Exploring ND-011992, a quinazoline-type inhibitor targeting quinone reductases and quinol oxidases.

Bacterial energy metabolism has become a promising target for next-generation tuberculosis chemotherapy. One strategy to hamper ATP production is to inhibit the respiratory oxidases. The respiratory chain of Mycobacterium tuberculosis comprises a cytochrome bcc:aa3 and a cytochrome bd ubiquinol oxidase that require a combined approach to block their activity. A quinazoline-type compound called ND-011992 has previously been reported to ineffectively inhibit bd oxidases, but to act bactericidal in combination with inhibitors of cytochrome bcc:aa3 oxidase. Due to the structural similarity of ND-011992 to quinazoline-type inhibitors of respiratory complex I, we suspected that this compound is also capable of blocking other respiratory chain complexes. Here, we synthesized ND-011992 and a bromine derivative to study their effect on the respiratory chain complexes of Escherichia coli. And indeed, ND-011992 was found to inhibit respiratory complex I and bo3 oxidase in addition to bd-I and bd-II oxidases. The IC50 values are all in the low micromolar range, with inhibition of complex I providing the lowest value with an IC50 of 0.12 µM. Thus, ND-011992 acts on both, quinone reductases and quinol oxidases and could be very well suited to regulate the activity of the entire respiratory chain.

Mutations in respiratory complex I promote antibiotic persistence through alterations in intracellular acidity and protein synthesis.

Antibiotic persistence describes the presence of phenotypic variants within an isogenic bacterial population that are transiently tolerant to antibiotic treatment. Perturbations of metabolic homeostasis can promote antibiotic persistence, but the precise mechanisms are not well understood. Here, we use laboratory evolution, population-wide sequencing and biochemical characterizations to identify mutations in respiratory complex I and discover how they promote persistence in Escherichia coli. We show that persistence-inducing perturbations of metabolic homeostasis are associated with cytoplasmic acidification. Such cytoplasmic acidification is further strengthened by compromised proton pumping in the complex I mutants. While RpoS regulon activation induces persistence in the wild type, the aggravated cytoplasmic acidification in the complex I mutants leads to increased persistence via global shutdown of protein synthesis. Thus, we propose that cytoplasmic acidification, amplified by a compromised complex I, can act as a signaling hub for perturbed metabolic homeostasis in antibiotic persisters.

Structure of the peripheral arm of a minimalistic respiratory complex I.

Respiratory complex I drives proton translocation across energy-transducing membranes by NADH oxidation coupled with (ubi)quinone reduction. In humans, its dysfunction is associated with neurodegenerative diseases. The Escherichia coli complex represents the structural minimal form of an energy-converting NADH:ubiquinone oxidoreductase. Here, we report the structure of the peripheral arm of the E. coli complex I consisting of six subunits, the FMN cofactor, and nine iron-sulfur clusters at 2.7 Å resolution obtained by cryo electron microscopy. While the cofactors are in equivalent positions as in the complex from other species, individual subunits are adapted to the absence of supernumerary proteins to guarantee structural stability. The catalytically important subunits NuoC and D are fused resulting in a specific architecture of functional importance. Striking features of the E. coli complex are scrutinized by mutagenesis and biochemical characterization of the variants. Moreover, the arrangement of the subunits sheds light on the unknown assembly of the complex.

Biochemical consequences of two clinically relevant ND-gene mutations in Escherichia coli respiratory complex I.

NADH:ubiquinone oxidoreductase (respiratory complex I) plays a major role in energy metabolism by coupling electron transfer from NADH to quinone with proton translocation across the membrane. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction that manifest in a wide variety of neurodegenerative diseases. Seven subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations discovered in mitochondria from patients' tissues. However, whether or how these genetic aberrations affect complex I at a molecular level is unknown. Here, we used Escherichia coli as a model system to biochemically characterize two mutations that were found in mtDNA of patients. The V253AMT-ND5 mutation completely disturbed the assembly of complex I, while the mutation D199GMT-ND1 led to the assembly of a stable complex capable to catalyze redox-driven proton translocation. However, the latter mutation perturbs quinone reduction leading to a diminished activity. D199MT-ND1 is part of a cluster of charged amino acid residues that are suggested to be important for efficient coupling of quinone reduction and proton translocation. A mechanism considering the role of D199MT-ND1 for energy conservation in complex I is discussed.

A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation With Electron Transfer in E. coli Respiratory Complex I.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with the translocation of protons across the membrane, thus contributing to the protonmotive force. Complex I has an overall L-shaped structure with a peripheral arm catalyzing electron transfer and a membrane arm engaged in proton translocation. Although both reactions are arranged spatially separated, they are tightly coupled by a mechanism that is not fully understood. Using redox-difference UV-vis spectroscopy, an unknown redox component was identified in Escherichia coli complex I as reported earlier. A comparison of its spectrum with those obtained for different quinone species indicates features of a quinol anion. The re-oxidation kinetics of the quinol anion intermediate is significantly slower in the D213GH variant that was previously shown to operate with disturbed quinone chemistry. Addition of the quinone-site inhibitor piericidin A led to strongly decreased absorption peaks in the difference spectrum. A hypothesis for a mechanism of proton-coupled electron transfer with the quinol anion as catalytically important intermediate in complex I is discussed.

Mass Photometry of Membrane Proteins.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

Quantifying the heterogeneity of macromolecular machines by mass photometry.

Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.

Visualizing the movement of the amphipathic helix in the respiratory complex I using a nitrile infrared probe and SEIRAS.

Conformational movements play an important role in enzyme catalysis. Respiratory complex I, an L-shaped enzyme, connects electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation through its membrane arm by a coupling mechanism still under debate. The amphipathic helix across the membrane arm represents a unique structural feature. Here, we demonstrate a new way to study conformational changes by introducing a small and highly flexible nitrile infrared (IR) label to this helix to visualize movement with surface-enhanced IR absorption spectroscopy. We find that labeled residues K551CL and Y590CL move to a more hydrophobic environment upon NADH reduction of the enzyme, likely as a response to the reorganization of the antiporter-like subunits in the membrane arm.

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.

Reduction of the off-pathway iron-sulphur cluster N1a of Escherichia coli respiratory complex I restrains NAD+ dissociation.

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. The reaction starts with NADH oxidation by a flavin cofactor followed by transferring the electrons through a chain of seven iron-sulphur clusters to quinone. An eighth cluster called N1a is located proximally to flavin, but on the opposite side of the chain of clusters. N1a is strictly conserved although not involved in the direct electron transfer to quinone. Here, we show that the NADH:ferricyanide oxidoreductase activity of E. coli complex I is strongly diminished when the reaction is initiated by an addition of ferricyanide instead of NADH. This effect is significantly less pronounced in a variant containing N1a with a 100 mV more negative redox potential. Detailed kinetic analysis revealed that the reduced activity is due to a lower dissociation constant of bound NAD+. Thus, reduction of N1a induces local structural rearrangements of the protein that stabilise binding of NAD+. The variant features a considerably enhanced production of reactive oxygen species indicating that bound NAD+ represses this process.

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.