The structure of eukaryotic and prokaryotic complex I.

The structures of the NADH dehydrogenases from Bos taurus and Aquifex aeolicus have been determined by 3D electron microscopy, and have been analyzed in comparison with the previously determined structure of Complex I from Yarrowia lipolytica. The results show a clearly preserved domain structure in the peripheral arm of complex I, which is similar in the bacterial and eukaryotic complex. The membrane arms of both eukaryotic complexes show a similar shape but also significant differences in distinctive domains. One of the major protuberances observed in Y. lipolytica complex I appears missing in the bovine complex, while a protuberance not found in Y. lipolytica connects in bovine complex I a domain of the peripheral arm to the membrane arm. The structural similarities of the peripheral arm agree with the common functional principle of all complex Is. The differences seen in the membrane arm may indicate differences in the regulatory mechanism of the enzyme in different species.

Site-directed mutagenesis of cytochrome c oxidase reveals two acidic residues involved in the binding of cytochrome c.

Site-directed mutagenesis in subunit II of the cytochrome c oxidase (haem aa3) from Paracoccus denitrificans reveals that two carboxylic residues, Glu-246 and Asp-206 (corresponding to 198 and 158 in the bovine subunit II), are involved in the binding of cytochrome c. Spectrophotometric and polarographic measurements with the isolated enzymes of both mutant strains show a strongly reduced activity compared to wild-type oxidase, with the overall catalytic capacity (kcat/KM) of both mutants decreased about 8-fold. EPR spectra reveal no significant differences between the wild-type and the mutant enzymes, indicating that neither residue contributes significantly to the structure of the CuA centre. We conclude that Glu-246 and Asp-206 constitute an essential part of the binding site for cytochrome c.

Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica.

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.

The NADH oxidation domain of complex I: do bacterial and mitochondrial enzymes catalyze ferricyanide reduction similarly?

The hexammineruthenium (HAR) and ferricyanide reductase activities of Complex I (H+-translocating NADH:ubiquinone reductase) from Paracoccus denitrificans and bovine heart mitochondria were studied. The rates of HAR reduction are high, and its steady-state kinetics is similar in both P. denitrificans and bovine Complex I. The deamino-NADH:HAR reductase activity of Complex I from both sources is significantly higher than the respective activity in the presence of NADH. The HAR reductase activity of the bacterial and mitochondrial Complex I is similarly and strongly pH dependent. The pK(a) of this activity could not be determined, however, due to low stability of the enzymes at pH values above 8.0. In contrast to the high similarity between bovine and P. denitrificans Complex I as far as HAR reduction is concerned, the ferricyanide reductase activity of the bacterial enzyme is much lower than in mitochondria. Moreover, ferricyanide reduction in P. denitrificans, but not bovine mitochondria, is partially sensitive to dicyclohexylcarbodiimide (T. Yagi, Biochemistry 26 (1987) 2822-2828). On the other hand, the inhibition of ferricyanide reduction by high concentration of NADH, a typical phenomenon in bovine Complex I, is much weaker in the bacterial enzyme. The functional differences between the two enzymes might be linked to the properties of their binuclear Fe-S clusters.

Binding of detergents and inhibitors to bovine complex I - a novel purification procedure for bovine complex I retaining full inhibitor sensitivity.

Mitochondrial complex I exhibits some peculiar and poorly understood features regarding the effects of detergents on activity and sensitivity to hydrophobic inhibitors that are not seen with other membrane complexes using ubiquinone as a substrate. Therefore, we investigated the interaction of complex I from bovine heart mitochondria with different types of detergents by monitoring activity, degree of inhibition and inhibitor binding in the presence of increasing concentrations of detergent. It is shown that apart from their nature as solubilizing and delipidating agents the polyoxyethylene-ether detergents Triton X-100, Brij-35 and Thesit act as specific inhibitors of complex I and compete with classical complex I inhibitors for a common binding domain. These findings were used to develop a novel large-scale chromatographic procedure for isolation of inhibitor-sensitive NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria. The enzyme was purified by selective solubilization in Triton X-100 and subsequent hydroxylapatite, ion-exchange and gel-exclusion chromatography. By switching detergents from Triton X-100 to dodecylmaltoside after hydroxylapatite chromatography the procedure yields highly pure, monodisperse and fully inhibitor-sensitive enzyme.

Purification of the 45 kDa, membrane bound NADH dehydrogenase of Escherichia coli (NDH-2) and analysis of its interaction with ubiquinone analogues.

The NADH:ubiquinone reductase (NDH-2) of Escherichia coli was expressed as a His-tagged protein, extracted from the membrane fraction using detergent and purified by chromatography. The His-tagged NDH-2 was highly active and catalyzed NADH oxidation by ubiquinone-1 at rates over two orders of magnitude higher than previously reported. The purified, His-tagged NDH-2, like native NDH-2, did not oxidize deamino-NADH. Steady-state kinetics were used to analyze the enzyme's activity in the presence of different electron acceptors. High V(max) and low K(m) values were only found for hydrophobic ubiquinone analogues, particularly ubiquinone-2. These findings strongly support the notion that NDH-2 is a membrane bound enzyme, despite the absence of predicted transmembrane segments in its primary structure. The latter observation is in agreement with possible evolutionary relation between NDH-2 and water-soluble enzymes such as dihydrolipoamide dehydrogenase. There is currently no clear indication of how NDH-2 binds to biological membranes.

Electron entry in a CuA mutant of cytochrome c oxidase from Paracoccus denitrificans. Conclusive evidence on the initial electron entry metal center.

A cytochrome c oxidase subunit II C216S mutant from Paracoccus denitrificans in which the CuA site was changed by site-directed mutagenesis to a mononuclear copper site [Zickermann, V., Wittershagen, A., Kolbesen, B.O. and Ludwig, B. Biochemistry 36 (1997) 3232-3236] was investigated by stopped-flow spectroscopy. Contrary to the behavior of the wild type enzyme, in this mutant cytochrome a cannot be reduced by excess cytochrome c in the millisecond time scale in which cytochrome c oxidation is observed. The results conclusively identify and establish CuA as the initial electron entry site in cytochrome c oxidase. Partial rapid reduction (ca. 20%) of the modified CuA site suggests that the mononuclear copper ion has a redox potential ca. 100 mV lower than the wild type, and that internal electron transfer to cytochrome a is > or = 10(3)-fold slower than with the wild type enzyme.

Cluster N1 of complex I from Yarrowia lipolytica studied by pulsed EPR spectroscopy.

After reduction with nicotinamide adenine dinucleotide (NADH), NADH:ubiquinone oxidoreductase (complex I) of the strictly aerobic yeast Yarrowia lipolytica shows clear signals from five different paramagnetic iron-sulfur (FeS) clusters (N1-N5) which can be detected using electron paramagnetic resonance (EPR) spectroscopy. The ligand environment and the assignment of several FeS clusters to specific binding motifs found in several subunits of the complex are still under debate. In order to characterize the hyperfine interaction of the surrounding nuclei with FeS cluster N1, one- and two-dimensional electron spin echo envelope modulation experiments were performed at a temperature of 30 K. At this temperature only cluster N1 contributes to the overall signal in a pulsed EPR experiment. The hyperfine and quadrupole tensors of a nitrogen nucleus and the isotropic and dipolar hyperfine couplings of two sets of protons could be determined by numerical simulation of the one- and two-dimensional spectra. The values obtained are in perfect agreement with a ferredoxin-like binding structure by four cysteine amino acid residues and allow the assignment of the nitrogen couplings to a backbone nitrogen nucleus and the proton couplings to the beta-protons of the bound cysteine residues.

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme.

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.

Structure-function relationships in mitochondrial complex I of the strictly aerobic yeast Yarrowia lipolytica.

The obligate aerobic yeast Yarrowia lipolytica has been established as a powerful model system for the analysis of mitochondrial complex I. Using a combination of genomic and proteomic approaches, a total of 37 subunits was identified. Several of the accessory subunits are predicted to be STMD (single transmembrane domain) proteins. Site-directed mutagenesis of Y. lipolytica complex I has provided strong evidence that a significant part of the ubiquinone reducing catalytic core resides in the 49 kDa and PSST subunits and can be modelled using X-ray structures of distantly related enzymes, i.e. water-soluble [NiFe] hydrogenases from Desulfovibrio spp. Iron-sulphur cluster N2, which is related to the hydrogenase proximal cluster, is directly involved in quinone reduction. Mutagenesis of His226 and Arg141 of the 49 kDa subunit provided detailed insight into the structure-function relationships around cluster N2. Overall, our findings suggest that proton pumping by complex I employs long-range conformational interactions and ubiquinone intermediates play a critical role in this mechanism.

Transformation of the CuA redox site in cytochrome c oxidase into a mononuclear copper center.

Subunit II of the aa3 type cytochrome c oxidase contains a binuclear copper center (CuA) which functions as the entry point for electrons donated by cytochrome c. We have introduced site-specific mutations in residues liganding the CuA center in the oxidase of the bacterium Paracoccus denitrificans; the purified, fully assembled enzyme complexes were analyzed by various techniques, including EPR, optical spectroscopy, and total-reflection X-ray fluorescence spectrometry, to determine metal to protein ratios. In the C216S mutant, the binuclear CuA site is transformed into a mononuclear copper center. In contrast to wild type, the C216S mutant does no longer exhibit the characteristic absorption band in the near-infrared region of the optical spectrum that has been assigned to CuA. These major changes in the CuA site of this mutant correlate with an almost complete loss in catalytic activity.

Analysis of the pathogenic human mitochondrial mutation ND1/3460, and mutations of strictly conserved residues in its vicinity, using the bacterium Paracoccus denitrificans.

The human mitochondrial ND1/3460 mutation changes Ala52 to Thr in the ND1 subunit of Complex I, and causes Leber's hereditary optic neuropathy (LHON) [Huoponen et al. (1991) Am. J. Hum. Genet. 48, 1147]. We have used a bacterial counterpart of Complex I, NDH-1 from Paracoccus denitrificans, for studying the effect of mutations in the ND1 subunit on the enzymatic activity. The LHON mutation as well as several other mutations in strictly conserved amino acids in its vicinity were introduced into the NQO8 subunit of NDH-1, a bacterial homologue of ND1. The enzymatic activity of the mutants in the presence of hexammineruthenium (rotenone-insensitive) and ubiquinone-1 (rotenone-sensitive) were assayed. In addition, the kinetics of the interaction of selected mutant enzymes with ubiquinone-1, ubiquinone-2, and decylubiquinone was studied. The results suggest that the mutated residues play an important role in ubiquinone reduction by Complex I.

Mutagenesis of three conserved Glu residues in a bacterial homologue of the ND1 subunit of complex I affects ubiquinone reduction kinetics but not inhibition by dicyclohexylcarbodiimide.

Steady-state kinetics of the H(+)-translocating NADH:ubiquinone reductase (complex I) were analyzed in membrane samples from bovine mitochondria and the soil bacterium Paracoccus denitrificans. In both enzymes the calculated K(m) values, in the membrane lipid phase, for four different ubiquinone analogues were in the millimolar range. Both the structure and size of the hydrophobic side chain of the acceptor affected its affinity for complex I. The ND1 subunit of bovine complex I is a mitochondrially encoded protein that binds the inhibitor dicyclohexylcarbodiimide (DCCD) covalently [Yagi and Hatefi (1988) J. Biol. Chem. 263, 16150-16155]. The NQO8 subunit of P. denitrificans complex I is a homologue of ND1, and within it three conserved Glu residues that could bind DCCD, E158, E212, and E247, were changed to either Asp or Gln and in the case of E212 also to Val. The DCCD sensitivity of the resulting mutants was, however, unaffected by the mutations. On the other hand, the ubiquinone reductase activity of the mutants was altered, and the mutations changed the interactions of complex I with short-chain ubiquinones. The implications of the results for the location of the ubiquinone reduction site in this enzyme are discussed.

Exploring the catalytic core of complex I by Yarrowia lipolytica yeast genetics.

We have developed Yarrowia lipolytica as a model system to study mitochondrial complex I that combines the application of fast and convenient yeast genetics with efficient structural and functional analysis of its very stable complex I isolated by his-tag affinity purification with high yield. Guided by a structural model based on homologies between complex I and [NiFe] hydrogenases mutational analysis revealed that the 49 kDa subunit plays a central functional role in complex I. We propose that critical parts of the catalytic core of complex I have evolved from the hydrogen reactive site of [NiFe] hydrogenases and that iron-sulfur cluster N2 resides at the interface between the 49 kDa and PSST subunits. These findings are in full agreement with the "semiquinone switch" mechanism according to which coupling of electron and proton transfer in complex I is achieved by a single integrated pump comprising cluster N2, the binding site for substrate ubiquinone, and a tightly bound quinone or quinoid group.

Perturbation of the CuA site in cytochrome-c oxidase of Paracoccus denitrificans by replacement of Met227 with isoleucine.

Subunit II of cytochrome-c oxidase contains a redox centre, CuA, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site-directed mutant of cytochrome-c oxidase from Paracoccus denitrificans in which the CuA site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady-state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the CuA site can be observed in the EPR and near-infrared optical spectra. It is concluded that in the mutant CuA is still binuclear but that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5)....Cu(1.5)] centre of the wild type into a localized [Cu(I)....Cu(II)] system. Changes in the overall kinetics of the mutant are correlated with a diminished electron transfer rate between CuA and heme alpha.