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.