Expression of human electron transfer flavoprotein-ubiquinone oxidoreductase from a baculovirus vector: kinetic and spectral characterization of the human protein.
Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulphur flavoprotein and a component of an electron-transfer system that links 10 different mitochondrial flavoprotein dehydrogenases to the mitochondrial bc1 complex via electron transfer flavoprotein (ETF) and ubiquinone. ETF-QO is an integral membrane protein, and the primary sequences of human and porcine ETF-QO were deduced from the sequences of the cloned cDNAs. We have expressed human ETF-QO in Sf9 insect cells using a baculovirus vector. The cDNA encoding the entire protein, including the mitochondrial targeting sequence, was present in the vector. We isolated a membrane-bound form of the enzyme that has a molecular mass identical with that of the mature porcine protein as determined by SDS/PAGE and has an N-terminal sequence that is identical with that predicted for the mature holoenzyme. These data suggest that the heterologously expressed ETF-QO is targeted to mitochondria and processed to the mature, catalytically active form. The detergent-solubilized protein was purified by ion-exchange and hydroxyapatite chromatography. Absorption and EPR spectroscopy and redox titrations are consistent with the presence of flavin and iron-sulphur centres that are very similar to those in the equivalent porcine and bovine proteins. Additionally, the redox potentials of the two prosthetic groups appear similar to those of the other eukaryotic ETF-QO proteins. The steady-state kinetic constants of human ETF-QO were determined with ubiquinone homologues, a ubiquinone analogue, and with human wild-type ETF and a Paracoccus-human chimaeric ETF as varied substrates. The results demonstrate that this expression system provides sufficient amounts of human ETF-QO to enable crystallization and mechanistic investigations of the iron-sulphur flavoprotein.
Simkovic
Martin
M
Department of Pediatrics, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
Degala
Gregory D
GD
Eaton
Sandra S
SS
Frerman
Frank E
FE
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D044927:Electron-Transferring Flavoproteins; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D044927:Electron-Transferring Flavoproteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D044943:Fatty Acid Desaturases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase