Mechanisms of Energy Transduction by Charge Translocating Membrane Proteins.

Life relies on the constant exchange of different forms of energy, i.e., on energy transduction. Therefore, organisms have evolved in a way to be able to harvest the energy made available by external sources (such as light or chemical compounds) and convert these into biological useable energy forms, such as the transmembrane difference of electrochemical potential (ΔµÌƒ). Membrane proteins contribute to the establishment of ΔµÌƒ by coupling exergonic catalytic reactions to the translocation of charges (electrons/ions) across the membrane. Irrespectively of the energy source and consequent type of reaction, all charge-translocating proteins follow two molecular coupling mechanisms: direct- or indirect-coupling, depending on whether the translocated charge is involved in the driving reaction. In this review, we explore these two coupling mechanisms by thoroughly examining the different types of charge-translocating membrane proteins. For each protein, we analyze the respective reaction thermodynamics, electron transfer/catalytic processes, charge-translocating pathways, and ion/substrate stoichiometries.

In Silico Discovery of a Substituted 6-Methoxy-quinalidine with Leishmanicidal Activity in Leishmania infantum.

There is an urgent need for the discovery of new antileishmanial drugs with a new mechanism of action. Type 2 NADH dehydrogenase from Leishmania infantum (LiNDH2) is an enzyme of the parasite's respiratory system, which catalyzes the electron transfer from NADH to ubiquinone without coupled proton pumping. In previous studies of the related NADH: ubiquinone oxidoreductase crystal structure from Saccharomyces cerevisiae, two ubiquinone-binding sites (UQI and UQII) were identified and shown to play an important role in the NDH-2-catalyzed oxidoreduction reaction. Based on the available structural data, we developed a three-dimensional structural model of LiNDH2 using homology detection methods and performed an in silico virtual screening campaign to search for potential inhibitors targeting the LiNDH2 ubiquinone-binding site 1-UQI. Selected compounds displaying favorable properties in the computational screening experiments were assayed for inhibitory activity in the structurally similar recombinant NDH-2 from S. aureus and leishmanicidal activity was determined in the wild-type axenic amastigotes and promastigotes of L. infantum. The identified compound, a substituted 6-methoxy-quinalidine, showed promising nanomolar leishmanicidal activity on wild-type axenic promastigotes and amastigotes of L. infantum and the potential for further development.

The key role of glutamate 172 in the mechanism of type II NADH:quinone oxidoreductase of Staphylococcus aureus.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane bound enzymes that deliver electrons to the respiratory chain by oxidation of NADH and reduction of quinones. In this way, these enzymes also contribute to the regeneration of NAD+, allowing several metabolic pathways to proceed. As for the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, the enzymatic mechanism of NDH-2s is still little explored and elusive. In this work we addressed the role of the conserved glutamate 172 (E172) residue in the enzymatic mechanism of NDH-2 from Staphylococcus aureus. We aimed to test our earlier hypothesis that E172 plays a key role in proton transfer to allow the protonation of the quinone. For this we performed a complete biochemical characterization of the enzyme's variants E172A, E172Q and E172S. Our steady state kinetic measurements show a clear decrease in the overall reaction rate, and our substrate interaction studies indicate the binding of the two substrates is also affected by these mutations. Interestingly our fast kinetic results show quinone reduction is more affected than NADH oxidation. We have also determined the X-ray crystal structure of the E172S mutant (2.55Ǻ) and compared it with the structure of the wild type (2.32Ǻ). Together these results support our hypothesis for E172 being of central importance in the catalytic mechanism of NDH-2, which may be extended to other members of the tDBDF superfamily.

Exploring membrane respiratory chains.

Acquisition of energy is central to life. In addition to the synthesis of ATP, organisms need energy for the establishment and maintenance of a transmembrane difference in electrochemical potential, in order to import and export metabolites or to their motility. The membrane potential is established by a variety of membrane bound respiratory complexes. In this work we explored the diversity of membrane respiratory chains and the presence of the different enzyme complexes in the several phyla of life. We performed taxonomic profiles of the several membrane bound respiratory proteins and complexes evaluating the presence of their respective coding genes in all species deposited in KEGG database. We evaluated 26 quinone reductases, 5 quinol:electron carriers oxidoreductases and 18 terminal electron acceptor reductases. We further included in the analyses enzymes performing redox or decarboxylation driven ion translocation, ATP synthase and transhydrogenase and we also investigated the electron carriers that perform functional connection between the membrane complexes, quinones or soluble proteins. Our results bring a novel, broad and integrated perspective of membrane bound respiratory complexes and thus of the several energetic metabolisms of living systems. 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.

Substrate-Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and responsible for the maintenance of NADH/NAD(+) balance in cells. NDH-2s are the only enzymes with NADH dehydrogenase activity present in the respiratory chain of many pathogens, and thus, they were proposed as suitable targets for antimicrobial therapies. In addition, NDH-2s were also considered key players for the treatment of complex I-related neurodegenerative disorders. In this work, we explored substrate-protein interaction in NDH-2 from Escherichia coli (EcNDH-2) combining surface-enhanced infrared absorption spectroscopic studies with electrochemical experiments, fluorescence spectroscopy assays, and quantum chemical calculations. Because of the specific stabilization of substrate complexes of EcNDH-2 immobilized on electrodes, it was possible to demonstrate the presence of two distinct substrate binding sites for NADH and the quinone and to identify a bound semiprotonated quinol as a catalytic intermediate.

Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction.

A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.

Type II NADH:quinone oxidoreductase family: phylogenetic distribution, structural diversity and evolutionary divergences.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins, crucial for the catabolic metabolism, because they contribute to the maintenance of the NADH/NAD+ balance. In several pathogenic bacteria and protists, NDH-2s are the only enzymes performing respiratory NADH:quinone oxidoreductase activity. For this reason and for being considered absent in mammals, NDH-2s were proposed as suitable targets for novel antimicrobial therapies. We selected all sequences of genes encoding NDH-2s from fully sequenced genomes present in the KEGG database. These genes were present in 61% of the 1805 species belonging to Eukarya (83%), Bacteria (60%) and Archaea (32%). Notably sequences from mammal species including humans were retrieved in our selection as NDH-2s. The data obtained and the already available information allowed systematizing several properties of NDH-2s: (i) the existence of additional sequence motifs with putative regulatory functions, (ii) specificity towards NADH or NADPH and (iii) the type of quinone binding motif. We observed that NDH-2 family distribution is not congruent with the taxonomic tree, suggesting different origins for the eukaryotic sequences and possible lateral gene transfer among prokaryotes. We note the absence of genes coding for NDH-2 in anaerobic phyla and the presence of multiple copies in several genomes, specifically in cyanobacteria. These observations inspired us to propose a metabolic hypothesis for the appearance of NDH-2s.

The two alternative NADH:quinone oxidoreductases from Staphylococcus aureus: two players with different molecular and cellular roles.

Staphylococcus aureus is an opportunistic pathogen that has emerged as a major public health threat due to the increased incidence of its drug resistance. S. aureus presents a remarkable capacity to adapt to different niches due to the plasticity of its energy metabolism. In this work, we investigated the energy metabolism of S. aureus, focusing on the alternative NADH:quinone oxidoreductases, NDH-2s. S. aureus presents two genes encoding NDH-2s (NDH-2A and NDH-2B) and lacks genes coding for Complex I, the canonical respiratory NADH:quinone oxidoreductase. This observation makes the action of NDH-2s crucial for the regeneration of NAD+ and, consequently, for the progression of metabolism. Our study involved the comprehensive biochemical characterization of NDH-2B and the exploration of the cellular roles of NDH-2A and NDH-2B, utilizing knockout mutants (Δndh-2a and Δndh-2b). We show that NDH-2B uses NADPH instead of NADH, does not establish a charge-transfer complex in the presence of NADPH, and its reduction by this substrate is the catalytic rate-limiting step. In the case of NDH-2B, the reduction of the flavin is inherently slow, and we suggest the establishment of a charge transfer complex between NADP+ and FADH2, as previously observed for NDH-2A, to slow down quinone reduction and, consequently, prevent the overproduction of reactive oxygen species, which is potentially unnecessary. Furthermore, we observed that the lack of NDH-2A or NDH-2B impacts cell growth, volume, and division differently. The absence of these enzymes results in distinct metabolic phenotypes, emphasizing the unique cellular roles of each NDH-2 in energy metabolism.IMPORTANCEStaphylococcus aureus is an opportunistic pathogen, posing a global challenge in clinical medicine due to the increased incidence of its drug resistance. For this reason, it is essential to explore and understand the mechanisms behind its resistance, as well as the fundamental biological features such as energy metabolism and the respective players that allow S. aureus to live and survive. Despite its prominence as a pathogen, the energy metabolism of S. aureus remains underexplored, with its respiratory enzymes often escaping thorough investigation. S. aureus bioenergetic plasticity is illustrated by its ability to use different respiratory enzymes, two of which are investigated in the present study. Understanding the metabolic adaptation strategies of S. aureus to bioenergetic challenges may pave the way for the design of therapeutic approaches that interfere with the ability of the pathogen to successfully adapt when it invades different niches within its host.

The plethora of membrane respiratory chains in the phyla of life.

The diversity of microbial cells is reflected in differences in cell size and shape, motility, mechanisms of cell division, pathogenicity or adaptation to different environmental niches. All these variations are achieved by the distinct metabolic strategies adopted by the organisms. The respiratory chains are integral parts of those strategies especially because they perform the most or, at least, most efficient energy conservation in the cell. Respiratory chains are composed of several membrane proteins, which perform a stepwise oxidation of metabolites toward the reduction of terminal electron acceptors. Many of these membrane proteins use the energy released from the oxidoreduction reaction they catalyze to translocate charges across the membrane and thus contribute to the establishment of the membrane potential, i.e. they conserve energy. In this work we illustrate and discuss the composition of the respiratory chains of different taxonomic clades, based on bioinformatic analyses and on biochemical data available in the literature. We explore the diversity of the respiratory chains of Animals, Plants, Fungi and Protists kingdoms as well as of Prokaryotes, including Bacteria and Archaea. The prokaryotic phyla studied in this work are Gammaproteobacteria, Betaproteobacteria, Epsilonproteobacteria, Deltaproteobacteria, Alphaproteobacteria, Firmicutes, Actinobacteria, Chlamydiae, Verrucomicrobia, Acidobacteria, Planctomycetes, Cyanobacteria, Bacteroidetes, Chloroflexi, Deinococcus-Thermus, Aquificae, Thermotogae, Deferribacteres, Nitrospirae, Euryarchaeota, Crenarchaeota and Thaumarchaeota.

Regulation of the mechanism of Type-II NADH: Quinone oxidoreductase from S. aureus.

Type-II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains and the only enzymes with NADH:quinone oxidoreductase activity expressed in Staphylococcus aureus (S. aureus), one of the most common causes of clinical infections. NDH-2s are members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, having a flavin adenine dinucleotide, FAD, as prosthetic group and NAD(P)H as substrate. The establishment of a Charge-Transfer Complex (CTC) between the isoalloxazine ring of the reduced flavin and the nicotinamide ring of NAD+ in NDH-2 was described, and in this work we explored its role in the kinetic mechanism using different electron donors and electron acceptors. We observed that CTC slows down the rate of the second half reaction (quinone reduction) and determines the effect of HQNO, an inhibitor. Also, protonation equilibrium simulations clearly indicate that the protonation probability of an important residue for proton transfer to the active site (D302) is influenced by the presence of the CTC. We propose that CTC is critical for the overall mechanism of NDH-2 and possibly relevant to keep a low quinol/quinone ratio and avoid excessive ROS production in vivo.

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family.

Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus.

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents. In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0.873 Å. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 81.8, b = 86.0, c = 269.9 Å, contained four monomers per asymmetric unit and diffracted to a resolution of 3.32 Å. A molecular-replacement solution was obtained and model building and refinement are currently under way.