Hanns Weiss

The proton-pumping respiratory complex I of bacteria and mitochondria and its homologue in chloroplasts

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 the synthesis of ATP. Closely related forms of this complex exist in the mitochondria of eucaryotes and in the plasma membranes of purple bacteria. The minimal structural framework common to the mitochondrial and the bacterial complex is composed of 14 polypeptides with 1 FMN and 6–8 iron‐sulfur clusters as prosthetic groups. The mitochondrial complex contains many accessory subunits for which no homologous counterparts exist in the bacterial complex. Genes for 11 of the 14 minimal subunits are also found in the plastidial DNA of plants and in the genome of cyanobacteria. However, genes encoding the 3 subunits of the NADH dehydrogenase part of complex I are apparently missing in these species. The possibility is discussed that chloroplasts and cyanobacteria contain a complex I equipped with a different electron input device. This complex may work as a NAD(P)H: or a ferrodoxin:plastoquinone oxidoreductase participating in cyclic electron transport during photosynthesis.

Three-dimensional structure of ubiquinol: Cytochrome C reductase from Neurospora mitochondria determined by electron microscopy of membrane crystals

Abstract Membrane crystals have been prepared from mitochondrial ubiquinol: cytochrome c reductase by mixing the enzyme-Triton complex with phospholipid-Triton micelles and subsequently removing the Triton. The electron micrographs of the negatively stained crystals diffract to 2·5 nm, with unit cell dimensions of 13·7 nm by 17·4 nm. The enzyme is arranged in a two-sided plane group P 22 1 2 1 , i.e. alternate molecules span the bilayer in an up and down manner. By combining tilted views of the membrane crystals, a low-resolution three-dimensional structure of the enzyme has been calculated. The structure shows that the enzyme is a dimer, the monomers being related by a 2-fold axis running perpendicular to the membrane. The monomeric units of the enzyme are elongated, extending approximately 15 nm across the membrane. The protein is unequally distributed with about 30% of the total mass located in the bilayer, 50% in a section which extends 7 nm from one side of the bilayer and 20% in a section which extends 3 nm from the opposite side of the bilayer. The two monomeric units are in contact only in the membraneous section. This structure is compared with a model of the enzyme which is derived from biochemical properties of the isolated subunits.

Assembly of NADH: Ubiquinone reductase (complex I) in Neurospora mitochondria: Independent pathways of nuclear-encoded and mitochondrially encoded subunits

NADH:ubiquinone reductase, the respiratory chain complex I of mitochondria, consists of some 25 nuclear-encoded and seven mitochondrially encoded subunits, and contains as redox groups one FMN, probably one internal ubiquinone and at least four iron-sulphur clusters. We are studying the assembly of the enzyme in Neurospora crassa. The flux of radioactivity in cells that were pulse-labelled with [35S]methionine was followed through immunoprecipitable assembly intermediates into the holoenzyme. Labelled polypeptides were observed to accumulate transiently in a Mr 350,000 intermediate complex. This complex contains all mitochondrially encoded subunits of the enzyme as well as subunits encoded in the nucleus that have no homologous counterparts in a small, merely nuclear-encoded form of the NADH:ubiquinone reductase made by Neurospora crassa cells poisoned with chloramphenicol. With regard to their subunit compositions, the assembly intermediate and small NADH:ubiquinone reductase complement each other almost perfectly to give the subunit composition of the large complex I. These results suggest that two pathways exist in the assembly of complex I that independently lead to the preassembly of two major parts, which subsequently join to form the complex. One preassembled part is related to the small form of NADH:ubiquinone reductase and contributes most of the nuclear-encoded subunits, FMN, three iron-sulphur clusters and the site for the internal ubiquinone. The other part is the assembly intermediate and contributes all mitochondrially encoded subunits, one iron-sulphur cluster and the catalytic site for the substrate ubiquinone. We discuss the results with regard to the evolution of the electron pathway through complex I.

Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein.

The nuclear genes (acp-1, ACP 1) encoding the mitochondrial acyl carrier protein were disrupted in Neurospora crassa and Saccharomyces cerevisiae. In N. crassa acp-1 is a peripheral subunit of the respiratory NADH: ubiquinone oxidoreductase (complex I). S. cerevisiae lacks complex I and its ACP1 appears to be located in the mitochondrial matrix. The loss of acp-1 in N. crassa causes two biochemical lesions. Firstly, the peripheral part of complex I is not assembled, and the membrane part is not properly assembled. The respiratory ubiquinol: cytochrome c oxidose (complex IV) are made in normal amounts. Secondly, the lysophospholipid content of mitochondrial membranes is increased four-fold. In S. cerevisiae, the loss of aCP1 leads to a pleiotropic respiratory deficient phenotype.

Low-resolution structural studies of mitochondrial ubiquinol: Cytochrome c reductase in detergent solutions by neutron scattering

Mitochondrial ubiquinol:cytochrome c reductase (Mr approximately 600,000) was cleaved into a complex (Mr approximately 280,000) of the subunits III (cytochrome b), IV (cytochrome c1) and VI to IX, a complex (Mr approximately 300,000) of the subunits I and II, and the single subunit V (iron-sulphur subunit, Mr approximately 25,000). Neutron scattering was applied to the whole enzyme, the cytochrome bc1 complex, both in hydrogenated and deuterated alkyl (phenyl) polyoxyethylene detergents, and the complex of subunits I and II in detergent-free solution. The neutron parameters were compared with the structures of the enzyme and the cytochrome bc1 complex previously determined by electron microscopy. Using the method of hard spheres, comparison of the calculated and experimental radius of gyration implies that the length of the enzyme across the bilayer or the detergent micelle is between 150 and 175 A and of the cytochrome bc1 complex between 90 and 115 A. The subunit topography was confirmed. The cleavage plane between the cytochrome bc1 complex and the complex of subunits I and II lies at the centre of the enzyme and runs parallel to the membrane just outside the bilayer. The detergent uniformly surrounds the protein as a belt, which is displaced by 30 to 40 A from the protein centre of the enzyme and by about 20 A from the protein centre of the cytochrome bc1 complex. The low protein matchpoint of the whole enzyme as compared to the subunit complexes is accounted for in terms of the non-exchange of about 30 to 60% of the exchangeable protons within the intact enzyme. Polar residues are, on average, at the protein surface and non-polar residues and polar residues with non-exchanged protons are buried within the enzyme.

Three-dimensional structure of NADH: Ubiquinone reductase (complex I) from Neurospora mitochondria determined by electron microscopy of membrane crystals☆

NADH: ubiquinone reductase (electron transfer complex I) has been isolated from Neurospora crassa mitochondria as a monodisperse protein-phospholipid-Triton X-100 complex (1:0.04:0.15, by weight). The enzyme is in the monomeric state, has a protein molecular weight of 610,000 and consists of about 25 different subunits. Membrane crystals of the enzyme complex have been prepared by adding mixed phospholipid-Triton X-100 micelles and then removing the Triton by dialysis. Diffraction patterns of the negatively stained membrane crystals extend to about 3.9 nm, with a unit cell size of 19 nm X 38 nm and gamma = 90 degrees. The two-sided plane group packing corresponding to pgg is p22(1)2(1). By combining four sets of tilted views, a low-resolution three-dimensional structure of the protein has been calculated. The structure shows that NADH: ubiquinone reductase extends 15 nm across the membrane, projecting 9 nm from one membrane side and 1 nm from the opposite side. Only about one-third of the total protein mass is located in the membrane. The structure of NADH: ubiquinone reductase is compared with that of ubiquinol: cytochrome c reductase determined by electron microscopy of membrane crystals.

Characterization of assembly intermediates of NADH : ubiquinone oxidoreductase (complex I) accumulated in Neurospora mitochondria by gene disruption

NADH:ubiquinone oxidoreductase, the respiratory chain complex I of mitochondria, is an assembly of some 25 nuclear-encoded and 7 mitochondrially encoded subunits. The complex has an overall L-shaped structure formed by a peripheral arm and an elongated membrane arm. The peripheral arm containing one FMN and at least three iron-sulphur clusters constitutes the NADH dehydrogenase segment of the electron pathway. The membrane arm with at least one iron-sulphur cluster constitutes the ubiquinone reducing segment. We are studying the assembly of the complex in Neurospora crassa. By disrupting the gene of a nuclear-encoded subunit of the membrane arm a mutant was generated that cannot form complex I. The mutant rather pre-assembles the peripheral arm with all redox groups and the ability to catalyse NADH oxidation by artificial electron acceptors. The final assembly of the membrane arm is blocked in the mutant leading to accumulation of complementary assembly intermediates. One intermediate is associated with a protein that is not present in the fully assembled complex I. The results demonstrate that the two arms of complex I are assembled independently on separate pathways, and gave a first insight into the assembly pathway of the membrane arm. It is also shown for the first time that the obligate aerobic fungus N. crassa can grow and respire without an intact complex I. Gene replacement in this fungus is therefore a tool for investigation of this complex.

Two genes of the putative mitochondrial fatty acid synthase in the genome of Saccharomyces cerevisiae.

Abstract In order to find further genes of the mitochondrial fatty acid synthase, we searched the genome of Saccharomyces cerevisiae for sequences that are homologous to conserved regions of bacterial fatty acid synthase genes. We found the gene products of ORF YKL055c (EMBL Accession No. X75781) and of YOR221C (EMBL Accession No. X92441) to be homologous to bacterial 3-oxoacyl-(acyl carrier protein) reductases and to malonyl-CoA:ACP-transferases, respectively. We disrupted these two genes which in both cases led to a respiratory deficient phenotype, as is the case for the genes encoding a mitochondrial acyl carrier protein and a β-ketoacyl-ACP synthase. We propose to call the above mentioned genes OAR1[3-oxo-acyl-(acyl carrier protein) reductase] and MCT1 (malonyl-CoA:ACP transferase). They are presumed to be part of a type-II mitochondrial fatty acid synthase, a relic of the endosymbiontic origin of mitochondria, delivering substrates for phospholipid re-modelling and/or repair.

Bacterial NADH-quinone oxidoreductases: Iron-sulfur clusters and related problems

Many bacteria contain proton-translocating membrane-bound NADH-quinone oxidoreductases (NDH-1), which demonstrate significant genetic, spectral, and kinetic similarity with their mitochondrial counterparts. This review is devoted to the comparative aspects of the ironsulfur cluster composition of NDH-1 from the most well-studied bacterial systems to date.:Paracoccus denitrificans, Rhodobacter sphaeroides, Escherichia coli, andThermus thermophilus. These bacterial systems provide useful models for the study of coupling Site I and contain all the essential parts of the electron-transfer and proton-translocating machinery of their eukaryotic counterparts.

The same domain motif for ubiquinone reduction in mitochondrial or chloroplast NADH dehydrogenase and bacterial glucose dehydrogenase

The respiratory chain NADH:ubiquinone oxidoreductase (NADH dehydrogenase or Complex I) of mitochondria comprises some 30 different subunits, and one FMN and 4 or 5 iron‐sulfur clusters as internal redox groups. The bacterial glucose dehydrogenase, which oxidizes glucose to gluconolactone in the periplasmatic space and transfers the electrons to ubiquinone, is a single polypeptide chain with pyrolloquinoline quinone as the only redox group. We report here that the two different enzymes have the same ubiquinone binding domain motif and we discuss the predicted membrane folding of this domain with regard to its role in the proton translocating function of the two enzymes.