Supramolecular organization of bacterial aerobic respiratory chains: From cells and back.

Aerobic respiratory chains from all life kingdoms are composed by several complexes that have been deeply characterized in their isolated form. These membranous complexes link the oxidation of reducing substrates to the reduction of molecular oxygen, in a process that conserves energy by ion translocation between both sides of the mitochondrial or prokaryotic cytoplasmatic membranes. In recent years there has been increasing evidence that those complexes are organized as supramolecular structures, the so-called supercomplexes and respirasomes, being available for eukaryotes strong data namely obtained by electron microscopy and single particle analysis. A parallel study has been developed for prokaryotes, based on blue native gels and mass spectrometry analysis, showing that in these more simple unicellular organisms such supercomplexes also exist, involving not only typical aerobic-respiration associated complexes, but also anaerobic-linked enzymes. After a short overview of the data on eukaryotic supercomplexes, we will analyse comprehensively the different types of prokaryotic aerobic respiratory supercomplexes that have been thus far suggested, in both bacteria and archaea. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Prof Conrad Mullineaux.

The bc:caa3 supercomplexes from the Gram positive bacterium Bacillus subtilis respiratory chain: a megacomplex organization?

The respiratory chain of some prokaryotes was shown to be organized in supercomplexes. This association has been proposed to improve enzyme stability and the overall efficiency of the oxidative phosphorylation process. Here, we have revisited recent data on the supercomplexes of Bacillus subtilis respiratory chain, by means of 1D and 2D-BN-PAGE, sucrose gradient fractionation of solubilized membranes, and mass spectrometry analysis of BN-PAGE bands detected in gel for succinate and cytochrome c oxidoreductase activities. The cytochrome bc:caa3 oxygen oxidoreductase supercomplex was observed in different stoichiometries, (bc)4:(caa3)2, (bc)2:(caa3)4 and 2[(bc)2:(caa3)4], suggesting for the first time the string association model of supercomplexes in a Gram positive bacterium. In addition, the presence of a succinate:quinone oxidoreductase:nitrate reductase supercomplex was confirmed by the co-localized succinate:nitroblue tetrazolium and methylviologen:nitrate oxidoreductase activities detected in gel and corroborated by LC-MS/MS analysis.

The novel NhaE-type Na(+)/H (+) antiporter of the pathogenic bacterium Neisseria meningitidis.

Neisseria meningitidis is a pathogenic bacterium responsible for meningitis. The mechanisms underlying the control of Na(+) transmembrane movement, presumably important to pathogenicity, have been barely addressed. To elucidate the function of the components of the Na(+) transport system in N. meningitidis, an open reading frame from the genome of this bacterium displaying similarity with the NhaE type of Na(+)/H(+) antiporters was expressed in Escherichia coli and characterized for sodium transport ability. The N. meningitidis antiporter (NmNhaE) was able to complement an E. coli strain devoid of Na(+)/H(+) antiporters (KNabc) respecting the ability to grow in the presence of NaCl and LiCl. Ion transport assays in everted vesicles prepared from KNabc expressing NmNhaE from a plasmid confirmed its ability to translocate Na(+) and Li(+). Here is presented the characterization of the first NhaE from a pathogen, an important contribution to the comprehension of sodium ion metabolism in this kind of microorganisms.

Oxidative stress modulates the nitric oxide defense promoted by Escherichia coli flavorubredoxin.

Mammalian cells of innate immunity respond to pathogen invasion by activating proteins that generate a burst of oxidative and nitrosative stress. Pathogens defend themselves from the toxic compounds by triggering a variety of detoxifying enzymes. Escherichia coli flavorubredoxin is a nitric oxide reductase that is expressed under nitrosative stress conditions. We report that in contrast to nitrosative stress alone, exposure to both nitrosative and oxidative stresses abolishes the expression of flavorubredoxin. Electron paramagnetic resonance (EPR) experiments showed that under these conditions, the iron center of the flavorubredoxin transcription activator NorR loses the ability to bind nitric oxide. Accordingly, triggering of the NorR ATPase activity, a requisite for flavorubredoxin activation, was impaired by treatment of the protein with the double stress. Studies of macrophages revealed that the contribution of flavorubredoxin to the survival of E. coli depends on the stage of macrophage infection and that the lack of protection observed at the early phase is related to inhibition of NorR activity by the oxidative burst. We propose that the time-dependent activation of flavorubredoxin contributes to the adaptation of E. coli to the different fluxes of hydrogen peroxide and nitric oxide to which the bacterium is subjected during the course of macrophage infection.

The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes.

In spite of the large number of reports on the aerobic respiratory chain of Escherichia coli, from gene transcription regulation to enzyme kinetics and structural studies, an integrative perspective of this pathway is yet to be produced. Here, a multi-level analysis of the aerobic respiratory chain of E. coli was performed to find correlations between gene transcription, enzyme activity, growth dynamics, and supercomplex formation and composition. The transcription level of all genes encoding the aerobic respiratory chain of E. coli varied significantly in response to bacterial growth. Coordinated expression patterns were observed between the genes encoding NADH : quinone oxidoreductase and complex I (NDH-1), alternative NADH : quinone oxidoreductase (NDH-2) and cytochrome bdI, and also between sdhA and appC, encoding succinate dehydrogenase and cytochrome bdII, respectively. In general, the rates of the respiratory chain activities increased from mid-exponential to late-stationary phase, with no significant further variation occurring until the mid-stationary phase. Multi-level correlations between gene transcription, enzyme activity and growth dynamics were also found in this study. The previously reported NADH dehydrogenase and formate : oxygen oxidoreductase supercomplexes of E. coli were already assembled at mid-exponential phase and remained throughout growth. A new succinate oxidase supercomplex composed of succinate dehydrogenase and cytochrome bdII was identified, in agreement with the suggestion provided by the coordinated transcription of sdhA and appC.

Making European performance and impact assessment frameworks for research infrastructures glocal.

Sustainability of research infrastructures (RIs) is a big challenge for funders, stakeholders and operators, and the development and adoption of adequate management tools is a major concern, namely tools for monitoring and evaluating their performance and impact. BioData.pt is the Portuguese Infrastructure of Biological and Portuguese node of the European Strategy Forum on Research Infrastructures "Landmark" ELIXIR. The foundations of this national research infrastructure were laid under the "Building BioData.pt" project, for four years. During this period, performance and impact indicators were collected and analysed under the light of international guidelines for assessing the performance and impact of European research infrastructures produced by the European Strategy Forum on Research Infrastructures, the Organisation for Economic Co-operation and Development and the EU-funded RI-PATHS project. The exercise shared herein showed that these frameworks can be adopted by national RIs, with the necessary adaptations, namely to reflect the national landscape and specificity of activities, and can be powerful tools in supporting the management of RIs. "Not everything that counts can be counted, and not everything that can be counted, counts". Albert Einstein, Theoretical physicist and Nobel Prize winner.

Vitamin D-related polymorphisms and vitamin D levels as risk biomarkers of COVID-19 disease severity.

Vitamin D is a fundamental regulator of host defences by activating genes related to innate and adaptive immunity. Previous research shows a correlation between the levels of vitamin D in patients infected with SARS-CoV-2 and the degree of disease severity. This work investigates the impact of the genetic background related to vitamin D pathways on COVID-19 severity. For the first time, the Portuguese population was characterized regarding the prevalence of high impact variants in genes associated with the vitamin D pathways. This study enrolled 517 patients admitted to two tertiary Portuguese hospitals. The serum concentration of 25 (OH)D, was measured in the hospital at the time of patient admission. Genetic variants, 18 variants, in the genes AMDHD1, CYP2R1, CYP24A1, DHCR7, GC, SEC23A, and VDR were analysed. The results show that polymorphisms in the vitamin D binding protein encoded by the GC gene are related to the infection severity (p = 0.005). There is an association between vitamin D polygenic risk score and the serum concentration of 25 (OH)D (p = 0.04). There is an association between 25 (OH)D levels and the survival and fatal outcomes (p = 1.5e-4). The Portuguese population has a higher prevalence of the DHCR7 RS12785878 variant when compared with its prevalence in the European population (19% versus 10%). This study shows a genetic susceptibility for vitamin D deficiency that might explain higher severity degrees in COVID-19 patients. These results reinforce the relevance of personalized strategies in the context of viral diseases.Trial registration: NCT04370808.

Structure at 1.0 A resolution of a high-potential iron-sulfur protein involved in the aerobic respiratory chain of Rhodothermus marinus.

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus, a nonphotosynthetic organism from the Bacteroidetes/Chlorobi group, contains a high-potential iron-sulfur protein (HiPIP) that transfers electrons from a bc 1 analog complex to a caa 3 oxygen reductase. Here, we describe the crystal structure of the reduced form of R. marinus HiPIP, solved by the single-wavelength anomalous diffraction method, based on the anomalous scattering of the iron atoms from the [4Fe-4S]3+/2+ cluster and refined to 1.0 A resolution. This is the first structure of a HiPIP isolated from a nonphotosynthetic bacterium involved in an aerobic respiratory chain. The structure shows a similar environment around the cluster as the other HiPIPs from phototrophic bacteria, but reveals several features distinct from those of the other HiPIPs of phototrophic bacteria, such as a different fold of the N-terminal region of the polypeptide due to a disulfide bridge and a ten-residue-long insertion.

A novel nitroreductase of Staphylococcus aureus with S-nitrosoglutathione reductase activity.

In this report we show that inactivation of the putative nitroreductase SA0UHSC_00833 (ntrA) increases the sensitivity of Staphylococcus aureus to S-nitrosoglutathione (GSNO) and augments its resistance to nitrofurans. S. aureus NtrA is a bifunctional enzyme that exhibits nitroreductase and GSNO reductase activity. A phylogenetic analysis suggests that NtrA is a member of a novel family of nitroreductases that seems to play a dual role in vivo, promoting nitrofuran activation and protecting the cell against transnitrosylation.

New insights into type II NAD(P)H:quinone oxidoreductases.

Type II NAD(P)H:quinone oxidoreductases (NDH-2) catalyze the two-electron transfer from NAD(P)H to quinones, without any energy-transducing site. NDH-2 accomplish the turnover of NAD(P)H, regenerating the NAD(P)(+) pool, and may contribute to the generation of a membrane potential through complexes III and IV. These enzymes are usually constituted by a nontransmembrane polypeptide chain of approximately 50 kDa, containing a flavin moiety. There are a few compounds that can prevent their activity, but so far no general specific inhibitor has been assigned to these enzymes. However, they have the common feature of being resistant to the complex I classical inhibitors rotenone, capsaicin, and piericidin A. NDH-2 have particular relevance in yeasts like Saccharomyces cerevisiae and in several prokaryotes, whose respiratory chains are devoid of complex I, in which NDH-2 keep the balance and are the main entry point of electrons into the respiratory chains. Our knowledge of these proteins has expanded in the past decade, as a result of contributions at the biochemical level and the sequencing of the genomes from several organisms. The latter showed that most organisms contain genes that potentially encode NDH-2. An overview of this development is presented, with special emphasis on microbial enzymes and on the identification of three subfamilies of NDH-2.

Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers.

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

A novel type of monoheme cytochrome c: biochemical and structural characterization at 1.23 A resolution of rhodothermus marinus cytochrome c.

Monoheme cytochromes of the C-type are involved in a large number of electron transfer processes, which play an essential role in multiple pathways, such as respiratory chains, either aerobic or anaerobic, and the photosynthetic electron transport chains. This study reports the biochemical characterization and the crystallographic structure, at 1.23 A resolution, of a monoheme cytochrome c from the thermohalophilic bacterium Rhodothermus marinus. In addition to an alpha-helical core folded around the heme, common for this type of cytochrome, the X-ray structure reveals one unusual alpha-helix and a unique N-terminal extension, which wraps around the back of the molecule. Based on a thorough structural and amino acid sequence comparison, we propose R. marinus cytochrome c as the first characterized member of a new class of C-type cytochromes.

The anaerobe Desulfovibrio desulfuricans ATCC 27774 grows at nearly atmospheric oxygen levels.

Sulfate reducing bacteria of the Desulfovibrio genus are considered anaerobes, in spite of the fact that they are frequently isolated close to oxic habitats. However, until now, growth in the presence of high concentrations of oxygen was not reported for members of this genus. This work shows for the first time that the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is able to grow in the presence of nearly atmospheric oxygen levels. In addition, the activity and expression profile of several key enzymes was analyzed under different oxygen concentrations.

A ba3 oxygen reductase from the thermohalophilic bacterium Rhodothermus marinus.

The aerobic respiratory chain of the thermohalophilic bacterium Rhodothermus marinus has been extensively studied. In this study the isolation and characterization of a third oxygen reductase expressed in this organism are described. This newly isolated enzyme is a typical member of the type B family of haem-copper oxygen reductases, showing 43% amino acid sequence identity and 63% similarity with the ba3 oxygen reductase from Thermus thermophilus. It constitutes two subunits with apparent molecular masses of 42 and 38 kDa. It contains a low-spin B-type haem and a high-spin A-type haem. A stoichiometry of 1B: 1A haem per protein was obtained by spectral integration of UV-visible spectra. Metal analysis showed the presence of two iron and three copper ions, which is in agreement with the existence of a CuA centre. Taking advantage of having two spectroscopically distinct haems, the redox behaviour of the ba3 oxygen reductase was analysed and discussed in the framework of a model with interacting centres. Both haems, B and A, present two transitions, have unusually low reduction potentials of -65 mV and an interaction potential of -52.5 mV.

Crystallization and X-ray analysis of Rhodothermus marinus cytochrome c at 1.23 A resolution.

Cytochrome c from Rhodothermus marinus has been crystallized using the hanging-drop vapor-diffusion method in 30 % (w/v) polyethylene glycol 8K, 0.2 M ammonium sulfate, 8 % hexanediol and 50 mM sodium citrate pH 2.2. The crystals belong to space group P2(1). X-ray diffraction data were collected to 1.23 A resolution using synchrotron radiation and a wavelength of 0.93 A.

A nhaD Na+/H+ antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes.

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.

Structure at 1.3 A resolution of Rhodothermus marinus caa(3) cytochrome c domain.

The cytochrome c domain of subunit II from the Rhodothermus marinus caa(3) HiPIP:oxygen oxidoreductase, a member of the superfamily of heme-copper-containing terminal oxidases, was produced in Escherichia coli and characterised. The recombinant protein, which shows the same optical absorption and redox properties as the corresponding domain in the holo enzyme, was crystallized and its structure was determined to a resolution of 1.3 A by the multiwavelength anomalous dispersion (MAD) technique using the anomalous dispersion of the heme iron atom. The model was refined to final R(cryst) and R(free) values of 13.9% and 16.7%, respectively. The structure reveals the insertion of two short antiparallel beta-strands forming a small beta-sheet, an interesting variation of the classical all alpha-helical cytochrome c fold. This modification appears to be common to all known caa(3)-type terminal oxidases, as judged by comparative modelling and by analyses of the available amino acid sequences for these enzymes. This is the first high-resolution crystal structure reported for a cytochrome c domain of a caa(3)-type terminal oxidase. The R.marinus caa(3) uses HiPIP as the redox partner. The calculation of the electrostatic potential at the molecular surface of this extra C-terminal domain provides insights into the binding to its redox partner on one side and its interaction with the remaining subunit II on the other side.

A ferredoxin from the thermohalophilic bacterium Rhodothermus marinus.

A [3Fe-4S](1+/0) ferredoxin was isolated from the thermohalophilic and strict aerobic bacterium Rhodothermus marinus. It is a small protein, with an apparent molecular mass of 9 kDa. Its N-terminal amino acid sequence reveals the capability of binding two tetranuclear clusters. However, upon purification, it contains a single [3Fe-4S](1+/0), with an unusually low reduction potential of -650 mV, determined by cyclic voltammetry at pH 7.6. [1H]NMR spectroscopy shows that the protein contains a single, homogeneous, trinuclear centre. When purified under anaerobic conditions, the EPR [3Fe-4S](1+/0) centre signal is also observed. However, it can now be reduced by dithionite and a new signal attributed to a [4Fe-4S](2+/1+) cluster develops. This can also be observed upon reconstitution of the prosthetic groups. The function of this ferredoxin in R. marinus is still unknown but it is very sensitive to oxygen, an unexpected characteristic for a protein from an aerobic organism. The thermodynamic stability of the R. marinus ferredoxin was also investigated and was shown to be high. Thermal and chemical unfolding reactions appear as single, cooperative transitions. The midpoint (T(m)) for thermally induced unfolding is 102+/-2 degrees C (pH 7). Unfolding induced by the chemical denaturant guanidine hydrochloride (GuHCl) shows a transition midpoint at 5.0 M GuHCl (pH 7.0, 20 degrees C). The iron-sulfur cluster degrades upon polypeptide unfolding, resulting in an irreversible denaturation process.

The formate:oxygen oxidoreductase supercomplex of Escherichia coli aerobic respiratory chain.

The Escherichia coli formate:oxygen oxidoreductase supercomplex (FdOx) was investigated with respect to function and composition. Formate oxidoreductase activity was detected in blue native polyacrylamide gel electrophoresis (BN-PAGE) resolved membranes of E. coli, which were also capable of cyanide sensitive formate:oxygen oxidoreductase activity. The latter was compromised in strains devoid of specific oxygen reductases, particularly, in those devoid of cytochrome bo3 or bdI. A principal component analysis (PCA) integrating E. coli aerobic respiratory chain gene transcription, enzyme activity and growth dynamics was performed, correlating formate:oxygen oxidoreductase activity and the transcription of the genes encoding cytochromes bo3 and bdI, and corroborating previous evidence that associated these complexes in FdOx.

Supramolecular organizations in the aerobic respiratory chain of Escherichia coli.

The organization of respiratory chain complexes in supercomplexes has been shown in the mitochondria of several eukaryotes and in the cell membranes of some bacteria. These supercomplexes are suggested to be important for oxidative phosphorylation efficiency and to prevent the formation of reactive oxygen species. Here we describe, for the first time, the identification of supramolecular organizations in the aerobic respiratory chain of Escherichia coli, including a trimer of succinate dehydrogenase. Furthermore, two heterooligomerizations have been shown: one resulting from the association of the NADH:quinone oxidoreductases NDH-1 and NDH-2, and another composed by the cytochrome bo(3) quinol:oxygen reductase, cytochrome bd quinol:oxygen reductase and formate dehydrogenase (fdo). These results are supported by blue native-electrophoresis, mass spectrometry and kinetic data of wild type and mutant E . coli strains.