Cytochrome Bo Research Articles

Glucose-gated nanocoating endowing polyetheretherketone implants for enzymatic gas therapy to boost infectious diabetic osseointegration

The hyperglycemic micromilieu surrounding implants in diabetic patients leads to high failure rate of implantation and implant-associated infection. Carbon monoxide (CO) has been reported to combat infections; however, its on-demand liberation and the elucidation of the underlying antibacterial mechanism remain challenging. To address this issue, we develop a multipurpose orthopedic implant comprising polyetheretherketone, glucose oxidase (GOx), and manganese carbonyl nanocrystals (MnCO), serving as a glucose-gated nanocoating for enzymatic gas therapy to improve infectious diabetic osseointegration. The GOx acts as a glucose-actuated gate responsive to hyperglycemia, thereby delivering CO in situ triggered by the GOx-driven Fenton-like reaction of MnCO nanocrystals. The released CO considerably prevents bacterial multiplication by penetrating the membrane, binding to cytochrome bo3, and interfering with the respiratory chain in vitro. Furthermore, the engineered implant displays desired antibacterial properties and enhances osseointegration in vivo. Collectively, the orthopedic nanocoating implant is capable of delivering glucose-gated enzymatic gas therapy, promising for treating infectious diabetic bone defects.

Improved fermentative gamma-aminobutyric acid production from glucose by the inactivation of respiratory chain components NDH-I and Cytbo₃ in Escherichia coli

Gamma-aminobutyric acid (GABA), which is synthesized from l-glutamic acid via glutamate decarboxylase (Gad), is used as food, supplements, and biodegradable plastics. Our previous study demonstrated an Escherichia coli mutant (ΔΔ) strain, lacking type I NADH dehydrogenase (NDH-I) and cytochrome bo3 oxidase (Cytbo3), produced 7g/L glutamic acid on MS1 glucose-minimal medium. In this study, the ΔΔ strain was used for improving GABA production. A plasmid (pMBL19-gadB') expressing a mutated E.coli GadB (Glu89Gln/Δ452-466), retaining activity at neutral pH, was introduced into the ΔΔ strain and its parent strain (W1485). The ΔΔ strain carrying pMBL19-gadB' exhibited a twofold increase in GABA production compared to the W1485 strain carrying pMBL19-gadB'. Deleting the C-terminal (Δ471-511) of GadC antiporter in the ΔΔ strain further improved GABA yield by 1.5g/L when cultured in MS1 glucose-minimal medium. On the other hand, a large amount of glutamic acid produced by the ΔΔ strain was not fully converted to GABA, likely due to the inhibition of GadB activity by the accumulation of acetic acid. Although there is room for improvement, these results indicate the efficacy of the ΔNDH-IΔCytbo3 double mutation in augmenting GABA production.

Cryo-EM structure of cytochrome bo3 quinol oxidase assembled in peptidiscs reveals an “open” conformation for potential ubiquinone-8 release

Cytochrome bo3 quinol oxidase belongs to the heme‑copper-oxidoreductase (HCO) superfamily, which is part of the respiratory chain and essential for cell survival. While the reaction mechanism of cyt bo3 has been studied extensively over the last decades, specific details about its substrate binding and product release have remained unelucidated due to the lack of structural information. Here, we report a 2.8 Å cryo-electron microscopy structure of cyt bo3 from Escherichia coli assembled in peptidiscs. Our structural model shows a conformation for amino acids 1–41 of subunit I different from all previously published structures while the remaining parts of this enzyme are similar. Our new conformation shows a “U-shape” assembly in contrast to the transmembrane helix, named “TM0”, in other reported structural models. However, TM0 blocks ubiquinone-8 (reaction product) release, suggesting that other cyt bo3 conformations should exist. Our structural model presents experimental evidence for an “open” conformation to facilitate substrate/product exchange. This work helps further understand the reaction cycle of this oxidase, which could be a benefit for potential drug/antibiotic design for health science.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

The terminal oxidase cytochrome bd-I confers carbon monoxide resistance to Escherichia coli cells

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme‑copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 μM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 μM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 μM CO.

A 2.2 Å cryoEM structure of a quinol-dependent NO Reductase shows close similarity to respiratory oxidases

Quinol-dependent nitric oxide reductases (qNORs) are considered members of the respiratory heme-copper oxidase superfamily, are unique to bacteria, and are commonly found in pathogenic bacteria where they play a role in combating the host immune response. qNORs are also essential enzymes in the denitrification pathway, catalysing the reduction of nitric oxide to nitrous oxide. Here, we determine a 2.2 Å cryoEM structure of qNOR from Alcaligenes xylosoxidans, an opportunistic pathogen and a denitrifying bacterium of importance in the nitrogen cycle. This high-resolution structure provides insight into electron, substrate, and proton pathways, and provides evidence that the quinol binding site not only contains the conserved His and Asp residues but also possesses a critical Arg (Arg720) observed in cytochrome bo3, a respiratory quinol oxidase.

Monomer and dimer structures of cytochrome bo3 ubiquinol oxidase from Escherichia coli.

The Escherichia coli cytochrome bo3 ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in the E. coli aerobic respiratory chain. Despite many mechanistic studies, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts-the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of the E. coli cytochrome bo3 ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 and 3.46 Å, respectively. We have discovered that the protein can form a dimer with C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in the monomers, except the movement of a loop in subunit IV (residues 67-74).

Probing molecular interactions of semiquinone radicals at quinone reduction sites of cytochrome bc1 by X-band HYSCORE EPR spectroscopy and quantum mechanical calculations.

Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state via various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Qi) of cytochrome bc1 using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Qi to arginine) in which the SQ stability has previously been shown to markedly increase. The 14N region of the HYSCORE 2D spectrum for SQi in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome bo3. Parametrization of hyperfine and quadrupolar interactions of SQi with surrounding magnetic nuclei (1H, 14N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQi in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (1H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.

Quinol oxidases in the superfamily of heme-copper oxidoreductases and the cryoEM structure of the cytochrome bo3 ubiquinol oxidase from E. coli

Quinol oxidases in the superfamily of heme-copper oxidoreductases and the cryoEM structure of the cytochrome bo3 ubiquinol oxidase from E. coli

Detergent-Free Functionalization of Hybrid Vesicles with Membrane Proteins Using SMALPs.

Hybrid vesicles (HVs) that consist of mixtures of block copolymers and lipids are robust biomimetics of liposomes, providing a valuable building block in bionanotechnology, catalysis, and synthetic biology. However, functionalization of HVs with membrane proteins remains laborious and expensive, creating a significant current challenge in the field. Here, using a new approach of extraction with styrene-maleic acid (SMA), we show that a membrane protein (cytochrome bo3) directly transfers into HVs with an efficiency of 73.9 ± 13.5% without the requirement of detergent, long incubation times, or mechanical disruption. Direct transfer of membrane proteins using this approach was not possible into liposomes, suggesting that HVs are more amenable than liposomes to membrane protein incorporation from a SMA lipid particle system. Finally, we show that this transfer method is not limited to cytochrome bo3 and can also be performed with complex membrane protein mixtures.

Adaptation to Varying Salinity in Halomonas elongata: Much More Than Ectoine Accumulation.

The halophilic γ-proteobacterium Halomonas elongata DSM 2581T thrives at salt concentrations well above 10 % NaCl (1.7 M NaCl). A well-known osmoregulatory mechanism is the accumulation of the compatible solute ectoine within the cell in response to osmotic stress. While ectoine accumulation is central to osmoregulation and promotes resistance to high salinity in halophilic bacteria, ectoine has this effect only to a much lesser extent in non-halophiles. We carried out transcriptome analysis of H. elongata grown on two different carbon sources (acetate or glucose), and low (0.17 M NaCl), medium (1 M), and high salinity (2 M) to identify additional mechanisms for adaptation to high saline environments. To avoid a methodological bias, the transcripts were evaluated by applying two methods, DESeq2 and Transcripts Per Million (TPM). The differentially transcribed genes in response to the available carbon sources and salt stress were then compared to the transcriptome profile of Chromohalobacter salexigens, a closely related moderate halophilic bacterium. Transcriptome profiling supports the notion that glucose is degraded via the cytoplasmic Entner-Doudoroff pathway, whereas the Embden-Meyerhoff-Parnas pathway is employed for gluconeogenesis. The machinery of oxidative phosphorylation in H. elongata and C. salexigens differs greatly from that of non-halophilic organisms, and electron flow can occur from quinone to oxygen along four alternative routes. Two of these pathways via cytochrome bo' and cytochrome bd quinol oxidases seem to be upregulated in salt stressed cells. Among the most highly regulated genes in H. elongata and C. salexigens are those encoding chemotaxis and motility proteins, with genes for chemotaxis and flagellar assembly severely downregulated at low salt concentrations. We also compared transcripts at low and high-salt stress (low growth rate) with transcripts at optimal salt concentration and found that the majority of regulated genes were down-regulated in stressed cells, including many genes involved in carbohydrate metabolism, while ribosome synthesis was up-regulated, which is in contrast to what is known from non-halophiles at slow growth. Finally, comparing the acidity of the cytoplasmic proteomes of non-halophiles, extreme halophiles and moderate halophiles suggests adaptation to an increased cytoplasmic ion concentration of H. elongata. Taken together, these results lead us to propose a model for salt tolerance in H. elongata where ion accumulation plays a greater role in salt tolerance than previously assumed.

The Cpx Stress Response Regulates Turnover of Respiratory Chain Proteins at the Inner Membrane of Escherichia coli.

The Cpx envelope stress response is a major signaling pathway monitoring bacterial envelope integrity, activated both internally by excessive synthesis of membrane proteins and externally by a variety of environmental cues. The Cpx regulon is enriched with genes coding for protein folding and degrading factors, virulence determinants, and large envelope-localized complexes. Transcriptional repression of the two electron transport chain complexes, NADH dehydrogenase I and cytochrome bo3, by the Cpx pathway has been demonstrated, however, there is evidence that additional regulatory mechanisms exist. In this study, we examine the interaction between Cpx-regulated protein folding and degrading factors and the respiratory complexes NADH dehydrogenase I and succinate dehydrogenase in Escherichia coli. Here we show that the cellular need for Cpx-mediated stress adaptation increases when respiratory complexes are more prevalent or active, which is demonstrated by the growth defect of Cpx-deficient strains on media that requires a functional electron transport chain. Interestingly, deletion of several Cpx-regulated proteolytic factors and chaperones results in similar growth-deficient phenotypes. Furthermore, we find that the stability of the NADH dehydrogenase I protein complex is lower in cells with a functional Cpx response, while in its absence, protein turnover is impaired. Finally, we demonstrated that the succinate dehydrogenase complex has reduced activity in E. coli lacking the Cpx pathway. Our results suggest that the Cpx two-component system serves as a sentry of inner membrane protein biogenesis, ensuring the function of large envelope protein complexes and maintaining the cellular energy status of the cell.

Cryo-EM structures of Escherichia coli cytochrome bo3 reveal bound phospholipids and ubiquinone-8 in a dynamic substrate binding site

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3 ) have been determined by cryogenic electron microscopy (cryo-EM) in styrene-maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-Å resolution, respectively. The structures include the metal redox centers (heme b, heme o3 , and CuB), the redox-active cross-linked histidine-tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Genomic evolution of the class Acidithiobacillia: deep-branching Proteobacteria living in extreme acidic conditions

Members of the genus Acidithiobacillus, now ranked within the class Acidithiobacillia, are model bacteria for the study of chemolithotrophic energy conversion under extreme conditions. Knowledge of the genomic and taxonomic diversity of Acidithiobacillia is still limited. Here, we present a systematic analysis of nearly 100 genomes from the class sampled from a wide range of habitats. Some of these genomes are new and others have been reclassified on the basis of advanced genomic analysis, thus defining 19 Acidithiobacillia lineages ranking at different taxonomic levels. This work provides the most comprehensive classification and pangenomic analysis of this deep-branching class of Proteobacteria to date. The phylogenomic framework obtained illuminates not only the evolutionary past of this lineage, but also the molecular evolution of relevant aerobic respiratory proteins, namely the cytochrome bo3 ubiquinol oxidases.

Cardiolipin enhances the enzymatic activity of cytochrome bd and cytochrome bo3 solubilized in dodecyl-maltoside

Cardiolipin (CL) is a lipid that is found in the membranes of bacteria and the inner membranes of mitochondria. CL can increase the activity of integral membrane proteins, in particular components of respiratory pathways. We here report that CL activated detergent-solubilized cytochrome bd, a terminal oxidase from Escherichia coli. CL enhanced the oxygen consumption activity ~ twofold and decreased the apparent KM value for ubiquinol-1 as substrate from 95 µM to 35 µM. Activation by CL was also observed for cytochrome bd from two Gram-positive species, Geobacillus thermodenitrificans and Corynebacterium glutamicum, and for cytochrome bo3 from E. coli. Taken together, CL can enhance the activity of detergent-solubilized cytochrome bd and cytochrome bo3.

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I.

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Escherichia coli is engineered to grow on CO2 and formic acid.

We engineered Escherichia coli to grow on CO2 and formic acid alone by introducing the synthetic CO2 and formic acid assimilation pathway, expressing two formate dehydrogenase genes, fine-tuning metabolic fluxes and optimizing the levels of cytochrome bo3 and bd-I ubiquinol oxidase. Our engineered strain can grow to an optical density at 600 nm of 7.38 in 450 h, and shows promise as a platform strain growing on CO2 and formic acid alone.

Physiological Response of Escherichia coli W3110 and BL21 to the Aerobic Expression of Vitreoscilla Hemoglobin.

The aerobic growth and metabolic performance of Escherichia coli strains BL21 and W3110 were studied when the Vitreoscilla hemoglobin (VHb) was constitutively expressed in the chromosome. When VHb was expressed, acetate production decreased in both strains and was nearly eliminated in BL21. Transcriptional levels of the glyoxylate shunt genes decreased in both strains when VHb was expressed. However, higher transcription of the α-ketoglutarate dehydrogenase genes were observed for W3110, while for BL21 transcription levels decreased. VHb expression reduced the transcription of the cytochrome bo3 genes only in BL21. These results are useful for better selecting a production host.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane.

Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

The aerobic respiratory chain of Pseudomonas aeruginosa cultured in artificial urine media: Role of NQR and terminal oxidases.

Pseudomonas aeruginosa is a Gram-negative γ-proteobacterium that forms part of the normal human microbiota and it is also an opportunistic pathogen, responsible for 30% of all nosocomial urinary tract infections. P. aeruginosa carries a highly branched respiratory chain that allows the colonization of many environments, such as the urinary tract, catheters and other medical devices. P. aeruginosa respiratory chain contains three different NADH dehydrogenases (complex I, NQR and NDH-2), whose physiologic roles have not been elucidated, and up to five terminal oxidases: three cytochrome c oxidases (COx), a cytochrome bo3 oxidase (CYO) and a cyanide-insensitive cytochrome bd-like oxidase (CIO). In this work, we studied the composition of the respiratory chain of P. aeruginosa cells cultured in Luria Broth (LB) and modified artificial urine media (mAUM), to understand the metabolic adaptations of this microorganism to the growth in urine. Our results show that the COx oxidases play major roles in mAUM, while P. aeruginosa relies on CYO when growing in LB medium. Moreover, our data demonstrate that the proton-pumping NQR complex is the main NADH dehydrogenase in both LB and mAUM. This enzyme is resistant to HQNO, an inhibitory molecule produced by P. aeruginosa, and may provide an advantage against the natural antibacterial agents produced by this organism. This work offers a clear picture of the composition of this pathogen’s aerobic respiratory chain and the main roles that NQR and terminal oxidases play in urine, which is essential to understand its physiology and could be used to develop new antibiotics against this notorious multidrug-resistant microorganism.