The adaptive response to alternative carbon sources in the pathogen Candida albicans involves a remodeling of thiol- and glutathione-dependent redox status.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. During infection in vivo, Candida albicans must adapt to host microenvironments and this adaptive response is crucial for the survival of this organism, as it facilitates the effective assimilation of alternative carbon sources others than glucose. We performed a global proteomic analysis on the global changes in protein abundance in response to changes in micronutrient levels, and, in parallel, explored changes in the intracellular redox and metabolic status of the cells. We show here that each of the carbon sources considered - glucose, acetate and lactate - induces a unique pattern of response in C. albicans cells, and that some conditions trigger an original and specific adaptive response involving the adaptation of metabolic pathways, but also a complete remodeling of thiol-dependent antioxidant defenses. Protein S-thiolation and the overproduction of reduced glutathione are two components of the response to high glucose concentration. In the presence of acetate, glutathione-dependent oxidative stress occurs, reduced thiol groups bind to proteins, and glutathione is exported out of the cells, these changes probably being triggered by an increase in glutathione-S-transferases. Overall, our results suggest that the role of cellular redox status regulation and defenses against oxidative stress, including the thiol- and glutathione-dependent response, in the adaptive response of C. albicans to alternative carbon sources should be reconsidered.

Modulation of the specific glutathionylation of mitochondrial proteins in the yeast Saccharomyces cerevisiae under basal and stress conditions.

The potential biological consequences of oxidative stress and changes in glutathione levels include the oxidation of susceptible protein thiols and reversible covalent binding of glutathione to the -SH groups of proteins by S-glutathionylation. Mitochondria are central to the response to oxidative stress and redox signaling. It is therefore crucial to explore the adaptive response to changes in thiol-dependent redox status in these organelles. We optimized the purification protocol of glutathionylated proteins in the yeast Saccharomyces cerevisiae and present a detailed proteomic analysis of the targets of protein glutathionylation in cells undergoing constitutive metabolism and after exposure to various stress conditions. This work establishes the physiological importance of the glutathionylation process in S. cerevisiae under basal conditions and provides evidence for an atypical and unexpected cellular distribution of the process between the cytosol and mitochondria. In addition, our data indicate that each oxidative condition (diamide, GSSG, H2O2, or the presence of iron) elicits an adaptive metabolic response affecting specific mitochondrial metabolic pathways, mainly involved in the energetic maintenance of the cells. The correlation of protein modifications with intracellular glutathione levels suggests that protein deglutathionylation may play a role in protecting mitochondria from oxidative stress. This work provides further insights into the diversity of proteins undergoing glutathionylation and the role of this post-translational modification as a regulatory process in the adaptive response of the cell.

Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates.

The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation.

Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells.

Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15-30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H(2)O(2). On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression.

2-thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation.

Myeloperoxidase (MPO) is a prime candidate for promoting oxidative stress during inflammation. This abundant enzyme of neutrophils uses hydrogen peroxide to oxidize chloride to highly reactive and toxic chlorine bleach. We have identified 2-thioxanthines as potent mechanism-based inactivators of MPO. Mass spectrometry and x-ray crystal structures revealed that these inhibitors become covalently attached to the heme prosthetic groups of the enzyme. We propose a mechanism whereby 2-thioxanthines are oxidized, and their incipient free radicals react with the heme groups of the enzyme before they can exit the active site. 2-Thioxanthines inhibited MPO in plasma and decreased protein chlorination in a mouse model of peritonitis. They slowed but did not prevent neutrophils from killing bacteria and were poor inhibitors of thyroid peroxidase. Our study shows that MPO is susceptible to the free radicals it generates, and this Achilles' heel of the enzyme can be exploited to block oxidative stress during inflammation.

Comparative electrochemical study of superoxide reductases.

Superoxide reductases are involved in relevant biological electron transfer reactions related to protection against oxidative stress caused by reactive oxygen species. The electrochemical features of metalloproteins belonging to the three different classes of enzymes were studied by potentio-dynamic techniques (cyclic and square wave voltammetry): desulfoferrodoxin from Desulfovibrio vulgaris Hildenborough, class I superoxide reductases and neelaredoxin from Desulfovibrio gigas and Treponema pallidum, namely class II and III superoxide reductases, respectively. In addition, a small protein, designated desulforedoxin from D. gigas, which has high homology with the N-terminal domain of class I superoxide reductases, was also investigated. A comparison of the redox potentials and redox behavior of all the proteins is presented, and the results show that SOR center II is thermodynamically more stable than similar centers in different proteins, which may be related to an intramolecular electron transfer function.

Glutathione-dependent redox status of frataxin-deficient cells in a yeast model of Friedreich's ataxia.

Friedreich's ataxia is a neurodegenerative disease caused by reduced expression of the mitochondrial protein frataxin. The main phenotypic features of frataxin-deficient human and yeast cells include iron accumulation in mitochondria, iron-sulphur cluster defects and high sensitivity to oxidative stress. Glutathione is a major protective agent against oxidative damage and glutathione-related systems participate in maintaining the cellular thiol/disulfide status and the reduced environment of the cell. Here, we present the first detailed biochemical study of the glutathione-dependent redox status of wild-type and frataxin-deficient cells in a yeast model of the disease. There were five times less total glutathione (GSH+GSSG) in frataxin-deficient cells, imbalanced GSH/GSSG pools and higher glutathione peroxidase activity. The pentose phosphate pathway was stimulated in frataxin-deficient cells, glucose-6-phosphate dehydrogenase activity was three times higher than in wild-type cells and this was coupled to a defect in the NADPH/NADP(+) pool. Moreover, analysis of gene expression confirms the adaptative response of mutant cells to stress conditions and we bring evidence for a strong relation between the glutathione-dependent redox status of the cells and iron homeostasis. Dynamic studies show that intracellular glutathione levels reflect an adaptation of cells to iron stress conditions, and allow to distinguish constitutive stress observed in frataxin-deficient cells from the acute response of wild-type cells. In conclusion, our findings provide evidence for an impairment of glutathione homeostasis in a yeast model of Friedreich's ataxia and identify glutathione as a valuable indicator of the redox status of frataxin-deficient cells.

The adaptive response to iron involves changes in energetic strategies in the pathogen Candida albicans.

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases known as candidiasis. Its ability to grow in different morphological forms, such as yeasts or filamentous hyphae, contributes to its survival in diverse microenvironments. Iron uptake has been associated with virulence, and C. albicans has developed elaborate strategies for acquiring iron from its host. In this work, we analyze the metabolic changes in response to changes in iron content in the growth medium and compare C. albicans adaptation to the presence or absence of iron. Functional and morphological studies, correlated to a quantitative proteomic analysis, were performed to assess the specific pathways underlying the response to iron, both in the yeast and filamentous forms. Overall, the results show that the adaptive response to iron is associated with a metabolic remodeling affecting the energetic pathways of the pathogen. This includes changes in the thiol-dependent redox status, the activity of key mitochondrial enzymes and the respiratory chain. Iron deficiency stimulates bioenergetic pathways, whereas iron-rich condition is associated with greater biosynthetic needs, particularly in filamentous forms. Moreover, we found that C. albicans yeast cells have an extraordinary capability to adapt to changes in environmental conditions.

Dysfunction of mitochondrial Lon protease and identification of oxidized protein in mouse brain following exposure to MPTP: Implications for Parkinson disease.

Compelling evidence suggests that mitochondrial dysfunction leading to reactive oxygen species (ROS) production and protein oxidation could represent a critical event in the pathogenesis of Parkinson's disease (PD). Pioneering studies have shown that the mitochondrial matrix contains the Lon protease, which degrades oxidized, dysfunctional, and misfolded protein. Using the PD animal model of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) intoxication in mice, we showed that Lon protease expression increased in the ventral mesencephalon of intoxicated animals, concomitantly with the appearance of oxidized proteins and dopaminergic cell loss. In addition, we report that Lon is inactivated by ROS. Moreover, proteomic experiments provide evidence of carbonylation in α-ketoglutarate dehydrogenase (KGDH), aconitase or subunits of respiratory chain complexes. Lon protease inactivation upon MPTP treatment in mice raises the possibility that Lon protease dysfunction is an early event in the pathogenesis of PD.

Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays.

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P2(1) (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, beta = 106.9 degrees) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, beta = 104.9 degrees) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (lambda = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2(1) data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

Oxidation of Cu, Zn-superoxide dismutase by the myeloperoxidase/hydrogen peroxide/chloride system: functional and structural effects.

This study investigated the functional and structural effects of bovine Cu, Zn-superoxide dismutase (Cu, Zn-SOD) oxidation by the myeloperoxidase (MPO)/hydrogen peroxide (H2O2)/chloride system and reagent hypochlorous acid (HOCl). Exposure to HOCl led to a fast inactivation accompanied by structural alterations. The residual SOD activity depended on the reactants concentration ratio and on the exposure time. The concomitant high consumption of HOCl indicated the presence of multiple targets on the protein. As assessed by SDS/PAGE, HOCl caused the dissociation of the protein into protomers at 16 kDa stable to both SDS and reducing conditions. Results from isoelectric focusing gels showed that exposure to HOCl induced the formation of modified protein derivatives, with a more acidic net electric charge than the parent molecule, consistent with the presence of additional ions observed in the electrospray ionization mass spectra. The reaction of protein with HOCl resulted in changes in protein conformation as assessed by the UV fluorescence and oxidation of the unique methionine and tyrosine, chlorination of several lysines with formation of chloramines. There was no significant formation of dityrosine and carbonyl groups. Exposure to high levels of HOCl resulted in complete enzyme inactivation, loss of additional lysine, histidine and arginine residues and coincident detection of weakly bound zinc and copper using 4-pyridylazaresorcinol. Collectively, the results suggest that the decrease of the dismutase activity is probably related to both dissociation into protomers and unfolding due to extensive oxidative modifications of amino acids.

Tissue- and cell-specific mitochondrial defect in Parkin-deficient mice.

Loss of Parkin, encoded by PARK2 gene, is a major cause of autosomal recessive Parkinson's disease. In Drosophila and mammalian cell models Parkin has been shown in to play a role in various processes essential to maintenance of mitochondrial quality, including mitochondrial dynamics, biogenesis and degradation. However, the relevance of altered mitochondrial quality control mechanisms to neuronal survival in vivo is still under debate. We addressed this issue in the brain of PARK2-/- mice using an integrated mitochondrial evaluation, including analysis of respiration by polarography or by fluorescence, respiratory complexes activity by spectrophotometric assays, mitochondrial membrane potential by rhodamine 123 fluorescence, mitochondrial DNA content by real time PCR, and oxidative stress by total glutathione measurement, proteasome activity, SOD2 expression and proteins oxidative damage. Respiration rates were lowered in PARK2-/- brain with high resolution but not standard respirometry. This defect was specific to the striatum, where it was prominent in neurons but less severe in astrocytes. It was present in primary embryonic cells and did not worsen in vivo from 9 to 24 months of age. It was not associated with any respiratory complex defect, including complex I. Mitochondrial inner membrane potential in PARK2-/- mice was similar to that of wild-type mice but showed increased sensitivity to uncoupling with ageing in striatum. The presence of oxidative stress was suggested in the striatum by increased mitochondrial glutathione content and oxidative adducts but normal proteasome activity showed efficient compensation. SOD2 expression was increased only in the striatum of PARK2-/- mice at 24 months of age. Altogether our results show a tissue-specific mitochondrial defect, present early in life of PARK2-/- mice, mildly affecting respiration, without prominent impact on mitochondrial membrane potential, whose underlying mechanisms remain to be elucidated, as complex I defect and prominent oxidative damage were ruled out.

The yeast metacaspase is implicated in oxidative stress response in frataxin-deficient cells.

Friedreich ataxia is the most common recessive neurodegenerative disease and is caused by reduced expression of mitochondrial frataxin. Frataxin depletion causes impairment in iron-sulfur cluster and heme biosynthesis, disruption of iron homeostasis and hypersensitivity to oxidants. Currently no pharmacological treatment blocks disease progression, although antioxidant therapies proved to benefit patients. We show that sensitivity of yeast frataxin-deficient cells to hydrogen peroxide is partially mediated by the metacaspase. Metacaspase deletion in frataxin-deficient cells results in recovery of antioxidant capacity and heme synthesis. In addition, our results suggest that metacaspase is associated with mitochondrial respiration, intracellular redox control and genomic stability.

Gex1 is a yeast glutathione exchanger that interferes with pH and redox homeostasis.

In the yeast Saccharomyces cerevisiae, glutathione plays a major role in heavy metal detoxification and protection of cells against oxidative stress. We show that Gex1 is a new glutathione exchanger. Gex1 and its paralogue Gex2 belong to the major facilitator superfamily of transporters and display similarities to the Aft1-regulon family of siderophore transporters. Gex1 was found mostly at the vacuolar membrane and, to a lesser extent, at the plasma membrane. Gex1 expression was induced under conditions of iron depletion and was principally dependent on the iron-responsive transcription factor Aft2. However, a gex1Δ gex2Δ strain displayed no defect in known siderophore uptake. The deletion mutant accumulated intracellular glutathione, and cells overproducing Gex1 had low intracellular glutathione contents, with glutathione excreted into the extracellular medium. Furthermore, the strain overproducing Gex1 induced acidification of the cytosol, confirming the involvement of Gex1 in proton transport as a probable glutathione/proton antiporter. Finally, the imbalance of pH and glutathione homeostasis in the gex1Δ gex2Δ and Gex1-overproducing strains led to modulations of the cAMP/protein kinase A and protein kinase C1 mitogen-activated protein kinase signaling pathways.

Impaired nuclear Nrf2 translocation undermines the oxidative stress response in Friedreich ataxia.

BACKGROUND: Friedreich ataxia originates from a decrease in mitochondrial frataxin, which causes the death of a subset of neurons. The biochemical hallmarks of the disease include low activity of the iron sulfur cluster-containing proteins (ISP) and impairment of antioxidant defense mechanisms that may play a major role in disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We thus investigated signaling pathways involved in antioxidant defense mechanisms. We showed that cultured fibroblasts from patients with Friedreich ataxia exhibited hypersensitivity to oxidative insults because of an impairment in the Nrf2 signaling pathway, which led to faulty induction of antioxidant enzymes. This impairment originated from previously reported actin remodeling by hydrogen peroxide. CONCLUSIONS/SIGNIFICANCE: Thus, the defective machinery for ISP synthesis by causing mitochondrial iron dysmetabolism increases hydrogen peroxide production that accounts for the increased susceptibility to oxidative stress.

Overexpression of the yeast frataxin homolog (Yfh1): contrasting effects on iron-sulfur cluster assembly, heme synthesis and resistance to oxidative stress.

Friedreich's ataxia is generally associated with defects in [Fe-S] cluster assembly/stability and heme synthesis and strong susceptibility to oxidative stress. We used the yeast (Saccharomyces cerevisiae) model of Friedreich's ataxia to study the physiological consequences of modulating the expression of the frataxin gene (YFH1). We show that the number of frataxin molecules per wild-type cell varies from less than 200 to 1500 according to the iron concentration in the medium. Cells overexpressing YFH1 on a plasmid (2muYFH1; about 3500 molecules Yfh1/cell) took up more iron than wild-type cells and displayed defective [Fe-S] cluster assembly/stability in vivo. By contrast, endogenous mitochondrial iron was more available to ferrochelatase in 2muYFH1 cells than in wild-type cells, resulting in higher levels of heme synthesis in vitro. Frataxin overproduction resulted in a shift from frataxin trimers to frataxin oligomers of higher molecular mass in the mitochondrial matrix. Much fewer carbonylated proteins were present in 2muYFH1 cells, and these cells were more resistant to oxidizing agents than wild-type cells, which probably resulted from the lower production of hydrogen peroxide by the mitochondria of 2muYFH1 cells compared to wild-type cells. To our knowledge, this work is the first description where major frataxin-related phenotypes ([Fe-S] cluster assembly and heme synthesis) can be split in vivo, suggesting that frataxin has independent roles in both processes, and that the optimal conditions for these independent roles are different.

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 spirochete 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 kapp 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 kapp value of 108.8+/-12.0 min(-1), and was then assigned as the potential physiological electron donor in this organism.

The first crystal structure of class III superoxide reductase from Treponema pallidum.

Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 A resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the beta-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.

What is the ultimate fate of superoxide anion in vivo?

For three decades, oxidative stress and the role of reactive oxygen species in biology have been extensively studied. Recently, a new interest in these areas has emerged with the discovery of superoxide reductases, a family of familiar bacterial metalloenzymes whose heretofore unknown function has now been apparently revealed. In a series of experiments utilizing genetic, molecular biological, and biochemical methods, these enzymes have been shown to be physiologically competent at removing superoxide. The role of these enzymes and their biological relationship to the well-known superoxide dismutases is discussed.

Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin.

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.