The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first of the respiratory complexes providing the proton motive force which is essential for energy consuming processes like the synthesis of ATP. Homologues of this complex exist in bacteria, archaea, in mitochondria of eukaryotes and in chloroplasts of plants. The bacterial and mitochondrial complexes function as NADH dehydrogenase, while the archaeal complex works as F 420 H 2 dehydrogenase. The electron donor of the cyanobacterial and plastidal complex is not yet known. Despite the different electron input sites, 11 polypeptides constitute the structural framework for proton translocation and quinone binding in the complex of all three domains of life. Six of them are also present in a family of membrane-bound multisubunit [NiFe] hydrogenases. It is discussed that they build a module for electron transfer coupled to proton translocation. ß

The proton-translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550,000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N-terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetranuclear (N2, N3 and N4) iron-sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin-A-sensitive electron transfer from NADH to ubiquinone-2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X-100. One of these fragments is a water-soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron-sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR-detectable iron-sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.

both the mitochondrial and the bacterial complex throughout Abstract The proton-pumping NADH:ubiquinone oxidoreducthis review. tase, also called complex I, is the first of the respiratory corn-The structure of complex I is extraordinarily complex. The plexes providing the proton motive force which is essential for the mitochondrial complex comprises about 40 different polypepsynthesis of ATP. Closely related forms of this complex exist in the mitochondria of eucaryotes and in the plasma membranes of tides, that are of both nuclear and mitochondrial genetic origin purple bacteria. The minimal structural framework common to [1,3,6]. Bacteria contain a minimal form of complex I with fewer the mitochondrial and the bacterial complex is composed of 14 subunits but including homologues of all subunits of the mitopolypeptides with 1 FMN and 6-8 iron-sulfur clusters as proschondrial complex presumed to bind subtrates or prosthetic thetic groups. The mitochondrial complex contains many acces-

Hydrogenosomes are organelles that produce ATP and hydrogen, and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates. Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist. These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes. Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product--biochemical traits typical of anaerobic mitochondria. The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

The proton‐translocating NADH:ubiquinone oxidoreductase (complex I) was isolated from Escherichia coli by chromatographic steps performed in the presence of an alkylglucoside detergent at pH 6.0. The complex is obtained in a monodisperse state with a molecular mass of approximately 550000 Da and is composed of 14 subunits. The subunits were assigned to the 14 genes of the nuo operon, partly based on their N‐terminal sequences and partly on their apparent molecular masses. The preparation contains one noncovalently bound FMN/molecule. At least two binuclear (N1b and N1c) and three tetra‐nuclear (N2, N3 and N4) iron‐sulfur clusters were detected by EPR in the preparation when reduced with NADH. Their EPR characteristics remained mostly unaltered during the isolation process. After reconstitution in phospholipid membranes, the preparation catalyses piericidin‐A‐sensitive electron transfer from NADH to ubiquinone‐2 with Km values similar to those of complex I in cytoplasmic membranes but with only 10% of the Vmax value. The isolated complex I was cleaved into three fragments when the pH was raised from 6.0 to 7.5 and the detergent exchanged to Triton X‐100. One of these fragments is a water‐soluble NADH dehydrogenase fragment which is composed of three subunits bearing at least four iron‐sulfur clusters (N1b, N1c, N3 and N4) that can be reduced with NADH, one of them bearing FMN. The second, amphipathic, fragment, which is presumed to connect the NADH dehydrogenase fragment with the membrane, contains four subunits and at least one EPR‐detectable iron‐sulfur cluster whose spectral properties are reminiscent of the eucaryotic cluster N2. The third membrane fragment is composed of seven homologues of the mitochondrially encoded subunits of the eucaryotic complex I. This subunit arrangement coincidences to some extent with the order of the genes on the nuo operon. A topological model of the E. coli complex I is proposed.