Rubredoxin-oxygen Oxidoreductase Research Articles

Role of NorR-like transcriptional regulators under nitrosative stress of the δ-proteobacterium, Desulfovibrio gigas

NorR protein was shown to be responsible for the transcriptional regulation of flavorubredoxin and its associated oxidoreductase in Escherichia coli. Since Desulfovibrio gigas has a rubredoxin:oxygen oxidoreductase (ROO) that is involved in both oxidative and nitrosative stress response, a NorR-like protein was searched in D. gigas genome. We have found two putative norR coding units in its genome. To study the role of the protein designated as NorR1-like (NorR1L) in the presence of nitrosative stress, a norR1L null mutant of D. gigas was created and a phenotypic analysis was performed under the nitrosating agent GSNO. We show that under these conditions, the growth of both D. gigas mutants Δroo and ΔnorR1-like is impaired. In order to confirm that D. gigas NorR1-like may play identical function as the NorR of E. coli, we have complemented the E. coli ΔnorR mutant strain with the norR1-like gene and have evaluated growth when nitrosative stress was imposed. The growth phenotype of E. coli ΔnorR mutant strain was recovered under these conditions. We also found that induction of roo gene expression is completely abolished in the norR1L mutant strain of D. gigas subjected to nitrosative stress. It is identified in δ-proteobacteria, for the first time a transcription factor that is involved in nitrosative stress response and regulates the rd-roo gene expression.

Oxygen uptake rates in the hyperthermophilic anaerobe Thermotoga maritima grown in a bioreactor under controlled oxygen exposure: clues to its defence strategy against oxidative stress

A 2.3-L bioreactor was specially adapted to grow hyperthermophilic microorganisms under controlled conditions of temperature, pH, redox potential and dissolved O(2). Using this bioreactor regulated at 80°C and pH 7.0, we demonstrated that Thermotoga maritima recovered its growth despite being exposed to oxygen for a short time (30min with a maximum concentration of 23μM of dissolved oxygen). Under these conditions, we demonstrated that O(2) uptake rate, estimated at 73.6μmoles O(2)min(-1)g proteins(-1) for dissolved oxygen, was optimal and constant, when dissolved oxygen was present in a range of 22-5μM. Transcription analyses revealed that during short oxygen exposure, T. maritima expressed genes coding for enzymes to deal with O(2) and reactive oxygen species (ROS) such as peroxides. Thus, genes encoding ROS-scavenging systems, alkyl hydroperoxide reductase (ahp), thioredoxin-dependent thiol peroxidase (bcp 2) and to a lesser extent neelaredoxin (nlr) and rubrerythrin (rbr), were found to be upregulated during oxygen exposure. The oxygen reductase FprA, homologous to the rubredoxin-oxygen oxidoreductase (ROO) found in Desulfovibrio species, is proposed as a primary consumer of O(2) in T. maritima. Moreover, the expression of frpA was shown to depend on the redox (Eh) level of the culture medium.

Multiple antioxidant proteins protect Chlorobaculum tepidum against oxygen and reactive oxygen species

The genome of the green sulfur bacterium Chlorobaculum (Cba.) tepidum, a strictly anaerobic photolithoautotroph, is predicted to encode more than ten genes whose products are potentially involved in protection from reactive oxygen species and an oxidative stress response. The encoded proteins include cytochrome bd quinol oxidase, NADH oxidase, rubredoxin oxygen oxidoreductase, several thiol peroxidases, alkyl hydroperoxide reductase, superoxide dismutase, methionine sulfoxide reductase, and rubrerythrin. To test the physiological functions of some of these proteins, ten genes were insertionally inactivated. Wild-type Cba. tepidum cells were very sensitive to oxygen in the light but were remarkably resistant to oxygen in the dark. When wild-type and mutant cells were subjected to air for various times under dark or light condition, significant decreases in viability were detected in most of the mutants relative to wild type. Treatments with hydrogen peroxide (H(2)O(2)), tert-butyl hydroperoxide (t-BOOH) and methyl viologen resulted in more severe effects in most of the mutants than in the wild type. The results demonstrated that these putative antioxidant proteins combine to form an effective defense against oxygen and reactive oxygen species. Reverse-transcriptase polymerase chain reaction studies showed that the genes with functions in oxidative stress protection were constitutively transcribed under anoxic growth conditions.

Dioxygen and nitric oxide pathways and affinity to the catalytic site of rubredoxin:oxygen oxidoreductase from Desulfovibrio gigas

Rubredoxin:oxygen oxidoreductase (ROO) is the terminal oxidase of a soluble electron transfer chain found in Desulfovibrio gigas. This protein belongs to the flavodiiron family and was initially described as an oxygen reductase, converting this substrate to water and acting as an oxygen-detoxifying system. However, more recent studies evidenced also the ability for this protein to act as a nitric oxide reductase, suggesting an alternative physiological role. To clarify the apparent bifunctional nature of this protein, we performed molecular dynamics simulations of the protein, in different redox states, together with O(2) and NO molecules in aqueous solution. The two small molecules were parameterized using free-energy calculations of the hydration process. With these simulations we were able to identify specific protein paths that allow the diffusion of both these molecules through the protein towards the catalytic centers. Also, we have tried to characterize the preference of ROO towards the presence of O(2) and/or NO at the active site. By using free-energy simulations, we did not find any significant preference for ROO to accommodate both O(2) and NO. Also, from our molecular dynamics simulations we were able to identify similar diffusion profiles for both O(2) and NO molecules. These two conclusions are in good agreement with previous experimental works stating that ROO is able to catalyze both O(2) and NO.

The hyperthermophilic anaerobe Thermotoga Maritima is able to cope with limited amount of oxygen: insights into its defence strategies

Thermotoga maritima, an anaerobic hyperthermophilic bacterium, was found able to grow in the presence of low concentrations of oxygen of up to 0.5% (v/v). Differential proteomics and transcripts analysis by qRT-PCR were used to identify the defence strategies used by T. maritima to protect itself against oxygen. A flavoprotein, homologous to rubredoxin oxygen oxidoreductase was found to be overproduced when cells were cultured in oxidative conditions. The recombinant protein, produced in Escherichia coli, exhibited an oxygen reductase activity, which could account for the observed decrease in oxygen concentration during growth. The gene encoding this oxygen reductase belongs to a multicistronic unit that includes genes encoding proteins involved in exopolysaccharide biosynthesis, which may be related to a biofilm formation induced by the presence of oxygen. Enzymes involved in reactive oxygen species detoxification, iron-sulfur centre synthesis/repair and the cysteine biosynthesis pathway were also overproduced. All these enzymatic systems together contribute to the defence strategy of T. maritima against oxygen. Because of the position of T. maritima in deep branches of the phylogenetic tree, we suggest that these strategies can be considered as ancestral mechanisms first developed by anaerobic microorganisms on the early Earth to protect themselves against primary abiotic or biotic oxygen production.

Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 Å resolution

Rubredoxin ( D. g. Rd) is a small non-heme iron–sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D. g. The crystal structure of D. g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 Å resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and π–π interactions maintain the internal Rd folding. Multiple conformations of the iron–sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N H⋯SG of the iron–sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron–sulfur cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium.

Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O(2). Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Deltaroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O(2) reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

Update of the standard numbering scheme for class B beta-lactamases.

Update of the standard numbering scheme for class B beta-lactamases.

Docking and electron transfer studies between rubredoxin and rubredoxin:oxygen oxidoreductase.

The interaction and electron transfer (ET) between rubredoxin (Rd) and rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two proteins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals interactions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non-polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed.

Analysis of the Desulfovibrio gigas Transcriptional Unit Containing Rubredoxin (rd) and Rubredoxin-Oxygen Oxidoreductase (roo) Genes and Upstream ORFs

Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment of D. gigas chromosomal DNA containing the roo gene and the rubredoxin gene was sequenced. Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of rubredoxin. The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative −10 and −35 regulator regions of a ς70-type promoter, having similarity with E. coli ς70 promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study.

Paper Alert

Paper Alert

Structure of a dioxygen reduction enzyme from Desulfovibrio gigas.

Desulfovibrio gigas is a strict anaerobe that contains a well-characterized metabolic pathway that enables it to survive transient contacts with oxygen. The terminal enzyme in this pathway, rubredoxin:oxygen oxidoreductase (ROO) reduces oxygen to water in a direct and safe way. The 2.5 A resolution crystal structure of ROO shows that each monomer of this homodimeric enzyme consists of a novel combination of two domains, a flavodoxin-like domain and a Zn-beta-lactamase-like domain that contains a di-iron center for dioxygen reduction. This is the first structure of a member of a superfamily of enzymes widespread in strict and facultative anaerobes, indicating its broad physiological significance.

A DNA Fragment ofDesulfovibrio gigasGenome Containing Replication Origin Related Genes

The nucleotide sequence of a 10,772 base pair (bp) region from Desulfovibrio gigas genome was determined. This sequence, which is adjacent to the region containing the coding units for the metalloproteins rubredoxin-oxygen oxidoreductase (ROO) and rubredoxin, includes the flavodoxin gene. Additionally, it also contains four open reading frames (ORFs) related to genes frequently found in replication origin regions of prokaryotes. These hypothetical encoded polypeptides are: the response regulator proteins (PhoP and PhoR) from the phosphate regulon, a DNA partitioning protein and an asparagine synthetase.

Crystallization and preliminary diffraction data analysis of both single and pseudo-merohedrally twinned crystals of rubredoxin oxygen oxidoreductase from Desulfovibrio gigas.

Crystals of rubredoxin oxygen oxidoreductase have been obtained and characterized. They belong to space group P2(1)2(1)2, with unit-cell dimensions a = 88.24 (15), b = 101.25 (7), c = 90.80 (3) A. The homodimer (86 kDa) in the asymmetric unit is related by a non-crystallographic twofold rotation axis parallel to the ab 'diagonal' direction, as shown by the self-rotation maximum in the section with chi = 180 degrees. This pseudo-crystallographic symmetry element was also found to be the twinning axis of pseudo-merohedrally twinned crystals, leading to apparent pseudo-tetragonal P42(1)2 crystal symmetry.

Studies on the Redox Centers of the Terminal Oxidase fromDesulfovibrio gigas and Evidence for Its Interaction with Rubredoxin

Rubredoxin-oxygen oxidoreductase (ROO) is the final component of a soluble electron transfer chain that couples NADH oxidation to oxygen consumption in the anaerobic sulfate reducer Desulfovibrio gigas. It is an 86-kDa homodimeric flavohemeprotein containing two FAD molecules, one mesoheme IX, and one Fe-uroporphyrin I per monomer, capable of fully reducing oxygen to water. EPR studies on the native enzyme reveal two components with g values at approximately 2.46, 2.29, and 1.89, which are assigned to low spin hemes and are similar to the EPR features of P-450 hemes, suggesting that ROO hemes have a cysteinyl axial ligation. At pH 7.6, the flavin redox transitions occur at 0 +/- 15 mV for the quinone/semiquinone couple and at -130 +/- 15 mV for the semiquinone/hydroquinone couple; the hemes reduction potential is -350 +/- 15 mV. Spectroscopic studies provided unequivocal evidence that the flavins are the electron acceptor centers from rubredoxin, and that their reduction proceed through an anionic semiquinone radical. The reaction with oxygen occurs in the flavin moiety. These data are strongly corroborated by the finding that rubredoxin and ROO are located in the same polycistronic unit of D. gigas genome. For the first time, a clear role for a rubredoxin in a sulfate-reducing bacterium is presented.

Iron Uroporphyrin I and a Heme c-Derivative Are Prosthetic Groups in Desulfovibrio gigas Rubredoxin Oxidase 1

Rubredoxin-oxygen oxidoreductase (ROO) from the sulfate-reducing bacterium Desulfovibrio gigas, is an unusual terminal oxidase capable of reducing molecular O 2 to water. The nature of its heme prosthetic groups has been investigated. It was found to contain two distinct hemes. One is Fe uroporphyrin I. The second is closely related to heme c. It is an Fe protoporphyrin IX where two electrophilic groups have added covalently across the double-bonds of the former 3,8 vinyl groups (IUB-IUPAC Tetrapyrrole numbering). These covalent attachment bonds are not to the main polypeptide chain, but instead are to an unidentified component. This component is soluble in acidic acetone, but cannot be extracted into organic solvents that typically solubilize free heme groups. Preliminary evidence suggests that the covalent bonds are not thioether bonds, but they are acid labile.

Rubredoxin Oxidase, a New Flavo-Hemo-Protein, Is the Site of Oxygen Reduction to Water by the "Strict Anaerobe" Desulfovibrio gigas

A rubredoxin-oxygen oxidoreductase, a homodimer with a molecular weight of 43 kDa per monomer, was found to be a component of an electron transfer chain that couples the reduction of oxygen to water with NADH oxidation. This FAD-containing protein appears to contain a new type of heme group. The electron transfer chain is not inhibited by cyanide and azide. In contrast, CO decreases NADH oxidation rate and also induces release of the prosthetic groups from the native terminal reductase.