Rapid Electron Transfer to Electron-transferring Flavoprotein

Abstract

Crystal structures of protein complexes with electrontransferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165␤ in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165␤A ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165␤A ETF⅐medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF⅐ medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165␤ plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation. Human electron-transferring flavoprotein (ETF)1 is a ubiquitous electron carrier that interacts with at least 10 different dehydrogenases, some of which are structurally distinct (1). Electrons are shuttled to the respiratory chain via ETF and subsequently transferred to the membrane-bound ETF-ubiquinone oxidoreductase (1). ETF is a 63-kDa heterodimer containing one FAD and one AMP per dimer (2) and folds into three distinct domains; the FAD is bound non-covalently to domain II that sits in a shallow bowl created by domains I and III (2). Partners of ETF include the fatty-acyl-CoA dehydrogenases involved in ␤-oxidation of fatty acids, such as medium chain acyl-CoA dehydrogenase (MCAD). MCAD is a homotetrameric enzyme containing one FAD per 48-kDa monomer (3). Other ETF partners include dehydrogenases involved in amino acid catabolism and 1-carbon metabolism (e.g. isovaleryl-CoA dehydrogenase, dimethylglycine dehydrogenase) (1).Crystal structures of the trimethylamine dehydrogenase⅐2ETF complex from Methylophilus methylotrophus and the human MCAD⅐ETF complex (4, 5) reveal a dual interaction mode at the protein-protein interface. One interaction is centered on the conserved residue Leu-195␤ (human numbering) that serves as an anchor and docks onto a hydrophobic patch on the dehydrogenase partner surface. The second interaction is highly dynamic, with the ETF FAD domain-sampling conformations compatible with the protein-protein interface. Notwithstanding obvious differences in the structure of trimethylamine dehydrogenase and MCAD, the structure of the protein-protein interfaces of the trimethylamine dehydrogenase⅐2ETF and MCAD⅐ETF complexes are remarkably similar. Both ETF partners present a shallow concave surface with the redox cofactor buried beneath. A residue located near the center of this surface is proposed to interact transiently with the conserved residue Arg-249␣ (human numbering...

Studies on the Mechanism of Electron Bifurcation Catalyzed by Electron Transferring Flavoprotein (Etf) and Butyryl-CoA Dehydrogenase (Bcd) of Acidaminococcus fermentans

“…Different Etf Af -Bcd Af contact areas were tested, but no orientation was found that meets the 14 Å distance criteria for an appropriate electron transfer rate between ␣-FAD and Dh-FAD (42). Therefore, we suggest a reorientation of domain II analogous to that observed between human Etf in the isolated and in complex with MCAD (43). The distance between the two FAD can thus be shortened to about 10 Å.…”

“…These conformational changes are realistic because of the soft solvent-mediated domain II-(I ϩ III) interface and their realization in the related Etf-MCAD and Etf-TMADH systems (36,41). It might be triggered by productive NADH binding conveyed to domain II and/or by Bcd binding (43).…”

Kinetic and spectral properties of isovaleryl-CoA dehydrogenase and interaction with ligands

“…Reduction of the flavin upon substrate binding is expected to affect this π-π interaction. It has been implied in the MACD:ETF co-crystal structure study that the indole of this residue plays an undefined role in electron transfer between ACADs and ETF [24, 27]. However, a role in the mechanism of electron transfer seems dispensable since replacing IVD Trp166 with a Phe residue did not abolish transfer of electrons to ETF.…”

“…The co-crystallization of human medium chain acyl-CoA dehydrogenase (MCAD) with human ETF, has provided additional evidence for the ability of ETF to bind its ACAD partners in the absence of their acyl-CoA substrates [23]. Important insight into the molecular mechanism of electron transfer in the oxidative half-reaction has also been gained with the crystallization of MCAD and the ETF βE165A complex [24]. …”

The purified recombinant precursor of rat mitochondrial dimethylglycine dehydrogenase binds FAD via an autocatalytic reaction

“…More recently we have obtained the production in Escherichia coli and purification of rat Me 2 GlyDH as a recombinant active holoenzyme [27], thus providing the basis for further characterization of this enzyme, through kinetic, site-directed mutagenesis and structural studies. Indeed, the recombinant Me 2 GlyDH holoenzyme has been used as a redox partner of human electron-transferring flavoprotein in interprotein electron transfer studies [28].…”