Cbb3 Oxidase Research Articles

Genetic potential for aerobic respiration and denitrification in globally distributed respiratory endosymbionts

The endosymbiont Candidatus Azoamicus ciliaticola was proposed to generate ATP for its eukaryotic host, an anaerobic ciliate of the Plagiopylea class, fulfilling a function analogous to mitochondria in other eukaryotic cells. The discovery of this respiratory endosymbiosis has major implications for both evolutionary history and ecology of microbial eukaryotes. However, with only a single species described, knowledge of its environmental distribution and diversity is limited. Here we report four complete, circular metagenome assembled genomes (cMAGs) representing respiratory endosymbionts inhabiting groundwater in California, Ohio, and Germany. These cMAGs form two lineages comprising a monophyletic clade within the uncharacterized gammaproteobacterial order UBA6186, enabling evolutionary analysis of their key protein complexes. Strikingly, all four cMAGs encode a cytochrome cbb3 oxidase, which indicates that these endosymbionts have the capacity for aerobic respiration. Accordingly, we detect these respiratory endosymbionts in diverse habitats worldwide, thus further expanding the ecological scope of this respiratory symbiosis.

Multidimensional engineering of Rhodobacter sphaeroides for enhanced photo-fermentative hydrogen production

The photo-fermentative hydrogen production by purple non-sulfur photosynthetic bacteria (PNSB) is regarded as a promising approach for sustainable hydrogen production. In PNSB, biohydrogen synthesis is catalyzed by nitrogenase which requires two essential cofactors of adenosine triphosphate (ATP) and electron. To avoid the insufficient ATP supply that limits the nitrogenase activity for hydrogen production, we systematically engineered Rhodobacter sphaeroides to improve the hydrogen production by manipulating the light harvesting (LH) complexes, the electron transfer chain (ETC), the adenosine diphosphate (ADP) synthetic pathway, the F0F1-ATPase expression, and the nitrogenase expression. To improve the photosynthetic efficiency, we engineered the LH complexes by eliminating pucBA and overexpressing pufQ and spbA. Then, the ETC was boosted by deleting qxt-type oxidase and integrating the electron carrier cytochrome cy at the site of cyt cbb3 oxidase. In addition, we increased the supply of precursor ADP by overexpressing adk, and the abundance of F0F1-ATPase was efficiently enhanced through overexpressing atpXF. Further transposon engineering with the integration of nifHDK and atpXF resulted in a mutant YH27 with a substantial improvement of performance over the base strain, and its nitrogenase activity and hydrogen production were enhanced by 404 % and 93 %, respectively. Taken together, the multidimensional strategies enabled the engineered R. sphaeroides with a hydrogen productivity of 61.2 mL/(Lh) and a yield of 3.0 mol/mol acetate under the fed-batch condition, which represents one of the most efficient biohydrogen-producing strains.

Cold atmospheric pressure plasma-antibiotic synergy in Pseudomonas aeruginosa biofilms is mediated via oxidative stress response

Cold atmospheric-pressure plasma (CAP) has emerged as a potential alternative or adjuvant to conventional antibiotics for the treatment of bacterial infections, including those caused by antibiotic-resistant pathogens. The potential of sub-lethal CAP exposures to synergise conventional antimicrobials for the eradication of Pseudomonas aeruginosa biofilms is investigated in this study. The efficacy of antimicrobials following or in the absence of sub-lethal CAP pre-treatment in P. aeruginosa biofilms was assessed. CAP pre-treatment resulted in an increase in both planktonic and biofilm antimicrobial sensitivity for all three strains tested (PAO1, PA14, and PA10548), with both minimum inhibitory concentrations (MICs) and minimum biofilm eradication concentrations (MBECs) of individual antimicrobials, being significantly reduced following CAP pre-treatment of the biofilm (512-fold reduction with ciprofloxacin/gentamicin; and a 256-fold reduction with tobramycin). At all concentrations of antimicrobial used, the combination of sub-lethal CAP exposure and antimicrobials was effective at increasing time-to-peak metabolism, as measured by isothermal microcalorimetry, again indicating enhanced susceptibility. CAP is known to damage bacterial cell membranes and DNA by causing oxidative stress through the in situ generation of reactive oxygen and nitrogen species (RONS). While the exact mechanism is not clear, oxidative stress on outer membrane proteins is thought to damage/perturb cell membranes, confirmed by ATP and LDH leakage, allowing antimicrobials to penetrate the bacterial cell more effectively, thus increasing bacterial susceptibility. Transcriptomic analysis, reveals that cold-plasma mediated oxidative stress caused upregulation of P. aeruginosa superoxide dismutase, cbb3 oxidases, catalases, and peroxidases, and upregulation in denitrification genes, suggesting that P. aeruginosa uses these enzymes to degrade RONS and mitigate the effects of cold plasma mediated oxidative stress. CAP treatment also led to an increased production of the signalling molecule ppGpp in P. aeruginosa, indicative of a stringent response being established. Although we did not directly measure persister cell formation, this stringent response may potentially be associated with the formation of persister cells in biofilm cultures. The production of ppGpp and polyphosphate may be associated with protein synthesis inhibition and increase efflux pump activity, factors which can result in antimicrobial tolerance. The transcriptomic analysis also showed that by 6 h post-treatment, there was downregulation in ribosome modulation factor, which is involved in the formation of persister cells, suggesting that the cells had begun to resuscitate/recover. In addition, CAP treatment at 4 h post-exposure caused downregulation of the virulence factors pyoverdine and pyocyanin; by 6 h post-exposure, virulence factor production was increasing. Transcriptomic analysis provides valuable insights into the mechanisms by which P. aeruginosa biofilms exhibits enhanced susceptibility to antimicrobials. Overall, these findings suggest, for the first time, that short CAP sub-lethal pre-treatment can be an effective strategy for enhancing the susceptibility of P. aeruginosa biofilms to antimicrobials and provides important mechanistic insights into cold plasma-antimicrobial synergy. Transcriptomic analysis of the response to, and recovery from, sub-lethal cold plasma exposures in P. aeruginosa biofilms improves our current understanding of cold plasma biofilm interactions.

Vibrio cholerae requires oxidative respiration through the bd-I and cbb3 oxidases for intestinal proliferation.

Vibrio cholerae respires both aerobically and anaerobically and, while oxygen may be available to it during infection, other terminal electron acceptors are proposed for population expansion during infection. Unlike gastrointestinal pathogens that stimulate significant inflammation leading to elevated levels of oxygen or alternative terminal electron acceptors, V. cholerae infections are not understood to induce a notable inflammatory response. To ascertain the respiration requirements of V. cholerae during infection, we used Multiplex Genome Editing by Natural Transformation (MuGENT) to create V. cholerae strains lacking aerobic or anaerobic respiration. V. cholerae strains lacking aerobic respiration were attenuated in infant mice 105-fold relative to wild type, while strains lacking anaerobic respiration had no colonization defect, contrary to earlier work suggesting a role for anaerobic respiration during infection. Using several approaches, including one we developed for this work termed Comparative Multiplex PCR Amplicon Sequencing (CoMPAS), we determined that the bd-I and cbb3 oxidases are essential for small intestinal colonization of V. cholerae in the infant mouse. The bd-I oxidase was also determined as the primary oxidase during growth outside the host, making V. cholerae the only example of a Gram-negative bacterial pathogen in which a bd-type oxidase is the primary oxidase for energy acquisition inside and outside of a host.

The Response Regulator RegA Is a Copper Binding Protein That Covalently Dimerizes When Exposed to Oxygen.

In Rhodobacter capsulatus, the histidine kinase RegB is believed to phosphorylate its cognate transcriptional factor RegA only under anaerobic conditions. However, transcriptome evidence indicates that RegA regulates 47 genes involved in energy storage, energy production, signaling and transcription, under aerobic conditions. In this study, we provide evidence that RegA is a copper binding protein and that copper promotes the dimerization of RegA under aerobic conditions. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicates that RegA binds Cu1+ and Cu2+ in a 1:1 and 2:1 ratio, respectively. Through LC-MS/MS, ESI-MS and non-reducing SDS-PAGE gels, we show that Cu2+ stimulates disulfide bond formation in RegA at Cys156 in the presence of oxygen. Finally, we used DNase I footprint analysis to demonstrate that Cu2+-mediated covalent dimerized RegA is capable of binding to the ccoN promoter, which drives the expression of cytochrome cbb3 oxidase subunits. This study provides a new model of aerobic regulation of gene expression by RegA involving the formation of an intermolecular disulfide bond.

Comparative genomics of Photobacterium species from terrestrial and marine habitats.

Photobacterium (P.) is a genus widely studied in regards to its association with and ubiquitous presence in marine environments. However, certain species (P. phosphoreum, P. carnosum, P. iliopiscarium) have been recently described to colonize and spoil raw meats without a marine link. We have studied 27 strains from meat as well as 26 strains from marine environments in order to probe for intraspecies marine/terrestrial subpopulations and identify distinct genomic features acquired by environmental adaptation. We have conducted phylogenetic analysis (MLSA, ANI, fur, codon usage), search of plasmids (plasmidSPADES), phages (PHASTER), CRISPR-cas operons (CRISPR-finder) and secondary metabolites gene clusters (antiSMASH, BAGEL), in addition to a targeted gene search for specific pathways (e.g. TCA cycle, pentose phosphate, respiratory chain) and elements relevant for growth, adaptation and competition (substrate utilization, motility, bioluminescence, sodium and iron transport). P. carnosum appears as a conserved single clade, with one isolate from MAP fish clustering apart that doesn't, however, show distinct features that could indicate different adaptation. The species harbors genes for a wide carbon source utilization (glycogen/starch, maltose, pullulan, fucose) for colonization of diverse niches in its genome. P. phosphoreum is represented by two different clades on the phylogenetic analyses not correlating to their origin or distribution of other features analyzed that can be divided into two novel subspecies based on genome-wide values. A more diverse antimicrobial activity (sactipeptides, microcins), production of secondary metabolites (siderophores and arylpolyenes), stress response and adaptation (bioluminescence, sodium transporters, catalase, high affinity for oxygen cytochrome cbb3 oxidase, DMSO reductase and proton translocating NADH dehydrogenase) is predicted compared to the other species. P. iliopiscarium was divided into two clades based on source of isolation correlating with phylogeny and distribution of several traits. The species shows traits common to the other two species, similar carbon utilization/transport gene conservation as P. carnosum for the meat-isolated strains, and predicted utilization of marine-common DMSO and flagellar cluster for the sea-isolated strains. Results additionally suggest that photobacteria are highly prone to horizontal acquisition/loss of genetic material and genetic transduction, and that it might be a strategy for increasing the frequency of strain- or species-specific features that offers a growth/competition advantage.

The mechanism for oxygen reduction in the C family cbb3 cytochrome c oxidases - Implications for the proton pumping stoichiometry

Cytochrome c oxidases (CcOs) couple the exergonic reduction of molecular oxygen to proton pumping across the membrane in which they are embedded, thereby conserving a significant part of the free energy. The A family CcOs are known to pump four protons per oxygen molecule, while there is no consensus regarding the proton pumping stoichiometry for the C family cbb3 oxidases. Hybrid density functional theory is used here to investigate the catalytic mechanism for oxygen reduction in cbb3 oxidases. A surprising result is that the barrier for O O bond cleavage at the mixed valence reduction level seems to be too high compared to the overall reaction rate of the enzyme. It is therefore suggested that the O O bond is cleaved only after the first proton coupled reduction step, and that this reduction step most likely is not coupled to proton pumping. Furthermore, since the cbb3 oxidases have only one proton channel leading to the active site, it is proposed that the activated EH intermediate, suggested to be responsible for proton pumping in one of the reduction steps in the A family, cannot be involved in the catalytic cycle for cbb3, which results in the lack of proton pumping also in the E to R reduction step. In summary, the calculations indicate that only two protons are pumped per oxygen molecule in cbb3 oxidases. However, more experimental information on this divergent enzyme is needed, e.g. whether the flow of electrons resembles that in the other more well-studied CcO families.

Structure and redox properties of the diheme electron carrier cytochrome c4 from Pseudomonas aeruginosa

At low oxygen concentrations, respiration of Pseudomonas aeruginosa (Pa) and other bacteria relies on activity of cytochrome cbb3 oxidases. A diheme cytochrome c4 (cyt c4) donates electrons to Pa cbb3 oxidases to enable oxygen reduction and proton pumping by these enzymes. Given the importance of this redox pathway for bacterial pathogenesis, both cyt c4 and cbb3 oxidase are potential targets for new antibacterial strategies. The structural information about these two proteins, however, is scarce, and functional insights for Pa and other bacteria have been primarily drawn from analyses of the analogous system from Pseudomonas stutzeri (Ps). Herein, we describe characterization of structural and redox properties of cyt c4 from Pa. The crystal structure of Pa cyt c4 has revealed that this protein is organized in two monoheme domains. The interdomain interface is more hydrophobic in Pa cyt c4, and the protein surface does not show the dipolar distribution of charges found in Ps cyt c4. The reduction potentials of the two hemes are similar in Pa cyt c4 but differ by about 100 mV in Ps cyt c4. Analyses of structural models of these and other cyt c4 proteins suggest that multiple factors contribute to the potential difference of the two hemes in these proteins, including solvent accessibility of the heme group, the distribution of surface charges, and the nature of the interdomain interface. The distinct properties of cyt c4 proteins from closely-related Pa and Ps bacteria emphasize the importance of examining the cbb3/cyt c4 redox pathway in multiple species.

An Integrated Systems Approach Unveils New Aspects of Microoxia-Mediated Regulation in Bradyrhizobium diazoefficiens.

The adaptation of rhizobia from the free-living state in soil to the endosymbiotic state comprises several physiological changes in order to cope with the extremely low oxygen availability (microoxia) within nodules. To uncover cellular functions required for bacterial adaptation to microoxia directly at the protein level, we applied a systems biology approach on the key rhizobial model and soybean endosymbiont Bradyrhizobium diazoefficiens USDA 110 (formerly B. japonicum USDA 110). As a first step, the complete genome of B. diazoefficiens 110spc4, the model strain used in most prior functional genomics studies, was sequenced revealing a deletion of a ~202 kb fragment harboring 223 genes and several additional differences, compared to strain USDA 110. Importantly, the deletion strain showed no significantly different phenotype during symbiosis with several host plants, reinforcing the value of previous OMICS studies. We next performed shotgun proteomics and detected 2,900 and 2,826 proteins in oxically and microoxically grown cells, respectively, largely expanding our knowledge about the inventory of rhizobial proteins expressed in microoxia. A set of 62 proteins was significantly induced under microoxic conditions, including the two nitrogenase subunits NifDK, the nitrogenase reductase NifH, and several subunits of the high-affinity terminal cbb3 oxidase (FixNOQP) required for bacterial respiration inside nodules. Integration with the previously defined microoxia-induced transcriptome uncovered a set of 639 genes or proteins uniquely expressed in microoxia. Finally, besides providing proteogenomic evidence for novelties, we also identified proteins with a regulation similar to that of FixK2: transcript levels of these protein-coding genes were significantly induced, while the corresponding protein abundance remained unchanged or was down-regulated. This suggested that, apart from fixK2, additional B. diazoefficiens genes might be under microoxia-specific post-transcriptional control. This hypothesis was indeed confirmed for several targets (HemA, HemB, and ClpA) by immunoblot analysis.

The Bradyrhizobium diazoefficiens two-component system NtrYX has a key role in symbiotic nitrogen fixation of soybean plants and cbb3 oxidase expression in bacteroids

Bradyrhizobium diazoefficiens is a soil bacterium capable of establishing nitrogen-fixing symbiosis with soybean plants, an important crop for food production worldwide. This interaction is under the control of several regulatory factors that affect the bacterial efficiency to nodulate legume plants and fix nitrogen within root nodules. To date, the function of the NtrYX two-component system in B. diazoefficiens remains unclear. In this work, we investigated the role of NtrY in the symbiotic interaction of B. diazoefficiens with soybean plants. We constructed a non-polar mutant of the sensor protein NtrY of the system, and then analysed the symbiosis of this mutant with soybean plants. We found that the ntrY mutant was defective in plant dry weight, nitrogen content and nitrogenase activity. Haem c-staining of cbb3 oxidase components showed a clear reduction in the expression of this terminal oxidase in ntrY bacteroids with respect to wild type bacteroids. Such defect in cbb3 expression correlated with a decreased respiratory capacity of the bacteroids produced by the ntrY mutant. These results suggest that the role of B. diazoefficiens NtrYX in symbiotic nitrogen fixation might be a consequence of its involvement in cbb3 oxidase expression in bacteroids.

Gene expression of terminal oxidases in two marine bacterial strains exposed to nanomolar oxygen concentrations.

The final step of aerobic respiration is carried out by a terminal oxidase transporting electrons to oxygen (O2). Prokaryotes harbor diverse terminal oxidases that differ in phylogenetic origin, structure, biochemical function, and affinity for O2. Here we report on the expression of high-affinity (cytochrome cbb3 oxidase), low-affinity (cytochrome aa3 oxidase), and putative low-affinity (cyanide-insensitive oxidase (CIO)) terminal oxidases in the marine bacteria Idiomarina loihiensis L2-TR and Marinobacter daepoensis SW-156 upon transition to very low O2 concentrations (<200 nM), measured by RT-qPCR. In both strains, high-affinity cytochrome cbb3 oxidase showed the highest expression levels and was significantly up-regulated upon transition to low O2 concentrations. Low-affinity cytochrome aa3 oxidase showed very low transcription levels throughout the incubation. Surprisingly, however, it was also up-regulated upon transition to low O2 concentrations. In contrast, putative low-affinity CIO had much lower expression levels and markedly different regulation patterns between the two strains. These results demonstrate that exposure to low O2 concentrations regulates the gene expression of different types of terminal oxidases, but also that the type and magnitude of transcriptional response is species-dependent. Therefore, in situ transcriptome data cannot, without detailed knowledge of the transcriptional regulation of the species involved, be translated into relative respiratory activity.

Characterization of the Symbiotic Nitrogen-Fixing Common Bean Low Phytic Acid (lpa1) Mutant Response to Water Stress.

The common bean (Phaseolus vulgaris L.) low phytic acid (lpa1) biofortified genotype produces seeds with improved nutritional characteristics and does not display negative pleiotropic effects. Here we demonstrated that lpa1 plants establish an efficient nitrogen-fixing symbiosis with Rhizobium etli CE3. The lpa1 nodules showed a higher expression of nodule-function related genes than the nodules of the parental wild type genotype (BAT 93). We analyzed the response to water stress of lpa1 vs. BAT 93 plants grown under fertilized or under symbiotic N2-fixation conditions. Water stress was induced by water withholding (up to 14% soil moisture) to fertilized or R. etli nodulated plants previously grown with normal irrigation. The fertilized lpa1 plants showed milder water stress symptoms during the water deployment period and after the rehydration recovery period when lpa1 plants showed less biomass reduction. The symbiotic water-stressed lpa1 plants showed decreased nitrogenase activity that coincides with decreased sucrose synthase gene expression in nodules; lower turgor weight to dry weight (DW) ratio, which has been associated with higher drought resistance index; downregulation of carbon/nitrogen (C/N)-related and upregulation of stress-related genes. Higher expression of stress-related genes was also observed in bacteroids of stressed lpa1 plants that also displayed very high expression of the symbiotic cbb3 oxidase (fixNd).

Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model

The cbb3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus.

On the role of subunit M in cytochrome cbb3 oxidase

Cytochrome cbb3 (or C-type) oxidases are a highly divergent group and the least studied members of the heme-copper oxidases (HCOs) superfamily. HCOs couple the reduction of oxygen at the end of the respiratory chain to the active proton translocation across the membrane, contributing to establishment of an electrochemical gradient essential for ATP synthesis. Cbb3 oxidases exhibit unique structural and functional features and have an essential role in the metabolism of many clinically relevant human pathogens. Such characteristics make them a promising therapeutic target. Three subunits, N, O and P, comprise the core cbb3 complex, with N, the catalytic subunit, being highly conserved among all members of the HCO superfamily, including the A-type (aa3, mitochondrial-like) oxidases. An additional fourth subunit containing a single transmembrane (TM) helix was present in the first crystal structure of cbb3. This TM segment was recently proposed to be part of a novel protein CcoM, which was shown to have a putative role in the complex stability and assembly. In this work, we performed large-scale all-atom molecular dynamics simulations of the CcoNOPM complex to further characterize the interactions between subunit M and the core subunits and to determine whether the presence of the fourth subunit influences the water/proton channels previously described for the core complex. The previously proposed putative CcoNOPH complex is also assessed, and the potential functional redundancy of CcoM and CcoQ is discussed.

Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707.

Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs) using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(c)aa3 type oxidases (Caa3 and Ccaa3), two cbb3 type oxidases (Cbb31 and Cbb32), and one bd type cyanide-insensitive quinol oxidase (CIO). While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(c)aa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

Insights into proton translocation in cbb3 oxidase from MD simulations

Heme-copper oxidases are membrane protein complexes that catalyse the final step of the aerobic respiration, namely the reduction of oxygen to water. The energy released during catalysis is coupled to the active translocation of protons across the membrane, which contributes to the establishment of an electrochemical gradient that is used for ATP synthesis. The distinctive C-type (or cbb3) cytochrome c oxidases, which are mostly present in proteobacteria, exhibit a number of unique structural and functional features, including high catalytic activity at low oxygen concentrations. At the moment, the functioning mechanism of C-type oxidases, in particular the proton transfer/pumping mechanism presumably via a single proton channel, is still poorly understood. In this work we used all-atom molecular dynamics simulations and continuum electrostatics calculations to obtain atomic-level insights into the hydration and dynamics of a cbb3 oxidase. We provide the details of the water dynamics and proton transfer pathways for both the “chemical” and “pumped” protons, and show that formation of protonic connections is strongly affected by the protonation state of key residues, namely H243, E323 and H337.

The unusual redox properties of C-type oxidases

Cytochrome cbb3 (also known as C-type) oxidases belong to the family of heme-copper terminal oxidases which couple at the end of the respiratory chain the reduction of molecular oxygen into water and the pumping of protons across the membrane. They are expressed most often at low pressure of O2 and they exhibit a low homology of sequence with the cytochrome aa3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b3 associated with a CuB center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb3 oxidases from three organisms, Rhodobacter sphaeroides, Vibrio cholerae and Pseudomonas stutzeri by means of electrochemical and spectroscopic techniques. We show that the redox potential of the heme b3 exhibits a relatively low midpoint potential, as in related cytochrome c-dependent nitric oxide reductases. Potential implications for the coupled electron transfer and proton uptake mechanism of C-type oxidases are discussed.

Water dynamics and proton translocation in cytochrome cbb3 oxidase

Water dynamics and proton translocation in cytochrome cbb3 oxidase

The unusual redox properties of the cytochrome cbb3 oxidases from various organisms and their similarity to NO reductases

The unusual redox properties of the cytochrome cbb3 oxidases from various organisms and their similarity to NO reductases

Reduced expression of cytochrome oxidases largely explains cAMP inhibition of aerobic growth in Shewanella oneidensis

Inhibition of bacterial growth under aerobic conditions by elevated levels of cyclic adenosine 3′,5′-monophosphate (cAMP), first revealed more than 50 years ago, was attributed to accumulation of toxic methylglyoxal (MG). Here, we report a Crp-dependent mechanism rather than MG accumulation that accounts for the phenotype in Shewanella oneidensis, an emerging research model for the bacterial physiology. We show that a similar phenotype can be obtained by removing CpdA, a cAMP phosphodiesterase that appears more effective than its Escherichia coli counterpart. Although production of heme c and cytochromes c is correlated well with cAMP levels, neither is sufficient for the retarded growth. Quantities of overall cytochromes c increased substantially in the presence of elevated cAMP, a phenomenon resembling cells respiring on non-oxygen electron acceptors. In contrast, transcription of Crp-dependent genes encoding both cytochromes bd and cbb3 oxidases is substantially repressed under the same condition. Overall, our results suggest that cAMP of elevated levels drives cells into a low-energetic status, under which aerobic respiration is inhibited.