NADH-quinone 1 oxidoreductase (Complex I) isolated from bovine heart mitochondria was, until recently, the major source for the study of this most complicated energy transducing device in the mitochondrial respiratory chain. Complex I has been shown to contain 43 subunits and possesses a molecular mass of about 1 million. Recently, Complex I genes have been cloned and sequenced from several bacterial sources including Escherichia coli, Paracoccus denitrificans, Rhodobacter capsulatus and Thermus thermophilus HB-8. These enzymes are less complicated than the bovine enzyme, containing a core of 13 or 14 subunits homologous to the bovine heart Complex I. From this data, important clues concerning the subunit location of both the substrate binding site and intrinsic redox centers have been gleaned. Powerful molecular genetic approaches used in these bacterial systems can identify structure/function relationships concerning the redox components of Complex I. Site-directed mutants at the level of bacterial chromosomes and over-expression and purification of single subunits have allowed detailed analysis of the amino acid residues involved in ligand binding to several iron-sulfur clusters. Therefore, it has become possible to examine which subunits contain individual iron-sulfur clusters, their location within the enzyme and what their ligand residues are. The discovery of g=2.00 EPR signals arising from two distinct species of semiquinone (SQ) in the activated bovine heart submitochondrial particles (SMP) is another line of recent progress. The intensity of semiquinone signals is sensitive to DeltamicroH+ and is diminished by specific inhibitors of Complex I. To date, semiquinones similar to those reported for the bovine heart mitochondrial Complex I have not yet been discovered in the bacterial systems. This mini-review describes three aspects of the recent progress in the study of the redox components of Complex I: (A) the location of the substrate (NADH) binding site, flavin, and most of the iron-sulfur clusters, which have been identified in the hydrophilic electron entry domain of Complex I; (B) experimental evidence indicating that the cluster N2 is located in the amphipathic domain of Complex I, connecting the promontory and membrane parts. Very recent data is also presented suggesting that the cluster N2 may have a unique ligand structure with an atypical cluster-ligation sequence motif located in the NuoB (NQO6/PSST) subunit rather than in the long advocated NuoI (NQO9/TYKY) subunit. The latter subunit contains the most primordial sequence motif for two tetranuclear clusters; (C) the discovery of spin-spin interactions between cluster N2 and two distinct Complex I-associated species of semiquinone. Based on the splitting of the g1 signal of the cluster N2 and concomitant strong enhancement of the semiquinone spin relaxation, one semiquinone species was localized 8-11 A from the cluster N2 within the inner membrane on the matrix side (N-side). Spin relaxation of the other semiquinone species is much less enhanc...

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.

Deamino-NADH/ubiquinone 1 oxidoreductase activity in membrane preparations from Escherichia coli GR19N is 20-50% of NADH/ubiquinone 1 oxidoreductase activity. In comparison, membranes from E. coli IY91, which contain amplified levels of NADH dehydrogenase, exhibit about 100-fold higher NADH/ubiquinone 1 reductase activity but about 20-fold less deamino-NADH/ubiquinone 1 reductase activity. Deamino-NADH/ubiquinone 1 reductase is more sensitive than NADH/ubiquinone 1 reductase activity to inhibition by 3-undecyl-2-hydroxyl-1,4-naphthoquinone, piericidin A, or myxothiazol. Furthermore, GR19N membranes exhibit two apparent Kms for NADH but only one for deamino-NADH. Inside-out membrane vesicles from E. coli GR19N generate a H+ electrochemical gradient (interior positive and acid) during electron transfer from deamino-NADH to ubiquinone 1 that is large and stable relative to that observed with NADH as substrate. Generation of the H+ electrochemical gradient in the presence of deamino-NADH is inhibited by 3-undecyl-2-hydroxy-1,4-naphthoquinone and is not observed in IY91 membrane vesicles or in vesicles from GR19N that are deficient in deamino-NADH/ubiquinone 1 reductase activity. The data provide a strong indication that the E. coli aerobic respiratory chain contains two species of NADH dehydrogenases: (i) an enzyme (NADH dh I) that reacts with deamino-NADH or NADH whose turnover leads to generation of a H+ electrochemical gradient at a site between the primary dehydrogenase and ubiquinone and (ii) an enzyme (NADH dh II) that reacts with NADH exclusively whose turnover does not lead to generation of a H+ electrochemical gradient between the primary dehydrogenase and ubiquinone 1.

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.