The iron-sulfur cluster of electron transfer flavoprotein-ubiquinone oxidoreductase is the electron acceptor for electron transfer flavoprotein.

Electron transfer flavoprotein–ubiquinone oxidoreductase (ETF-QO) is responsible for linking electrons derived from the oxidation of fatty acids and some amino acids to the main mitochondrial respiratory chain (1). It is a monotopic membrane protein located on the inner mitochondria membrane facing into the mitochondrial matrix. ETF-QO has two redox-active cofactors, one [4Fe-4S]2+,1+ cluster and one flavin adenine dinucleotide (FAD). ETF-QO oxidizes electron transfer flavoprotein (ETF), a protein responsible for oxidizing nine flavoprotein dehydrogenases and two N-methyl dehydrogenases (2), and reduces ubiquinone (UQ). UQ then transfers the electrons to the cytochrome bc1 complex (complex III). Defects in ETF-QO, or its electron donor ETF, result in a metabolic disease known as multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric acidemia type II (GAII). The severity of this disease depends upon the particular mutation and can range from late-onset in the teenage years to death within the first days after birth (3, 4). It is important to understand better the physical properties of this enzyme because of its important role in oxidative metabolism and its link to GAII. The locations of the redox centers in the crystal structure of porcine ETF-QO provide insights into the possible mechanism of electron transfer in this enzyme (5). The FAD molecule is closer to the UQ than the [4Fe-4S]2+,1+ cluster (9.9 A as opposed to 18.8 A) and the cluster is closer to the surface of the protein than the flavin ring of FAD (5) (Figure 1). This suggests that the [4Fe-4S]2+,1+ cluster is responsible for accepting electrons from ETF and that the FAD is responsible for reducing UQ. The closest approach between the [4Fe-4S]2+,1+ cluster and the FAD is 11.5 A which is consistent with electron transfer between the two cofactors (6). There is 98% sequence identity between porcine and human ETF-QO and 67% sequence identity between human and Rhodobacter sphaeroides ETF-QO (N. J. Watmough, F. E. Frerman, and J. N. Butt, 2008, unpublished results). This high sequence similarity predicts that the tertiary structures are closely related (7) and that the mechanism of electron transfer is likely similar in all three enzymes. Figure 1 Crystal structure of porcine ETF-QO (pdb data bank: 2GMH) highlighting the positions of the three redox centers, [4Fe-4S]2+,1+ cluster (red and yellow), FAD (pink), and ubiquinone (blue). On the right the ribbon structure has been removed for clarity ... Recently the mutations Y501F, T525A, and Y501F/T525A were introduced into R. sphaeroides ETF-QO (8). These residues were chosen for mutation because the same residues (although numbered differently) hydrogen bond to the cysteine Sγ atoms that are ligated to the [4Fe-4S]2+,1+ cluster of porcine ETF-QO. The mutations eliminate this hydrogen bonding and thus change the redox potential of the [4Fe-4S]2+,1+ cluster, which in turn affects the activity of the enzyme. The [4Fe-4S]2+,1+ midpoint potentials were lowered by about 100 mV for either single mutation and by about 165 mV for the double mutant. The lower redox potentials of these mutants decreased the quinone reductase activity and the rates of disproportionation of ETF1e−compared to the wild-type enzyme (8). Both single mutations had similar impacts on activities. These results demonstrate that reduction of [4Fe-4S]2+,1+ is required for proper enzyme function. As expected these mutations caused no change in FAD midpoint potentials. In this study mutations were introduced to modulate the FAD redox potentials and investigate its importance for ETF-QO function. It has been shown that amino acids that form hydrogen bonding interactions with the isoalloxazine head group of a FAD molecule can modulate its redox potentials (9). The X-ray crystal structure of porcine ETF-QO (5) shows that the location of threonine 367 is suitable for formation of hydrogen bonds with the N1 and O2 of the FAD. In R. sphaeroides ETF-QO asparagine 338 is at the position equivalent to threonine 367 of the porcine enzyme. Therefore, it is proposed that this residue is in position to form hydrogen bonds with N1 and O2 of the FAD (Figure 2). The mutations N338A and N338T were introduced to eliminate or lower the energy of the hydrogen bond to the FAD respectively. The impact of these changes on FAD redox potential and enzyme activity was determined. Results from this study and the previous mutagenesis near the [4Fe-4S]2+,1+ cluster can be combined to give a description of the roles of the FAD and [4Fe-4S]2+,1+ cluster in ETF-QO electron transfer. Figure 2 Position of threonine 367 and the C2 oxygen of the FAD isoalloxazine group from the porcine ETF-QO crystal structure (pdb data bank: 2GMH). The corresponding amino acid in R. sphaeroides ETF-QO is asparagine 338 which is in position to interact with the ...