Structure of electron transfer flavoprotein-ubiquinone oxidoreductase and electron transfer to the mitochondrial ubiquinone pool

Abstract

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQbinding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membranebinding surface contains an ␣-helix and a ␤-hairpin, forming a hydrophobic plateau. The UQOflavin distance (8.5 Å) is shorter than the UQOcluster distance (18.8 Å), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD.fatty acid oxidation ͉ iron-sulfur flavoprotein ͉ mitochondrial respiratory chain ͉ membrane protein ͉ acyl-CoA dehydrogenases E lectron transfer f lavoprotein-ubiquinone oxidoreductase (ETF-QO) is an intrinsic membrane protein located in the inner mitochondrial membrane. It contains single equivalents of FAD and a [4Fe4S] 2ϩ,1ϩ cluster (1). The protein is the single input site to the main respiratory chain for electrons from nine flavoprotein acyl-CoA dehydrogenases and two N-methyl dehydrogenases (2, 3). The electron acceptor for the dehydrogenases is the ETF, which is the reductant of ETF-QO. ETF-QO is oxidized by the diffusible ubiquinone (UQ) pool that also is accessed by NADH-UQ oxidoreductase (Complex I), succinate-UQ oxidoreductase (Complex II), the flavin-linked glycerol-3-phosphate dehydrogenase, and dihydroorotate dehydrogenase, another flavin-linked UQ oxidoreductase (4). The ubiquinol product of these oxidoreductases transfers electrons to the bc 1 complex (Complex III). Thus, ETF and ETF-QO link the oxidation of fatty acids and some amino acids to the mitochondrial respiratory system, and the overall electron flow can be summarized as follows: Acyl-CoA 3 Acyl-CoA dehydrogenases 3 ETF 3 ETF-QO 3 UQ 3 Complex III. Inherited deficiencies of ETF-QO or ETF cause a metabolic disease, multiple acyl-CoA dehydrogenase deficiency, also known as glutaric acidemia type II (5). This metab...

Structure of glycerol-3-phosphate dehydrogenase, an essential monotopic membrane enzyme involved in respiration and metabolism

“…Structural evidence that this region is the UQ docking site is that a number of ␤-strands are found here, presenting a planar lipophilic surface, similar to that observed in electron-transfer flavoproteinubiquinone oxidoreductase (ETF-QO) (19). Additionally, the presence of a topological switch point, whereby two adjacent parallel ␤-strands form a substrate binding cleft with their respective loops, further signify a physiological binding site (20).…”

“…9). No other cofactors or metals seem to be required for GlpD activity nor metal clusters, such as FeS-clusters in the case of ETF-QO (19). Lack of additional cofactors suggests that electron transfer from FADH 2 to UQ may be mediated through protein residues or that a ping-pong type mechanism may function whereby the product, DHAP, first exits the cleft, permitting UQ access to FADH 2 for reduction.…”

Biochemical Characterization and Membrane Topology of Alg2 from Saccharomyces cerevisiae as a Bifunctional α1,3- and 1,6-Mannosyltransferase Involved in Lipid-linked Oligosaccharide Biosynthesis

“…All of the sugar residues are donated by sugar nucleotides. Alg2, a Bifunctional Mannosyltransferase insertion of monotopic proteins is often accomplished by hydrophobic helices interacting with one leaflet of the membrane (63)(64)(65)(66)(67)(68). Further work will be necessary to understand mechanistically how one and the same enzyme can accomplish catalyzing two different linkages and altering the specificity of the active site from the Man(␤1,4)GlcNAc 2 trisaccharide acceptor to the newly formed tetrasaccharide intermediate Man(␣1,3) Man(␤1,4)GlcNAc 2 .…”

Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO)

“…Both ETF-QO and ETF contain FAD in an extended conformation [11,55] and both stabilize an anionic semiquinone with very similar EPR line widths, 14.5 G for ETF [56] and 14.6 G for porcine ETF-QO [7].…”

“…Previously, the possibility of dipolar interaction between the anionic semiquinone and [4Fe-4S] + in ETF-QO was suggested [7] and it was noted that a small broadening along one principal axis of the [4Fe-4S] + signal at low temperature could be due to dipolar interaction with paramagnetic semiquinone [7]; however, the changes were too small to be conclusive. The crystal structure of porcine ETF-QO to 2.7-Å resolution shows that the distances of closest approach between the [4Fe-4S] + and the methyl group(8α) of the FAD isoalloxazine ring and between the [4Fe-4S] + and the bound benzoquinone ring of ubiquinone are 11.5 Å and 18.8 Å, respectively [11]. The point-dipole distance between the unpaired electrons is substantially larger than the distance of closest approach since the semiquinone unpaired spin density is distributed over the FAD molecule [12,13].…”