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The crystal structure of the human electron transferring flavoprotein (ETF)⅐medium chain acyl-CoA dehydrogenase (MCAD) complex reveals a dual mode of protein-protein interaction, imparting both specificity and promiscuity in the interaction of ETF with a range of structurally distinct primary dehydrogenases. ETF partitions the functions of partner binding and electron transfer between (i) the recognition loop, which acts as a static anchor at the ETF⅐MCAD interface, and (ii) the highly mobile redox active FAD domain. Together, these enable the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. Disorders in amino acid or fatty acid catabolism can be attributed to mutations at the protein-protein interface. Crucially, complex formation triggers mobility of the FAD domain, an induced disorder that contrasts with general models of protein-protein interaction by induced fit mechanisms. The subsequent interfacial motion in the MCAD⅐ETF complex is the basis for the interaction of ETF with structurally diverse protein partners. Solution studies using ETF and MCAD with mutations at the protein-protein interface support this dynamic model and indicate ionic interactions between MCAD Glu 212 and ETF Arg␣ 249 are likely to transiently stabilize productive conformations of the FAD domain leading to enhanced electron transfer rates between both partners.

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...

Amines are a carbon source for the growth of a number of bacterial species and they also play key roles in neurotransmission, cell growth and differentiation, and neoplastic cell proliferation. Enzymes have evolved to catalyse these reactions and these oxidoreductases can be grouped into the flavoprotein and quinoprotein families. The mechanism of amine oxidation catalysed by the quinoprotein amine oxidases is understood reasonably well and occurs through the formation of enzyme-substrate covalent adducts with TPQ (topaquinone), TTQ (tryptophan tryptophylquinone), CTQ (cysteine tryptophylquinone) and LTQ (lysine tyrosyl quinone) redox centres. Oxidation of amines by flavoenzymes is less well understood. The role of protein-based radicals and flavin semiquinone radicals in the oxidation of amines is discussed.

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