The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing

Abstract

The primary structure of the Fe-S subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A 1st c[complementary]DNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da [dalton] resulted for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with 4 Arg as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only 4 Cys of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cys 3 and 4 are unusually arranged in that they are separated by only 6 Pro. From sequence data the arrangement of the subunit in the membrane is deduced.