Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs.

Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. TCSs and sRNAs mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors to oxygen. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent mechanisms of control. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second requires only the very 5' end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

History of Maturation of Prokaryotic Molybdoenzymes-A Personal View.

In prokaryotes, the role of Mo/W enzymes in physiology and bioenergetics is widely recognized. It is worth noting that the most diverse family of Mo/W enzymes is exclusive to prokaryotes, with the probable existence of several of them from the earliest forms of life on Earth. The structural organization of these enzymes, which often include additional redox centers, is as diverse as ever, as is their cellular localization. The most notable observation is the involvement of dedicated chaperones assisting with the assembly and acquisition of the metal centers, including Mo/W-bisPGD, one of the largest organic cofactors in nature. This review seeks to provide a new understanding and a unified model of Mo/W enzyme maturation.

Electrochemical Kinetics Support a Second Coordination Sphere Mechanism in Metal-Based Formate Dehydrogenase.

Metal-based formate dehydrogenases are molybdenum or tungsten-dependent enzymes that catalyze the interconversion between formate and CO2 . According to the current consensus, the metal ion of the catalytic center in its active form is coordinated by 6 S (or 5 S and 1 Se) atoms, leaving no free coordination sites to which formate could bind to the metal. Some authors have proposed that one of the active site ligands decoordinates during turnover to allow formate binding. Another proposal is that the oxidation of formate takes place in the second coordination sphere of the metal. Here, we have used electrochemical steady-state kinetics to elucidate the order of the steps in the catalytic cycle of two formate dehydrogenases. Our results strongly support the "second coordination sphere" hypothesis.

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.

The radical SAM protein HemW is a heme chaperone.

Radical S-adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S-C(5') bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes. For example, heme chaperones insert heme into target proteins but have been studied only for the formation of cytochrome c-type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer but not for stable heme binding. A bacterial two-hybrid system screen identified bacterioferritins and the heme-containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain-containing 1 (RSAD1) stably bound heme. In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins.

Horizontal acquisition of a hypoxia-responsive molybdenum cofactor biosynthesis pathway contributed to Mycobacterium tuberculosis pathoadaptation.

The unique ability of the tuberculosis (TB) bacillus, Mycobacterium tuberculosis, to persist for long periods of time in lung hypoxic lesions chiefly contributes to the global burden of latent TB. We and others previously reported that the M. tuberculosis ancestor underwent massive episodes of horizontal gene transfer (HGT), mostly from environmental species. Here, we sought to explore whether such ancient HGT played a part in M. tuberculosis evolution towards pathogenicity. We were interested by a HGT-acquired M. tuberculosis-specific gene set, namely moaA1-D1, which is involved in the biosynthesis of the molybdenum cofactor. Horizontal acquisition of this gene set was striking because homologues of these moa genes are present all across the Mycobacterium genus, including in M. tuberculosis. Here, we discovered that, unlike their paralogues, the moaA1-D1 genes are strongly induced under hypoxia. In vitro, a M. tuberculosis moaA1-D1-null mutant has an impaired ability to respire nitrate, to enter dormancy and to survive in oxygen-limiting conditions. Conversely, heterologous expression of moaA1-D1 in the phylogenetically closest non-TB mycobacterium, Mycobacterium kansasii, which lacks these genes, improves its capacity to respire nitrate and grants it with a marked ability to survive oxygen depletion. In vivo, the M. tuberculosis moaA1-D1-null mutant shows impaired survival in hypoxic granulomas in C3HeB/FeJ mice, but not in normoxic lesions in C57BL/6 animals. Collectively, our results identify a novel pathway required for M. tuberculosis resistance to host-imposed stress, namely hypoxia, and provide evidence that ancient HGT bolstered M. tuberculosis evolution from an environmental species towards a pervasive human-adapted pathogen.

A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies.

Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido-proxyl-based spin label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.

Distribution and dynamics of OXPHOS complexes in the bacterial cytoplasmic membrane.

Oxidative phosphorylation (OXPHOS) is an essential process for most living organisms mostly sustained by protein complexes embedded in the cell membrane. In order to thrive, cells need to quickly respond to changes in the metabolic demand or in their environment. An overview of the strategies that can be employed by bacterial cells to adjust the OXPHOS outcome is provided. Regulation at the level of gene expression can only provide a means to adjust the OXPHOS outcome to long-term trends in the environment. In addition, the actual view is that bioenergetic membranes are highly compartmentalized structures. This review discusses what is known about the spatial organization of OXPHOS complexes and the timescales at which they occur. As exemplified with the commensal gut bacterium Escherichia coli, three levels of spatial organization are at play: supercomplexes, membrane microdomains and polar assemblies. This review provides a particular focus on whether dynamic spatial organization can fine-tune the OXPHOS through the definition of specialized functional membrane microdomains. Putative mechanisms responsible for spatio-temporal regulation of the OXPHOS complexes are discussed. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Probing the Menasemiquinone Binding Mode to Nitrate Reductase†A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling.

In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15 N selective labeling of His15 Nδ or Lys15 Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15 Nδ as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15 Nζ consistent with a through-space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2 H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2 H-and hence of the 1 H-methyl isotropic hyperfine coupling by 2 H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen-bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one-sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.

Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR, 14,15N HYSCORE, and DFT Study.

Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in 98Mo and 15N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively 15N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

Reductive activation of E. coli respiratory nitrate reductase.

Over the past decades, a number of authors have reported the presence of inactive species in as-prepared samples of members of the Mo/W-bisPGD enzyme family. This greatly complicated the spectroscopic studies of these enzymes, since it is impossible to discriminate between active and inactive species on the basis of the spectroscopic signatures alone. Escherichia coli nitrate reductase A (NarGHI) is a member of the Mo/W-bisPGD family that allows anaerobic respiration using nitrate as terminal electron acceptor. Here, using protein film voltammetry on NarGH films, we show that the enzyme is purified in a functionally heterogeneous form that contains between 20 and 40% of inactive species that activate the first time they are reduced. This activation proceeds in two steps: a non-redox reversible reaction followed by an irreversible reduction. By carefully correlating electrochemical and EPR spectroscopic data, we show that neither the two major Mo(V) signals nor those of the two FeS clusters that are the closest to the Mo center are associated with the two inactive species. We also conclusively exclude the possibility that the major "low-pH" and "high-pH" Mo(V) EPR signatures correspond to species in acid-base equilibrium.

Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site.

Quinones are essential building blocks of respiration, a universal process dedicated to efficient harvesting of environmental energy and its conversion into a transmembrane chemiosmotic potential. Quinones differentiate mostly by their midpoint redox potential. As such, γ-proteobacteria such as Escherichia coli are characterized by the presence of demethylmenaquinone (DMK) with an intermediate redox potential between low-potential (menaquinone) and high-potential (ubiquinone) quinones. In this study, we show that demethylmenaquinol (DMKH2) is a good substrate for nitrate reductase A (NarGHI) in nitrate respiration in E. coli. Kinetic studies performed with quinol analogs on NarGHI show that removal of the methyl group on the naphthoquinol ring impacts modestly the catalytic constant but not the KM. EPR-monitored redox titrations of NarGHI-enriched membrane vesicles reveal that endogeneous demethylmenasemiquinone (DMSK) intermediates are stabilized in the enzyme. The measured midpoint potential of the DMK/DMKH2 redox couple in NarGHI (E'm,7.5 (DMK/DMKH2) ~-70mV) is significantly lower than that previously measured for unbound species. High resolution pulsed EPR experiments demonstrate that DMSK are formed within the NarGHI quinol oxidation site. Overall, our results provide the first characterization of a protein-bound DMSK and allows for comparison for distinct use of three quinones at a single Q-site in NarGHI.

The prokaryotic Mo/W-bisPGD enzymes family: a catalytic workhorse in bioenergetic.

Over the past two decades, prominent importance of molybdenum-containing enzymes in prokaryotes has been put forward by studies originating from different fields. Proteomic or bioinformatic studies underpinned that the list of molybdenum-containing enzymes is far from being complete with to date, more than fifty different enzymes involved in the biogeochemical nitrogen, carbon and sulfur cycles. In particular, the vast majority of prokaryotic molybdenum-containing enzymes belong to the so-called dimethylsulfoxide reductase family. Despite its extraordinary diversity, this family is characterized by the presence of a Mo/W-bis(pyranopterin guanosine dinucleotide) cofactor at the active site. This review highlights what has been learned about the properties of the catalytic site, the modular variation of the structural organization of these enzymes, and their interplay with the isoprenoid quinones. In the last part, this review provides an integrated view of how these enzymes contribute to the bioenergetics of prokaryotes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Cardiolipin-based respiratory complex activation in bacteria.

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b(D) center and is responsible for structural adjustments of b(D) and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.

Spatiotemporal dynamics of the proton motive force on single bacterial cells.

Electrochemical gradients across biological membranes are vital for cellular bioenergetics. In bacteria, the proton motive force (PMF) drives essential processes like adenosine triphosphate production and motility. Traditionally viewed as temporally and spatially stable, recent research reveals a dynamic PMF behavior at both single-cell and community levels. Moreover, the observed lateral segregation of respiratory complexes could suggest a spatial heterogeneity of the PMF. Using a light-activated proton pump and detecting the activity of the bacterial flagellar motor, we perturb and probe the PMF of single cells. Spatially homogeneous PMF perturbations reveal millisecond-scale temporal dynamics and an asymmetrical capacitive response. Localized perturbations show a rapid lateral PMF homogenization, faster than proton diffusion, akin to the electrotonic potential spread observed in passive neurons, explained by cable theory. These observations imply a global coupling between PMF sources and consumers along the membrane, precluding sustained PMF spatial heterogeneity but allowing for rapid temporal changes.

A sulfurtransferase is essential for activity of formate dehydrogenases in Escherichia coli.

l-Cysteine desulfurases provide sulfur to several metabolic pathways in the form of persulfides on specific cysteine residues of an acceptor protein for the eventual incorporation of sulfur into an end product. IscS is one of the three Escherichia coli l-cysteine desulfurases. It interacts with FdhD, a protein essential for the activity of formate dehydrogenases (FDHs), which are iron/molybdenum/selenium-containing enzymes. Here, we address the role played by this interaction in the activity of FDH-H (FdhF) in E. coli. The interaction of IscS with FdhD results in a sulfur transfer between IscS and FdhD in the form of persulfides. Substitution of the strictly conserved residue Cys-121 of FdhD impairs both sulfur transfer from IscS to FdhD and FdhF activity. Furthermore, inactive FdhF produced in the absence of FdhD contains both metal centers, albeit the molybdenum cofactor is at a reduced level. Finally, FdhF activity is sulfur-dependent, as it shows reversible sensitivity to cyanide treatment. Conclusively, FdhD is a sulfurtransferase between IscS and FdhF and is thereby essential to yield FDH activity.

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).

Cardiolipin binding in bacterial respiratory complexes: structural and functional implications.

The structural and functional integrity of biological membranes is vital to life. The interplay of lipids and membrane proteins is crucial for numerous fundamental processes ranging from respiration, photosynthesis, signal transduction, solute transport to motility. Evidence is accumulating that specific lipids play important roles in membrane proteins, but how specific lipids interact with and enable membrane proteins to achieve their full functionality remains unclear. X-ray structures of membrane proteins have revealed tight and specific binding of lipids. For instance, cardiolipin, an anionic phospholipid, has been found to be associated to a number of eukaryotic and prokaryotic respiratory complexes. Moreover, polar and septal accumulation of cardiolipin in a number of prokaryotes may ensure proper spatial segregation and/or activity of proteins. In this review, we describe current knowledge of the functions associated with cardiolipin binding to respiratory complexes in prokaryotes as a frame to discuss how specific lipid binding may tune their reactivity towards quinone and participate to supercomplex formation of both aerobic and anaerobic respiratory chains. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Heme biosynthesis is coupled to electron transport chains for energy generation.

Cellular energy generation uses membrane-localized electron transfer chains for ATP synthesis. Formed ATP in turn is consumed for the biosynthesis of cellular building blocks. In contrast, heme cofactor biosynthesis was found driving ATP generation via electron transport after initial ATP consumption. The FMN enzyme protoporphyrinogen IX oxidase (HemG) of Escherichia coli abstracts six electrons from its substrate and transfers them via ubiquinone, cytochrome bo(3) (Cyo) and cytochrome bd (Cyd) oxidase to oxygen. Under anaerobic conditions electrons are transferred via menaquinone, fumarate (Frd) and nitrate reductase (Nar). Cyo, Cyd and Nar contribute to the proton motive force that drives ATP formation. Four electron transport chains from HemG via diverse quinones to Cyo, Cyd, Nar, and Frd were reconstituted in vitro from purified components. Characterization of E. coli mutants deficient in nar, frd, cyo, cyd provided in vivo evidence for a detailed model of heme biosynthesis coupled energy generation.