Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k _app values, 176.9±25.0 min⁻¹ in the case of the Tp/Tp electron transfer, 31.8±3.6 min⁻¹ for the Dg/Dg electron transfer, and 6.9±1.3 min⁻¹ for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron–sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k _app value of 108.8±12.0 min⁻¹, and was then assigned as the potential physiological electron donor in this organism.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

An iron tetrahydroporphyrin prosthetic group common to both assimilatory and dissimilatory sulfite reductases

The heme² chromophore of the “assimilatory” E. coli sulfite reductase is an iron-octacarboxylic tetrahydroporphyrin of the isobacteriochlorin type (1). Although the two “dissimilatory” sulfite reductases, desulfoviridin and desulforubidin, from the sulfate reducing bacteria Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain), have absorption spectra and reaction products which differ from those of E. coli sulfite reductase, the present studies indicate that they contain prosthetic groups with an organic structure closely similar or identical to that of the E. coli sulfite reductase heme. EPR spectra show high-spin ferriheme in all three enzymes. It is clear, however, that the prosthetic groups must reside in substantially different environments within their respective proteins.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Sco proteins are involved in electron transfer processes

Sco proteins are widespread in eukaryotic and in many prokaryotic organisms. They have a thioredoxin-like fold and bind a single copper(I) or copper(II) ion through a CXXXC motif and a conserved His ligand, with both tight and weak affinities. They have been implicated in the assembly of the Cu_A site of cytochrome c oxidase as copper chaperones and/or thioredoxins. In this work we have structurally characterized a Sco domain which is naturally fused with a typical electron transfer molecule, i.e., cytochrome c, in Pseudomonas putida. The thioredoxin-like Sco domain does not bind copper(II), binds copper(I) with weak affinity without involving the conserved His, and has redox properties consisting of a thioredoxin activity and of the ability of reducing copper(II) to copper(I), and iron(III) to iron(II) of the cytochrome c domain. These findings indicate that the His ligand coordination is the discriminating factor for introducing a metallochaperone function in a thioredoxin-like fold, typically responsible for electron transfer processes. A comparative structural analysis of the Sco domain from P. putida versus eukaryotic Sco proteins revealed structural determinants affecting the formation of a tight-affinity versus a weak-affinity copper binding site in Sco proteins.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c(3) is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c(3). This work presents the NMR assignment of the haem substituents in type I cytochrome c(3) isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c(3) belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

An assessment of growth yields and energy coupling inDesulfovibrio

The yield coefficients forDesulfovibrio vulgaris andD. gigas varied with the electron donoracceptor combinations and with the bacterial strain. The only evidence for electron transport coupled formation of adenosine triphosphate (ATP) was with sulfate as the electron acceptor. WithD. vulgaris the ATP formation coupling to electron flow with pyruvate oxidation was 1:4 electrons and with lactate oxidation was 1:8 electrons. WithD. gigas these ratios were 1:8 electrons and 1:16 electrons for the oxidation of pyruvate and lactate. The clearest resolution of energy coupling was withD. vulgaris growing on formatesulfate medium where 2 ATP appear to be formed with the transfer of electrons from formate to adenosine phosphosulfate and one ATP with the transfer of electrons from formate to sulfite.     

Electrochemical and structural characterization of Azotobacter vinelandii flavodoxin II

Azotobacter vinelandii flavodoxin II serves as a physiological reductant of nitrogenase, the enzyme system mediating biological nitrogen fixation. Wildtype A. vinelandii flavodoxin II was electrochemically and crystallographically characterized to better understand the molecular basis for this functional role. The redox properties were monitored on surfactant‐modified basal plane graphite electrodes, with two distinct redox couples measured by cyclic voltammetry corresponding to reduction potentials of ?483 ± 1 mV and ?187 ± 9 mV (vs. NHE) in 50 mM potassium phosphate, 150 mM NaCl, pH 7.5. These redox potentials were assigned as the semiquinone/hydroquinone couple and the quinone/semiquinone couple, respectively. This study constitutes one of the first applications of surfactant‐modified basal plane graphite electrodes to characterize the redox properties of a flavodoxin, thus providing a novel electrochemical method to study this class of protein. The X‐ray crystal structure of the flavodoxin purified from A. vinelandii was solved at 1.17 Å resolution. With this structure, the native nitrogenase electron transfer proteins have all been structurally characterized. Docking studies indicate that a common binding site surrounding the Fe‐protein [4Fe:4S] cluster mediates complex formation with the redox partners Mo‐Fe protein, ferredoxin I, and flavodoxin II. This model supports a mechanistic hypothesis that electron transfer reactions between the Fe‐protein and its redox partners are mutually exclusive.     

Orange protein from <Emphasis Type="Italic">Desulfovibrio alaskensis</Emphasis> G20: insights into the Mo–Cu cluster protein-assisted synthesis

A novel metalloprotein containing a unique [S₂MoS₂CuS₂MoS₂]^3? cluster, designated as Orange Protein (ORP), was isolated for the first time from Desulfovibrio gigas, a sulphate reducer. The orp operon is conserved in almost all sequenced Desulfovibrio genomes and in other anaerobic bacteria, however, so far D. gigas ORP had been the only ORP characterized in the literature. In this work, the purification of another ORP isolated form Desulfovibrio alaskensis G20 is reported. The native protein is monomeric (12443.8 ± 0.1 Da by ESI–MS) and contains also a MoCu cluster with characteristic absorption bands at 337 and 480 nm, assigned to S–Mo charge transfer bands. Desulfovibrio alaskensis G20 recombinant protein was obtained in the apo-form from E. coli. Cluster reconstitution studies and UV–visible titrations with tetrathiomolybdate of the apo-ORP incubated with Cu ions indicate that the cluster is incorporated in a protein metal-assisted synthetic mode and the protein favors the 2Mo:1Cu stoichiometry. In Desulfovibrio alaskensis G20, the orp genes are encoded by a polycistronic unit composed of six genes whereas in Desulfovibrio vulgaris Hildenborough the same genes are organized into two divergent operons, although the composition in genes is similar. The gene expression of ORP (Dde_3198) increased 6.6 ± 0.5 times when molybdate was added to the growth medium but was not affected by Cu(II) addition, suggesting an involvement in molybdenum metabolism directly or indirectly in these anaerobic bacteria.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

The relationship between hydrogen metabolism,sulfate reduction and nitrogen fixation in sulfate reducers

Summary Hydrogenase and nitrogenase activities of sulfate-reducing bacteria allow their adaptation to different nutritional habits even under adverse conditions. These exceptional capabilities of adaptation are important factors in the understanding of their predominant role in problems related to anaerobic metal corrosion. Although the D₂–H⁺ exchange reaction indicated thatDesulfovibrio desulfuricans strain Berre-Sol andDesulfovibrio gigas hydrogenases were reversible, the predominant activity in vivo was hydrogen uptake. Hydrogen production was restricted to some particular conditions such as sulfate or nitrogen starvation. Under diazotrophic conditions, a transient hydrogen evolution was followed by uptake when dinitrogen was effectively fixed. In contrast, hydrogen evolution proceeded when acetylene was substituted as the nitrogenase substrate. Hydrogen can thus serve as an electron donor in sulfate reduction and nitrogen metabolism.     

An immune complex assay for SV40 T antigen.

The two forms of ferredoxin from $\text{Desulfovibrio gigas}$, Fd I and Fd II, are studied by differential pulse polarography 1. Fd I and Fd II give one well defined peak corresponding to $\text{E}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= ?0.33}\text{and}\text{?0.35}$ V (vs. the hydrogen electrode) respectively, at c > 5 μM. The influence of the concentration on the peak potentials Ep and the peak heights i_p is examined. The denaturation of the two forms of ferredoxin is studied by polarography in conjunction with UV spectrophotometry. Two new peaks at negative potentials before the reduction of the solvent are observed in denaturated proteins.     

New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two iron-centre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 × 10? M?1 · s?1 and 1.3 × 10? M?1 · s?1 for SORFe(IIII)-Fe(II) and for SORFe(IIII)-Fe(III) forms respectively, and 3.2 × 10? M?1 · s?1 for the SORZn(II)-Fe(III) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.     

Enhanced Electron Transfer by Unsaturated Fatty Acids and Superoxide Dismutase

We have previously shown that unsaturated fatty acids (UFA) facilitate electron transfer between iron centers such as ferrous iron and ferricytochrome C. Extending this concept to a more physiologic model of fatty acids associated with proteins, we find that electron transfer is also enhanced in this model. While investigating whether free superoxide was involved in this electron transfer, we discovered that superoxide dismutase (SOD) enchanced the electron transfer. While the mechanism of electron transfer is unknown, the above findings are consistent with UFA and SOD participating in membrane redox systems.     

Enzymatic activity mastered by altering metal coordination spheres

Metalloenzymes control enzymatic activity by changing the characteristics of the metal centers where catalysis takes place. The conversion between inactive and active states can be tuned by altering the coordination number of the metal site, and in some cases by an associated conformational change. These processes will be illustrated using heme proteins (cytochrome c nitrite reductase, cytochrome c peroxidase and cytochrome cd ₁ nitrite reductase), non-heme proteins (superoxide reductase and [NiFe]-hydrogenase), and copper proteins (nitrite and nitrous oxide reductases) as examples. These examples catalyze electron transfer reactions that include atom transfer, abstraction and insertion.     

A new strain of Desulfovibrio gigas isolated from a sewage plant

Summary A new strain of Desulfovibrio gigas was isolated from sludge of a sewage plant. The medium contained ethanol, sulfate, minerals, some vitamins but no source of combined nitrogen. Several enrichment cultures of sulfate reducers could be obtained by employing the method to sludge samples from various sewage plants. With respect to their morphology, the dominating bacteria were of the Desulfovibrio gigas type.     

Effect of redox mediators on the flash-induced membrane potential generation in Mn-depleted photosystem II core particles

An electrometrical technique was used to investigate flash-induced electron transfer reactions between Mn-depleted spinach photosystem II core particles incorporated into liposomes and redox mediators. Besides the fast increase in the transmembrane electric potential difference associated with electron transfer between the redox active tyrosine (Y_Z) and the primary quinone acceptor Q_A, an additional electrogenic phase was observed in the presence of N,N,N′N′-tetramethyl-p-phenylenediamine and 2,6-dichlorophenol-indophenol. The latter phase is attributed to vectorial electron transfer from the redox dye(s) to the protein-embedded Y_Z. The data obtained suggest an electrically isolated location of the Y_Z from the external water phase.     

Localization of hydrogenase in Desulfovibrio gigas cells

The localization of hydrogenase protein in Desulfovibrio gigas cells grown either in lactate-sulfate or hydrogen-sulfate media, has been investigated by subcellular fractionation with immunoblotting and by electron microscopic immunocytochemistry. Subcellular fractionation experiments suggest that no integral membrane-bound hydrogenase is present in D. gigas. About 40% of the hydrogenase activity could be extracted by treatment of D. gigas cells with Tris-EDTA buffer. The rest of the soluble hydrogenase activity (50%) was found in the soluble fraction which was obtained after disruption of Tris-EDTA extracted cells and high speed centrifugation. Both soluble hydrogenase fractions purified to homogeneity showed identical molecular properties including the N-terminal aminoacid sequences of their large and small subunits. Polyacrylamide gel electrophoresis of the proteins of the subcellular fractions revealed a single band of hydrogenase activity exhibiting the same mobility as purified D. gigas hydrogenase. Western blotting carried out on these subcellular fractions revealed crossreactivity with the antibodies raised against (NiFe) hydrogenase. The lack of crossreactivity with antibodies against (FE) or (NiFeSe) hydrogenases, indicated that only (NiFe) type hydrogenase is present in D. gigas.Immunocytolocalization in ultrathin frozen sections of D. gigas cells grown either in lactate-sulfate, pyruvate-sulfate or hydrogen-sulfate media showed only a (NiFe) hydrogenase located in the periplasmic space. The bioenergetics of D. gigas are discussed in the light of these findings.     

Horizontal Transfer of Two Operons Coding for Hydrogenases Between Bacteria and Archaea

Using a phylogenetic approach, we discovered three putative horizontal transfers between bacterial and archaeal species involving large clusters of genes. One transfer involves an operon of 13 genes, called mbx, wich probably was transferred into the genome of Thermotoga maritima from a species belonging or close to the Pyrococcus genus. The two others implied an operon of six genes, called ech, transferred independently to the genomes of Thermoanaerobacter tengcongensis and Desulfovibrio gigas, from a species belonging or close to the Methanosarcina genus. All these transfers affected operons coding for multisubunit membrane-bound (NiFe) hydrogenases involved in the energy metabolism of the donor genomes. The functionality of the transferred operons has not been experimentally demonstrated for T. maritima, whereas in D. gigas and T. tengcongensis the encoded multisubunit hydrogenase could have a role in energy conservation. This report adds several cases of horizontal gene transfers among hydrogenases already described.Reviewing Editor: Dr. Siv Andersson     

Molecular cloning of the gene encoding flavoredoxin, a flavoprotein from Desulfovibrio gigas

Sulfate-reducing bacteria are rich in unique redox proteins and electron carriers that participate in a variety of essential pathways. Several studies have been carried out to characterize these proteins, but the structure and function of many are poorly understood. Many Desulfovibrio species can grow using hydrogen as the sole energy source, indicating that the oxidation of hydrogen with sulfite as the terminal electron acceptor is an energy-conserving mechanism. Flavoredoxin is an FMN-binding protein isolated from the sulfate-reducing bacteria Desulfovibrio gigas that participates in the reduction of bisulfite from hydrogen. Here we report the cloning and sequencing of the flavoredoxin gene. The derived amino acid sequence exhibits similarity to several flavoproteins which are members of a new family of flavin reductases suggested to bind FMN in a novel mode.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.