The Iron−Sulfur Cluster of Electron Transfer Flavoprotein−Ubiquinone Oxidoreductase Is the Electron Acceptor for Electron Transfer Flavoprotein

Abstract

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the ubiquinone-pool. It contains one [4Fe-4S]2+,1+ and one FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. In the porcine protein, threonine 367 is hydrogen bonded to N1 and O2 of the flavin ring of the FAD. The analogous site in Rhodobacter sphaeroides ETF-QO is asparagine 338. Mutations N338T and N338A were introduced into the R. sphaeroides protein by site-directed mutagenesis to determine the impact of hydrogen bonding at this site on redox potentials and activity. The mutations did not alter the optical spectra, EPR g-values, spin-lattice relaxation rates, or the [4Fe-4S]2+,1+ to FAD point-dipole interspin distances. The mutations had no impact on the reduction potential for the iron-sulfur cluster, which was monitored by changes in the continuous wave EPR signals of the [4Fe-4S]+ at 15 K. For the FAD semiquinone, significantly different potentials were obtained by monitoring the titration at 100 or 293 K. Based on spectra at 293 K the N338T mutation shifted the first and second midpoint potentials for the FAD from +47 mV and −30 mV for wild type to −11 mV and −19 mV, respectively. The N338A mutation decreased the potentials to −37 mV and −49 mV. Lowering the midpoint potentials resulted in a decrease in the quinone reductase activity and negligible impact on disproportionation of ETF1e− catalyzed by ETF-QO. These observations indicate that the FAD is involved in electron transfer to ubiquinone, but not in electron transfer from ETF to ETF-QO. Therefore the iron-sulfur cluster is the immediate acceptor from ETF.

A Photolyase-Like Protein from Agrobacterium tumefaciens with an Iron-Sulfur Cluster

“…These authors showed that CryB comprises, in addition to FAD, another cofactor that is different from other flavins or MTHF but the cofactor remained unidentified. The absorption spectrum of PhrB also resembles that of the electron transfer flavoprotein-ubiquinone oxidoreductase [41]. This flavoprotein contains FAD and a (4Fe-4S) cluster as cofactors, which both contribute to the absorption in the spectral region of interest [41].…”

An Electron-bifurcating Caffeyl-CoA Reductase

“…This extension can be found in the large Etf subunit of other anaerobic bacteria such as Clostridium saccharolyticum and was proposed for several other organisms by comparing genomic sequences (41). In the mitochondrial electron transfer flavoprotein:ubiquinone oxidoreductase, a [4Fe-4S] cluster is the immediate acceptor for electrons derived from the Etf protein (45,46). Thus, the iron-sulfur cluster in EtfA from A. woodii may be involved in electron transport, possibly in the intramolecular electron transfer from the Etf protein subunit to the caffeyl-CoA reductase subunit inside the complex.…”

Identification of the 2-Hydroxyglutarate and Isovaleryl-CoA Dehydrogenases as Alternative Electron Donors Linking Lysine Catabolism to the Electron Transport Chain ofArabidopsisMitochondria

“…In addition, the functional role of both ETFQO and ETF itself was recently established via a characterization of allelic T-DNA mutants of the ETFQO protein and of the b-subunit of ETF (Ishizaki et al, , 2006. The latter studies demonstrated the formation of a similar complex in plants as those previously characterized in microbial and mammalian systems (Frerman, 1988;Frerman and Goodman, 2001;Swanson et al, 2008). Moreover, metabolic profiling of leaves from the wild type and all mutants following transfer to extended periods of darkness revealed a dramatic decline in sugar levels.…”