Three-dimensional structure of human electron transfer flavoprotein to 2.1-Å resolution

Abstract

Mammalian electron transfer f lavoproteins (ETF) are heterodimers containing a single equivalent of f lavin adenine dinucleotide (FAD). They function as electron shuttles between primary f lavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the ␣ subunit and the third domain is made up entirely by the ␤ subunit. The N-terminal portion of the ␣ subunit and the majority of the ␤ subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the ␣ subunit. Alignment of all the known sequences for the ETF ␣ subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of ␣T266, suggesting why the pathogenic mutation, ␣T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between ␣H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

Extensive Domain Motion and Electron Transfer in the Human Electron Transferring Flavoprotein·Medium Chain Acyl-CoA Dehydrogenase Complex

“…Comparison of the complex crystal structure with the available crystal structures for the uncomplexed protein partners (6,18) reveals no significant differences for the MCAD structures. The recognition loop of ETF has, however, adopted a slightly different backbone conformation with more extensive rear- Leu␤ 195 Is Essential for Complex Formation-The ETF mutant L␤195A leads to no significant rearrangement of the recognition loop, as determined by the 1.8 Å crystal structure of this mutant protein (data not shown); yet this mutant is severely impaired in complex formation with MCAD.…”

“…ETFs are heterodimeric proteins that fold into three distinct domains; the FAD is bound noncovalently to domain II, which sits in a shallow bowl created by domains I and III (6). Recent models constructed using the known crystal structures of human ETF and its partner human MCAD have suggested that electron transfer from MCAD to ETF requires rotational movement of the ETF FAD domain (7).…”

Electron Transfer and Conformational Change in Complexes of Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

“…Dynamic deactivation of excited FAD by the adenosine moiety is well known for FAD in aqueous solution (32). However, the crystal structures of human (24) and Paracoccus denitrificans (33) ETF, and the model of M. methylotrophus ETF (25), indicate the adenosine moiety does not contact the isoalloxazine ring in ETF-bound FAD. The quenching group is therefore derived from the protein and not the FAD cofactor itself.…”

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

“…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).…”