Bovine mitochondrial NADH-ubiquinone reductase (complex I), the first enzyme in the electron-transport chain, is a membrane-bound assembly of more than 30 different proteins, and the flavoprotein (FP) fraction, a water-soluble assembly of the 51-, 24-, and 10-kDa subunits, retains some of the catalytic properties of the enzyme. The 51-kDa subunit binds the substrate NAD(H) and probably contains both the cofactor, FMN, and also a tetranuclear iron-sulfur center, while a binuclear iron-sulfur center is located in the 24- or 10-kDa proteins. The 75-kDa subunit is the largest of the six proteins in the iron-sulfur protein (IP) fraction, and its sequence indicates that it too contains iron-sulfur clusters. Partial protein sequences have been determined at the N-terminus and at internal sites in the 51-kDa subunit, and the corresponding cDNA encoding a precursor of the protein has been isolated by using a novel strategy based on the polymerase chain reaction. The mature protein is 444 amino acids long. Its sequence, and those of the 24- and 75-kDa subunits, shows that mitochondrial complex I is related to a soluble NAD-reducing hydrogenase from the facultative chemolithotroph Alcaligenes eutrophus H16. This enzyme has four subunits, alpha, beta, gamma, and delta, and the alpha gamma dimer is an NADH oxidoreductase that contains FMN. The gamma-subunit is related to residues 1-240 of the 75-kDa subunit of complex I, and the alpha-subunit sequence is a fusion of homologues of the 24- and 51-kDa subunits, in the order N- to C-terminal. The most highly conserved regions are in the 51-kDa subunit and probably form parts of nucleotide binding sites for NAD(H) and FMN. Another conserved region surrounds the sequence motif CysXXCysXXCys, which is likely to provide three of the four ligands of a 4Fe-4S center, possibly that known as N-3. Characteristic ligands for a second 4Fe-4S center are conserved in the 75-kDa and gamma-subunits. This relationship with the bacterial enzyme implies that the 24- and 51-kDa subunits, together with part of the 75-kDa subunit, constitute a structural unit in mitochondrial complex I that is concerned with the first steps of electron transport.

The 24-kDa subunit of mitochondrial NADH-ubiquinone reductase (complex I) is an iron-sulfur protein that is present in the flavoprotein or NADH dehydrogenase II subcomplex. It is a nuclear gene product and is imported into the organelle. A group of human patients with mitochondrial myopathy have been shown to have reduced levels of subunits of complex I in skeletal muscle mitochondria, and in one patient the 24-kDa subunit appears to be absent (Schapira et al., 1988). To investigate the genetic basis of this type of myopathy, cDNA clones have been isolated from a bovine library derived from heart and liver mRNA by hybridization with two mixtures of 48 synthetic oligonucleotides 17 bases in length that were designed on the basis of known protein sequences. The recombinant DNA sequence has been determined, and it encodes a precursor of the mature 24-kDa protein. The N terminus of the mature protein is preceded by a presequence of 32 amino acids that has properties that are characteristic of mitochondrial import sequences. The sequence of the mature protein deduced from the cDNA contains a segment of nine amino acids that was not determined in an earlier partial protein sequence analysis. The bovine clone has been employed as a hybridization probe to identify cDNA clones of the human homologue of the 24-kDa protein. Its DNA sequence has also been determined, and it codes for a protein that is closely related to the bovine protein.(ABSTRACT TRUNCATED AT 250 WORDS)

In cattle, 7 of the 30 or more subunits of the respiratory enzyme NADH:ubiquinone reductase (complex I) are encoded in mitochondrial DNA, and potential genes (open reading frames, orfs) for related proteins are found in the chloroplast genomes of Marchantia polymorpha and Nicotiana tabacum. Homologues of the nuclear-coded 49- and 23-kDa subunits are also coded in chloroplast DNA, and these orfs are clustered with four of the homologues of the mammalian mitochondrial genes. These findings have been taken to indicate that chloroplasts contain a relative of complex I. The present work provides further support. The 30-kDa subunit of the bovine enzyme is a component of the iron-sulfur protein fraction. Partial protein sequences have been determined, and synthetic oligonucleotide mixtures based on them have been employed as hybridization probes to identify cognate cDNA clones from a bovine library. Their sequences encode the mitochondrial import precursor of the 30-kDa subunit. The mature protein of 228 amino acids contains a segment of 57 amino acids which is closely related to parts of proteins encoded in orfs 169 and 158 in the chloroplast genomes of M. polymorpha and N. tabacum. Moreover, the chloroplast orfs are found near homologues of the mammalian mitochondrial genes for subunit ND3. Therefore, the plant chloroplast genomes have at least two separate clusters of potential genes encoding homologues of subunits of mitochondrial complex I. The bovine 30-kDa subunit has no extensive sequences of hydrophobic amino acids that could be folded into membrane-spanning alpha-helices, and although it contains two cysteine residues, there is no clear evidence in the sequence that it is an iron-sulfur protein.