Molecular cloning and expression of a cDNA encoding human electron transfer flavoprotein‐ubiquinone oxidoreductase

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Abstract
Electron‐transfer flavoprotein‐ubiquinone oxidoreductase (ETF‐QO) in the inner mitochondrial membrane accepts electrons from electron‐transfer flavoprotein which is located in the mitochondrial matrix and reduces ubiquinone in the mitochondrial membrane. The two redox centers in the protein, FAD and a [4Fe4S]+2,+1 cluster, are present in a 64‐kDa monomer. We cloned several cDNA sequences encoding the majority of porcine ETF‐QO and used these as probes to clone a full‐length human ETF‐QO cDNA. The deduced human ETF‐QO sequence predicts a protein containing 617 amino acids (67 kDa), two domains associated with the binding of the AMP moiety of the FAD prosthetic group, two membrane helices and a motif containing four cysteine residues that is frequently associated with the liganding of ferredoxin‐like iron‐sulfur clusters. A cleavable 33‐amino‐acid sequence is also predicted at the amino terminus of the 67‐kDa protein which targets the protein to mitochondria. In vitro transcription and translation yielded a 67‐kDa immunoprecipitable product as predicted from the open reading frame of the cDNA. The human cDNA was expressed in Saccharomyces cerevisiae, which does not normally synthesize the protein. The ETF‐QO is synthesized as a 67‐kDa precursor which is targeted to mitochondria and processed in a single step to a 64‐kDa mature form located in the mitochondrial membrane. The detergent‐solubilized protein transfers electrons from ETF to the ubiquinone homolog, Q1, indicating that both the FAD and iron‐sulfur cluster are properly inserted into the heterologously expressed protein.

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