Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.

Respiratory complex I plays a central role in cellular energy metabolism coupling NADH oxidation to proton translocation. In humans its dysfunction is associated with degenerative diseases. Here we report the structure of the electron input part of Aquifex aeolicus complex I at up to 1.8 Å resolution with bound substrates in the reduced and oxidized states. The redox states differ by the flip of a peptide bond close to the NADH binding site. The orientation of this peptide bond is determined by the reduction state of the nearby [Fe-S] cluster N1a. Fixation of the peptide bond by site-directed mutagenesis led to an inactivation of electron transfer and a decreased reactive oxygen species (ROS) production. We suggest the redox-gated peptide flip to represent a previously unrecognized molecular switch synchronizing NADH oxidation in response to the redox state of the complex as part of an intramolecular feed-back mechanism to prevent ROS production.

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.

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.

Summary 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.

Edited by Peter BrzezinskiCytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)-and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme.

Streptomyces diastatochromogenes Tü6028 is known to produce the polyketide antibiotic polyketomycin. The deletion of the pokOIV oxygenase gene led to a non-polyketomycin-producing mutant. Instead, novel compounds were produced by the mutant, which have not been detected before in the wild type strain. Four different compounds were identified and named foxicins A–D. Foxicin A was isolated and its structure was elucidated as an unusual nitrogen-containing quinone derivative using various spectroscopic methods. Through genome mining, the foxicin biosynthetic gene cluster was identified in the draft genome sequence of S. diastatochromogenes. The cluster spans 57 kb and encodes three PKS type I modules, one NRPS module and 41 additional enzymes. A foxBII gene-inactivated mutant of S. diastatochromogenes Tü6028 ΔpokOIV is unable to produce foxicins. Homologous fox biosynthetic gene clusters were found in more than 20 additional Streptomyces strains, overall in about 2.6% of all sequenced Streptomyces genomes. However, the production of foxicin-like compounds in these strains has never been described indicating that the clusters are expressed at a very low level or are silent under fermentation conditions. Foxicin A acts as a siderophore through interacting with ferric ions. Furthermore, it is a weak inhibitor of the Escherichia coli aerobic respiratory chain and shows moderate antibiotic activity. The wide distribution of the cluster and the various properties of the compound indicate a major role of foxicins in Streptomyces strains.

Formate hydrogenlyase (FHL) is the main hydrogen‐producing enzyme complex in enterobacteria. It converts formate to CO2 and H2 via a formate dehydrogenase and a [NiFe]‐hydrogenase. FHL and complex I are evolutionarily related and share a common core architecture. However, complex I catalyses the fundamentally different electron transfer from NADH to quinone and pumps protons. The catalytic FHL subunit, HycE, resembles NuoCD of Escherichia coli complex I; a fusion of NuoC and NuoD present in other organisms. The C‐terminal domain of HycE harbours the [NiFe]‐active site and is similar to other hydrogenases, while this domain in NuoCD is involved in quinone binding. The N‐terminal domains of these proteins do not bind cofactors and are not involved in electron transfer. As these N‐terminal domains are separate proteins in some organisms, we removed them in E. coli and observed that both FHL and complex I activities were essentially absent. This was due to either a disturbed assembly or to complex instability. Replacing the N‐terminal domain of HycE with a 180 amino acid E. coli NuoC protein fusion did not restore activity, indicating that the domains have complex‐specific functions. A FHL complex in which the N‐ and C‐terminal domains of HycE were physically separated still retained most of its FHL activity, while the separation of NuoCD abolished complex I activity completely. Only the FHL complex tolerates physical separation of the HycE domains. Together, the findings strongly suggest that the N‐terminal domains of these proteins are key determinants in complex assembly.