The 75-kDa subunit of complex I (NADH:ubiquinone oxidoreductase) from bovine heart mitochondria is its largest subunit and is a component of the iron–sulfur (IP) fragment of the enzyme. It is encoded in nuclear DNA and is imported into the organelle. Protein sequences have been determined at the N-terminus of the intact protein and on fragments generated by partial cleavage with cyanogen bromide and with Staphylococcus aureus protease V8. Parts of these data have been used to design two mixtures of oligonucleotides 17 bases long, containing 192 and 256 different sequences, which have been synthesized and used as hybridization probes for identification of cognate cDNA clones. Two different but overlapping clones have been isolated, and the sequences of the cloned DNAs have been determined. Together they code for a precursor of the 75-kDa subunit of complex I. The mature protein is 704 amino acids in length, has a calculated molecular mass of 75961 daltons, and contains no segments of sequence that could be folded into hydrophobic α-helixes of sufficient length to span the inner membrane of the mitochondrion. Its precursor has an N-terminal extension of 23 amino acids to specify its import into the mitochondrion from the cytoplasm. Seventeen cysteine residues are dispersed throughout the 75-kDa subunit; some of them are close to each other in the sequence in three separate groups and, by analogy with other iron–sulfur proteins, could be involved in iron–sulfur clusters. The sequence of the 75-kDa subunit of complex I is not related closely to any known protein sequence, and the protein is not, as has been proposed, the major human biliary cirrhosis autoantigen.
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