Respiratory complex I with charge symmetry in the membrane arm pumps protons.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Characterisation of the redox centers of ethylbenzene dehydrogenase.

Ethylbenzene dehydrogenase (EbDH), the initial enzyme of anaerobic ethylbenzene degradation from the beta-proteobacterium Aromatoleum aromaticum, is a soluble periplasmic molybdenum enzyme consisting of three subunits. It contains a Mo-bis-molybdopterin guanine dinucleotide (Mo-bis-MGD) cofactor and an 4Fe-4S cluster (FS0) in the α-subunit, three 4Fe-4S clusters (FS1 to FS3) and a 3Fe-4S cluster (FS4) in the ß-subunit and a heme b cofactor in the γ-subunit. Ethylbenzene is hydroxylated by a water molecule in an oxygen-independent manner at the Mo-bis-MGD cofactor, which is reduced from the MoVI to the MoIV state in two subsequent one-electron steps. The electrons are then transferred via the Fe-S clusters to the heme b cofactor. In this report, we determine the midpoint redox potentials of the Mo-bis-MGD cofactor and FS1-FS4 by EPR spectroscopy, and that of the heme b cofactor by electrochemically induced redox difference spectroscopy. We obtained relatively high values of > 250 mV both for the MoVI-MoV redox couple and the heme b cofactor, whereas FS2 is only reduced at a very low redox potential, causing magnetic coupling with the neighboring FS1 and FS3. We compare the results with the data on related enzymes and interpret their significance for the function of EbDH.

The cbb3-type cytochrome oxidase assembly factor CcoG is a widely distributed cupric reductase.

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb3-type cytochrome c oxidase (cbb3-Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb3-Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.

Recent Advances in Structural Studies of Cytochrome bd and Its Potential Application as a Drug Target.

Cytochrome bd is a triheme copper-free terminal oxidase in membrane respiratory chains of prokaryotes. This unique molecular machine couples electron transfer from quinol to O2 with the generation of a proton motive force without proton pumping. Apart from energy conservation, the bd enzyme plays an additional key role in the microbial cell, being involved in the response to different environmental stressors. Cytochrome bd promotes virulence in a number of pathogenic species that makes it a suitable molecular drug target candidate. This review focuses on recent advances in understanding the structure of cytochrome bd and the development of its selective inhibitors.

A Cobalamin-Dependent Radical SAM Enzyme Catalyzes the Unique Cα -Methylation of Glutamine in Methyl-Coenzyme†M Reductase.

Methyl-coenzyme M reductase, which is responsible for the production of the greenhouse gas methane during biological methane formation, carries several unique posttranslational amino acid modifications, including a 2-(S)-methylglutamine. The enzyme responsible for the Cα -methylation of this glutamine is not known. Herein, we identify and characterize a cobalamin-dependent radical SAM enzyme as the glutamine C-methyltransferase. The recombinant protein from Methanoculleus thermophilus binds cobalamin in a base-off, His-off conformation and contains a single [4Fe-4S] cluster. The cobalamin cofactor cycles between the methyl-cob(III)alamin, cob(II)alamin and cob(I)alamin states during catalysis and produces methylated substrate, 5'-deoxyadenosine and S-adenosyl-l-homocysteine in a 1 : 1 : 1 ratio. The newly identified glutamine C-methyltransferase belongs to the class B radical SAM methyltransferases known to catalyze challenging methylation reactions of sp3 -hybridized carbon atoms.

Structural Basis for Inhibition of ROS-Producing Respiratory Complex I by NADH-OH.

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a central role in cellular energy metabolism. As a major source of reactive oxygen species (ROS) it affects ageing and mitochondrial dysfunction. The novel inhibitor NADH-OH specifically blocks NADH oxidation and ROS production by complex I in nanomolar concentrations. Attempts to elucidate its structure by NMR spectroscopy have failed. Here, by using X-ray crystallographic analysis, we report the structure of NADH-OH bound in the active site of a soluble fragment of complex I at 2.0 Šresolution. We have identified key amino acid residues that are specific and essential for binding NADH-OH. Furthermore, the structure sheds light on the specificity of NADH-OH towards the unique Rossmann-fold of complex I and indicates a regulatory role in mitochondrial ROS generation. In addition, NADH-OH acts as a lead-structure for the synthesis of a novel class of ROS suppressors.

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton-electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.

Photolysis of Caged Inositol Pyrophosphate InsP8 Directly Modulates Intracellular Ca2+ Oscillations and Controls C2AB Domain Localization.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living ß-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts ß-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH:ubiquinone oxidoreductase (complex I).

The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.

CyaC, a redox-regulated adenylate cyclase of Sinorhizobium meliloti with a quinone responsive diheme-B membrane anchor domain.

The nucleotide cyclase CyaC of Sinorhizobium meliloti is a member of class III adenylate cyclases (AC), a diverse group present in all forms of life. CyaC is membrane-integral by a hexahelical membrane domain (6TM) with the basic topology of mammalian ACs. The 6TM domain of CyaC contains a tetra-histidine signature that is universally present in the membrane anchors of bacterial diheme-B succinate-quinone oxidoreductases. Heterologous expression of cyaC imparted activity for cAMP formation from ATP to Escherichia coli, whereas guanylate cyclase activity was not detectable. Detergent solubilized and purified CyaC was a diheme-B protein and carried a binuclear iron-sulfur cluster. Single point mutations in the signature histidine residues caused loss of heme-B in the membrane and loss of AC activity. Heme-B of purified CyaC could be oxidized or reduced by ubiquinone analogs (Q0 or Q0 H2 ). The activity of CyaC in bacterial membranes responded to oxidation or reduction by Q0 and O2 , or NADH and Q0 H2 respectively. We conclude that CyaC-like membrane anchors of bacterial ACs can serve as the input site for chemical stimuli which are translated by the AC into an intracellular second messenger response.

Stabilization of the Highly Hydrophobic Membrane Protein, Cytochrome bd Oxidase, on Metallic Surfaces for Direct Electrochemical Studies.

The cytochrome bd oxidase catalyzes the reduction of oxygen to water in bacteria and it is thus an interesting target for electrocatalytic studies and biosensor applications. The bd oxidase is completely embedded in the phospholipid membrane. In this study, the variation of the surface charge of thiol-modified gold nanoparticles, the length of the thiols and the other crucial parameters including optimal phospholipid content and type, have been performed, giving insight into the role of these factors for the optimal interaction and direct electron transfer of an integral membrane protein. Importantly, all three tested factors, the lipid type, the electrode surface charge and the thiol length mutually influenced the stability of films of the cytochrome bd oxidase. The best electrocatalytic responses were obtained on the neutral gold surface when the negatively charged phosphatidylglycerol (PG) was used and on the charged gold surface when the zwitterionic phosphatidylethanolamine (PE) was used. The advantages of the covalent binding of the membrane protein to the electrode surface over the non-covalent binding are also discussed.

Charge transfer through a fragment of the respiratory complex I and its regulation: an atomistic simulation approach.

We simulate electron transfer within a fragment of the NADH:ubiquinone oxidoreductase (respiratory complex I) of the hyperthermophilic bacterium Aquifex aeolicus. We apply molecular dynamics simulations, thermodynamic integration, and a thermodynamic network least squares analysis to compute two key parameters of Marcus' theory of charge transfer, the thermodynamic driving force and the reorganization energy. Intramolecular contributions to the Gibbs free energy differences of electron and hydrogen transfer processes, ΔG, are accessed by calibrating against experimental redox titration data. This approach permits the computation of the interactions between the species NAD+, FMNH2, N1a-, and N3-, and the construction of a free energy surface for the flow of electrons within the fragment. We find NAD+ to be a strong candidate for the regulation of charge transfer.

An oxygenase that forms and deoxygenates toxic epoxide.

Catabolism may give rise to toxic intermediates that compromise cell vitality, such as epoxide formation in the recently elucidated and apparently universal bacterial coenzyme A (CoA)-dependent degradation of phenylacetic acid. This compound is central to the catabolism of a variety of aromatics, such as phenylalanine, lignin-related compounds or environmental contaminants. The key phenylacetyl-CoA monooxygenase (epoxidase) of the pathway, PaaABCE, is also connected to the production of various primary and secondary metabolites, as well as to the virulence of certain pathogens. However, the enzyme complex has so far not been investigated in detail. Here we characterize the bacterial multicomponent monooxygenase PaaABCE that, surprisingly, not only transforms phenylacetyl-CoA into its ring-1,2-epoxide, but also mediates the NADPH-dependent removal of the epoxide oxygen, regenerating phenylacetyl-CoA with formation of water. We provide evidence for a catalytic di-iron centre that is probably the key to the unprecedented deoxygenation of an organic compound by an oxygenase. Presumably, the bifunctionality is vital to avoid toxic intracellular epoxide levels if the subsequent catabolic steps are impeded. Our data suggest that detoxification is assisted by two thioesterases (PaaI and PaaY) forming non-reactive breakdown products. Hence, PaaABCE may harbour an intrinsic escape mechanism from its own toxic product and represents the archetype of a bifunctional oxygenase/deoxygenase. Analogous reactions may possibly be catalysed by other di-iron epoxidases.

Significance of [2Fe-2S] Cluster N1a for Electron Transfer and Assembly of Escherichia coli Respiratory Complex I.

NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with proton translocation across the membrane. NADH reduces a noncovalently bound FMN, and the electrons are transported further to the quinone reduction site by a 95 Å long chain of seven iron-sulfur (Fe-S) clusters. Binuclear Fe-S cluster N1a is not part of this long chain but is located within electron transfer distance on the opposite site of FMN. The relevance of N1a to the mechanism of complex I is not known. To elucidate its role, we individually substituted the cysteine residues coordinating N1a of Escherichia coli complex I by alanine and serine residues. The mutations led to a significant loss of the NADH oxidase activity of the mutant membranes, while the amount of the complex was only slightly diminished. N1a could not be detected by electron paramagnetic resonance spectroscopy, and unexpectedly, the content of binuclear cluster N1b located on a neighboring subunit was significantly decreased. Because of the lack of N1a and the partial loss of N1b, the variants did not survive detergent extraction from the mutant membranes. Only the C97AE variant retained N1a and was purified by chromatographic steps. The preparation showed a slightly diminished NADH/ferricyanide oxidoreductase activity, while the NADH:decyl-ubiquinone oxidoreductase activity was not affected. N1a of this preparation showed unusual spectroscopic properties indicating a different ligation. We discuss whether N1a is involved in the physiological electron transfer reaction.

Assembly of the Escherichia coli NADH:ubiquinone oxidoreductase (respiratory complex I).

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone with the translocation of four protons across the membrane. The Escherichia coli complex I is made up of 13 different subunits encoded by the so-called nuo-genes. The electron transfer is catalyzed by nine cofactors, a flavin mononucleotide and eight iron-sulfur (Fe/S)-clusters. The individual subunits and the cofactors have to be assembled together in a coordinated way to guarantee the biogenesis of the active holoenzyme. Only little is known about the assembly of the bacterial complex compared to the mitochondrial one. Due to the presence of so many Fe/S-clusters the assembly of complex I is intimately connected with the systems responsible for the biogenesis of these clusters. In addition, a few other proteins have been reported to be required for an effective assembly of the complex in other bacteria. The proposed role of known bacterial assembly factors is discussed and the information from other bacterial species is used in this review to draw an as complete as possible model of bacterial complex I assembly. In addition, the supramolecular organization of the complex in E. coli is briefly described. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof. Conrad Mullineaux.

Infrared spectroscopic studies on reaction induced conformational changes in the NADH ubiquinone oxidoreductase (complex I).

Redox-dependent conformational changes are currently discussed to be a crucial part of the reaction mechanism of the respiratory complex I. Specialized difference Fourier transform infrared techniques allow the detection of side-chain movements and minute secondary structure changes. For complex I, (1)H/(2)H exchange kinetics of the amide modes revealed a better accessibility of the backbone in the presence of NADH and quinone. Interestingly, the presence of phospholipids, that is crucial for the catalytic activity of the isolated enzyme complex, changes the overall conformation. When comparing complex I samples from different species, very similar electrochemically induced FTIR difference spectra and very similar rearrangements are reported. Finally, the information obtained with variants and from Zn(2+) inhibited samples for the conformational reorganization of complex I upon electron transfer are discussed in this review. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

The multitude of iron-sulfur clusters in respiratory complex I.

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. Complex I contains one non-covalently bound flavin mononucleotide and, depending on the species, up to ten iron-sulfur (Fe/S) clusters as cofactors. The reason for the presence of the multitude of Fe/S clusters in complex I remained enigmatic for a long time. The question was partly answered by investigations on the evolution of the complex revealing the stepwise construction of the electron transfer domain from several modules. Extension of the ancestral to the modern electron input domain was associated with the acquisition of several Fe/S-proteins. The X-ray structure of the complex showed that the NADH oxidation-site is connected with the quinone-reduction site by a chain of seven Fe/S-clusters. Fast enzyme kinetics revealed that this chain of Fe/S-clusters is used to regulate electron-tunneling rates within the complex. A possible function of the off-pathway cluster N1a is discussed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Cysteine scanning reveals minor local rearrangements of the horizontal helix of respiratory complex I.

The NADH:ubiquinone oxidoreductase, respiratory complex I, couples electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The complex consists of a peripheral arm catalyzing the redox reaction and a membrane arm catalyzing proton translocation. The membrane arm is almost completely aligned by a 110 Å unique horizontal helix that is discussed to transmit conformational changes induced by the redox reaction in a piston-like movement to the membrane arm driving proton translocation. Here, we analyzed such a proposed movement by cysteine-scanning of the helix of the Escherichia coli complex I. The accessibility of engineered cysteine residues and the flexibility of individual positions were determined by labeling the preparations with a fluorescent marker and a spin-probe, respectively, in the oxidized and reduced states. The differences in fluorescence labeling and the rotational flexibility of the spin probe between both redox states indicate only slight conformational changes at distinct positions of the helix but not a large movement.

Reactive Oxygen Species Production by Escherichia coli Respiratory Complex I.

Respiratory complex I couples the electron transfer exclusively from NADH to a quinone with the translocation of protons across the membrane. However, Escherichia coli adapts to imposed high cellular NADPH concentrations by selecting the mutations E183A(F) and E183G(F) that lead to a high catalytic efficiency of complex I with NADPH. Other mutations at position E183(F) resulting in an efficient NADPH oxidation were not selected. Here we show that the naturally occurring variants exhibit a remarkably low level of production of reactive oxygen species, a byproduct of NAD(P)H oxidation, that besides high catalytic efficiency might be favored by natural selection.

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation.

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.