Thorsten Friedrich

The respiratory complex I of bacteria, archaea and eukarya and its module common with membrane-bound multisubunit hydrogenases.

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 F420H2 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.

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.

An anaerobic mitochondrion that produces hydrogen.

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 NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli

-lactate dehydrogenase, which oxidize organic sub-strates and deliver the electrons to membrane boundubiquinone-8. The ubiquinol is then reoxidized byreductases, and the terminal oxidases. This exergonicprocess is coupled with the endergonic translocationof protons across the cytoplasmic membrane. Theresulting membrane potential is used to driveenergy-consuming processes like ATP synthesis, so-lute transport and flagellar motion 1,2 .wxThe number of NADH dehydrogenases and theirrole in energy transduction had been under debate fora long time. Today, it is widely accepted that E. colipossesses two different NADH dehydrogenases called

Complex I: a chimaera of a redox and conformation-driven proton pump?

From phylogenetic sequence analysis, it can be concluded that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) has evolved from preexisting modules for electron transfer and proton translocation. It is built up by a peripheral NADH dehydrogenase module, an amphipatic hydrogenase module, and a membrane-bound transporter module. These modules, or at least part of them, are also present in various other bacterial enzymes. It is assumed that they fulfill a similar function in complex I and related enzymes. Based on the function of the individual modules, it is possible to speculate about the mechanism of complex I. The hydrogenase module might work as a redox-driven proton pump, while the transporter module might act as a conformation-driven proton pump. This implies that complex I contains two energy-coupling sites. The NADH dehydrogenase module seems to be involved in electron transfer and not in proton translocation.

Identification and characterization of the tungsten-containing class of benzoyl-coenzyme A reductases

Aromatic compounds are widely distributed in nature and can only be biomineralized by microorganisms. In anaerobic bacteria, benzoyl-CoA (BCoA) is a central intermediate of aromatic degradation, and serves as substrate for dearomatizing BCoA reductases (BCRs). In facultative anaerobes, the mechanistically difficult reduction of BCoA to cyclohexa-1,5-dienoyl-1-carboxyl-CoA (dienoyl-CoA) is driven by a stoichiometric ATP hydrolysis, catalyzed by a soluble, three [4Fe-4S] cluster-containing BCR. In this work, an in vitro assay for BCR from the obligately anaerobic Geobacter metallireducens was established. It followed the reverse reaction, the formation of BCoA from dienoyl-CoA in the presence of various electron acceptors. The benzoate-induced activity was highly specific for dienoyl-CoA (Km = 24 ± 4 μM). The corresponding oxygen-sensitive enzyme was purified by several chromatographic steps with a 115-fold enrichment and a yield of 18%. The 185-kDa enzyme comprised 73- and 20-kDa subunits, suggesting an α2β2-composition. MS analysis revealed the subunits as products of the benzoate-induced bamBC genes. The αβ unit contained 0.9 W, 15 Fe, and 12.5 acid-labile sulfur. Results from EPR spectroscopy suggest the presence of one [3Fe-4S]0/+1 and three [4Fe-4S]+1/+2 clusters per αβ unit; oxidized BamBC exhibited an EPR signal typical for a W(V) species. The FeS clusters and the W- cofactor could only be fully reduced by dienoyl-CoA. BamBC represents the prototype of a previously undescribed class of dearomatizing BCRs that differ completely from the ATP-dependent enzymes from facultative anaerobes.

Menaquinone as pool quinone in a purple bacterium

Purple bacteria have thus far been considered to operate light-driven cyclic electron transfer chains containing ubiquinone (UQ) as liposoluble electron and proton carrier. We show that in the purple γ-proteobacterium Halorhodospira halophila, menaquinone-8 (MK-8) is the dominant quinone component and that it operates in the QB-site of the photosynthetic reaction center (RC). The redox potentials of the photooxidized pigment in the RC and of the Rieske center of the bc1 complex are significantly lower (Em = +270 mV and +110 mV, respectively) than those determined in other purple bacteria but resemble those determined for species containing MK as pool quinone. These results demonstrate that the photosynthetic cycle in H. halophila is based on MK and not on UQ. This finding together with the unusual organization of genes coding for the bc1 complex in H. halophila suggests a specific scenario for the evolutionary transition of bioenergetic chains from the low-potential menaquinones to higher-potential UQ in the proteobacterial phylum, most probably induced by rising levels of dioxygen 2.5 billion years ago. This transition appears to necessarily proceed through bioenergetic ambivalence of the respective organisms, that is, to work both on MK- and on UQ-pools. The establishment of the corresponding low- and high-potential chains was accompanied by duplication and redox optimization of the bc1 complex or at least of its crucial subunit oxidizing quinols from the pool, the Rieske protein. Evolutionary driving forces rationalizing the empirically observed redox tuning of the chain to the quinone pool are discussed.

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.

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.