Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a monotopic protein that is located in the inner mitochondrial membrane (1
). ETF-QO is the electron acceptor for electron transfer flavoprotein (ETF) which catalyzes the oxidation of nine flavoprotein dehydrogenases and two N-methyl dehydrogenases (2
). Ubiquinone is the physiological electron acceptor for ETF-QO and transfers the electrons to cytochrome bc1
complex (Complex III). ETF-QO is essential for the oxidation of fatty acids and some amino acids (1
). Inherited deficiencies of this protein result in a severe metabolic disease known as multiple acyl-CoA dehydrogenase deficiency or glutaric academia type II (5
). Interest in the pathophysiology of this metabolic disease and the role of the enzyme in oxidative metabolism motivates investigation of the kinetic and thermodynamic properties of ETF-QO.
ETF-QO contains a [4Fe-4S]2+,1+
cluster and a single equivalent of flavin adenine dinucleotide (FAD). Both centers are diamagnetic in the oxidized as-isolated enzyme. In the wild type protein the redox midpoint potentials of the two centers are similar and it is only possible to poise the system in a state where both the [4Fe-4S]+
cluster and the flavin are partially in the paramagnetic forms (4
). Enzymatic reduction of the wild type protein with octanoyl-CoA adds approximately two electrons to the system, distributed between the iron-sulfur cluster, the flavin semiquinone, and the flavin hydroquinone. Strong chemical reducing agents such as dithionite fully reduce the iron-sulfur cluster and reduce the flavin to hydroquinone. To elucidate the roles of the two redox active centers it would be helpful to selectively modify the redox potential of the iron-sulfur cluster. Some of the environmental factors that modulate redox potentials include solvent accessibility, hydrogen bonding to cysteine Sγ ligated to the cluster, backbone amide dipoles and local charge (6
). Computational methods predicting the effects of protein environment on the redox potentials of iron-sulfur clusters are now complemented by direct determination of cluster potentials in site directed mutants (7
). In particular, Denke et al
. have shown alteration of the midpoint potential and catalytic activity of the Rieske iron-sulfur protein by eliminating hydrogen bonds to the Sγ sulfur atoms bound to the cluster (8
Recently, the crystal structure of porcine ETF-QO (2.7 Å) was determined in the presence and absence of bound ubiquinone (UQ) (11
). The structure shows that the three functional domains, i.e., the iron-sulfur cluster, FAD, and ubiquinone domains, are closely packed and share structural elements. The spatial relationship between the flavin and the cluster are shown in . The distance of closest approach between the flavin (C8) and UQ (O2) is 9.9 Å, which is much shorter than the 18.8 Å distance between cluster (Fe3) and UQ (O2). A survey of intramolecular electron-transfer pathways in proteins concluded that the upper limit for direct transfer was 14 Å (12
). Thus, the shorter distance between the flavin and UQ suggests that the flavin is responsible for the reduction of UQ and not the cluster. There is no structural information available on the docking sites of ETF to ETF-QO, but the relative proximity of the cluster to the enzyme surface (~8 Å), as opposed to the flavin (>14 Å), suggests that the cluster might accept electrons from ETF. The midpoint potentials for flavin semiquinone (+28 mV) and cluster (+47 mV) are similar and the Fe3 atom of the cluster is 11.5 Å from the C8 atom in the isoalloxazine ring, consistent with rapid equilibration between the two centers (1
). Zhang et al.
) that the cluster may serve as a redox-poising or an electron storage site for the flavin as in NADH-UQ oxidoreductase (Complex I) (13
). The structure reveals that the cluster is supported through hydrogen bonds between Sγ atoms of the four cysteines and the polypeptide chain. Amino acids H503, T558, and Y533 in porcine ETF-QO form weak hydrogen bonds to cysteine Sγ that could modulate the redox potential of the iron-sulfur cluster, . These sites are of interest to investigate the impact that hydrogen bonding to the iron-sulfur cluster has on enzyme activity and electronic structure of the cluster. Porcine and human ETF-QO have 98% sequence identity, and human and R. sphaeroides
ETF-QO have 67% sequence identity (Watmough, N. J., Frerman, F. E., Butt, J. N. (2007), unpublished results), which predicts closely related tertiary structures (14
). The equivalents of porcine T558 and Y533 are Y501 and T525 in R. sphaeroides
ETF-QO. The mutations Y501F, T525A, and Y501F/T525A were introduced to eliminate the hydrogen bonds to cysteine Sγ atoms of the [4Fe-4S] cluster and determine the impact of the hydrogen bonds on redox potentials and activity.
Figure 1 Relative locations of the iron-sulfur cluster, the residues that were mutated, and the FAD based on the crystal structure of porcine ETF-QO (2GMH). The distance of closest approach between the cluster (Fe3) and the isoalloxazine ring (C8) of the FAD is (more ...)