Hybrid cluster proteins in a photosynthetic microalga.

Hybrid cluster proteins (HCPs) are metalloproteins characterized by the presence of an iron-sulfur-oxygen cluster. These proteins occur in all three domains of life. In eukaryotes, HCPs have so far been found only in a few anaerobic parasites and photosynthetic microalgae. With respect to all species harboring an HCP, the green microalga Chlamydomonas reinhardtii stands out by the presence of four HCP genes. The study of the gene and protein structures as well as the phylogenetic analyses strongly support a model in which the HCP family in the alga has emerged from a single gene of alpha proteobacterial origin and then expanded by several rounds of duplications. The spectra and redox properties of HCP1 and HCP3, produced heterologously in Escherichia coli, were analyzed by electron paramagnetic resonance spectroscopy on redox-titrated samples. Both proteins contain a [4Fe-4S]-cluster as well as a [4Fe-2O-2S]-hybrid cluster with paramagnetic properties related to those of HCPs from Desulfovibrio species. Immunoblotting experiments combined with mass spectrometry-based proteomics showed that both nitrate and darkness contribute to the strong upregulation of the HCP levels in C. reinhardtii growing under oxic conditions. The link to the nitrate metabolism is discussed in the light of recent data on the potential role of HCP in S-nitrosylation in bacteria.

Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.

Bottom-up proteomics is increasingly being used to characterize unknown environmental, clinical, and forensic samples. Proteomics-based bacterial identification typically proceeds by tabulating peptide "hits" (i.e., confidently identified peptides) associated with the organisms in a database; those organisms with enough hits are declared present in the sample. This approach has proven to be successful in laboratory studies; however, important research gaps remain. First, the common-practice reliance on unique peptides for identification is susceptible to a phenomenon known as signal erosion. Second, no general guidelines are available for determining how many hits are needed to make a confident identification. These gaps inhibit the transition of this approach to real-world forensic samples where conditions vary and large databases may be needed. In this work, we propose statistical criteria that overcome the problem of signal erosion and can be applied regardless of the sample quality or data analysis pipeline. These criteria are straightforward, producing a p-value on the result of an organism or toxin identification. We test the proposed criteria on 919 LC-MS/MS data sets originating from 2 toxins and 32 bacterial strains acquired using multiple data collection platforms. Results reveal a > 95% correct species-level identification rate, demonstrating the effectiveness and robustness of proteomics-based organism/toxin identification.

Desulfovibrio DA2_CueO is a novel multicopper oxidase with cuprous, ferrous and phenol oxidase activity.

Desulfovibrio sp. A2 is a novel Gram-negative sulfate-reducing bacterium that was isolated from sediments of the Norilsk mining/smelting area in Russia. The organism possesses a monocistronic operon encoding a 71 kDa periplasmic multicopperoxidase, which we call DA2_CueO. Histidine-tagged DA2_CueO expressed from a plasmid in Escherichia coli and purified by Ni-NTA affinity chromatography oxidizes Cu+ and Fe2+, and exhibits phenol oxidase activity with 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,3-dihydroxybenzoic acid and 2,6-dimethoxyphenol as substrates, using O2 as the oxidant. When expressed in an E. coli cueO knock-out strain, DA2_CueO exhibits phenol oxidase activity in vivo and enhances the copper tolerance of the strain. These findings indicate that the DA2_CueO gene of Desulfovibrio sp. A2 encodes a multicopperoxidase with a role in metal ion resistance. The enzyme displays some novel structural features, which are discussed.

Platinum Group Metal-free Catalysts for Hydrogen Evolution Reaction in Microbial Electrolysis Cells.

Hydrogen gas is a green energy carrier with great environmental benefits. Microbial electrolysis cells (MECs) can convert low-grade organic matter to hydrogen gas with low energy consumption and have gained a growing interest in the past decade. Cathode catalysts for the hydrogen evolution reaction (HER) present a major challenge for the development and future applications of MECs. An ideal cathode catalyst should be catalytically active, simple to synthesize, durable in a complex environment, and cost-effective. A variety of noble-metal free catalysts have been developed and investigated for HER in MECs, including Nickel and its alloys, MoS2 , carbon-based catalysts and biocatalysts. MECs in turn can serve as a research platform to study the durability of the HER catalysts. This personal account has reviewed, analyzed, and discussed those catalysts with an emphasis on synthesis and modification, system performance and potential for practical applications. It is expected to provide insights into the development of HER catalysts towards MEC applications.

Molecular Basis of C-N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase.

Deamination of choline catalyzed by the glycyl radical enzyme choline trimethylamine-lyase (CutC) has emerged as an important route for the production of trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial trimethylamine production.

Effects of Ag and Cu ions on the microbial corrosion of 316L stainless steel in the presence of Desulfovibrio sp.

The utilization of Ag and Cu ions to prevent both microbial corrosion and biofilm formation has recently increased. The emphasis of this study lies on the effects of Ag and Cu ions on the microbial corrosion of 316L stainless steel (SS) induced by Desulfovibrio sp. Electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization were used to analyze the corrosion behavior. The biofilm formation, corrosion products and Ag and Cu ions on the surfaces were investigated using scanning electron microscopy (SEM), energy dispersive X-ray spectrometry (EDS) and elemental mapping. Through circuit modeling, EIS results were used to interpret the physicoelectric interactions between the electrode, biofilm and culture interfaces. EIS results indicated that the metabolic activity of Desulfovibrio sp. accelerated the corrosion rate of SS in both conditions with and without ions. However, due to the retardation in the growth of Desulfovibrio sp. in the presence of Ag and Cu ions, significant decrease in corrosion rate was observed in the culture with the ions. In addition, SEM and EIS analyses revealed that the presence of the ions leads to the formation on the SS of a biofilm with different structure and morphology. Elemental analysis with EDS detected mainly sulfide- and phosphorous-based corrosion products on the surfaces.

Biosynthesis of CdS nanoparticles: A fluorescent sensor for sulfate-reducing bacteria detection.

CdS nanoparticles were synthesized with an environmentally friendly method by taking advantage of the characteristic metabolic process of sulfate-reducing bacteria (SRB), and used as fluorescence labels for SRB detection. The presence of CdS nanoparticles was observed within and immediately surrounded bacterial cells, indicating CdS nanoparticles were synthesized both intracellularly and extracellularly. Moreover, fluorescent properties of microbial synthesized CdS nanoparticles were evaluated for SRB detection, and a linear relationship between fluorescence intensity and the logarithm of bacterial concentration was obtained in the range of from 1.0×10(2) to 1.0×10(7)cfu mL(-1). The proposed SRB detection method avoided the use of biological bio-recognition elements which are easy to lose their specific recognizing abilities, and the bacterial detection time was greatly shortened compared with the widely used MPN method which would take up to 15 days to accomplish the detection process.

MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

MamA is a highly conserved protein found in magnetotactic bacteria (MTB), a diverse group of prokaryotes capable of navigating according to magnetic fields - an ability known as magnetotaxis. Questions surround the acquisition of this magnetic navigation ability; namely, whether it arose through horizontal or vertical gene transfer. Though its exact function is unknown, MamA surrounds the magnetosome, the magnetic organelle embedding a biomineralised nanoparticle and responsible for magnetotaxis. Several structures for MamA from a variety of species have been determined and show a high degree of structural similarity. By determining the structure of MamA from Desulfovibrio magneticus RS-1 using X-ray crystallography, we have opened up the structure-sequence landscape. As such, this allows us to perform structural- and phylogenetic-based analyses using a variety of previously determined MamA from a diverse range of MTB species across various phylogenetic groups. We found that MamA has remained remarkably constant throughout evolution with minimal change between different taxa despite sequence variations. These findings, coupled with the generation of phylogenetic trees using both amino acid sequences and 16S rRNA, indicate that magnetotaxis likely did not spread via horizontal gene transfer and instead has a significantly earlier, primordial origin.

[Mechanisms of electron transfer to insoluble terminal acceptors in chemoorganotrophic bacteria].

The mechanisms of electron transfer of association of chemoorganotrophic bacteria to the anode in microbial fuel cells are summarized in the survey. These mechanisms are not mutually exclusive and are divided into the mechanisms of mediator electron transfer, mechanisms of electron transfer with intermediate products of bacterial metabolism and mechanism of direct transfer of electrons from the cell surface. Thus, electron transfer mediators are artificial or synthesized by bacteria riboflavins and phenazine derivatives, which also determine the ability of bacteria to antagonism. The microorganisms with hydrolytic and exoelectrogenic activity are involved in electron transfer mechanisms that are mediated by intermediate metabolic products, which are low molecular carboxylic acids, alcohols, hydrogen etc. The direct transfer of electrons to insoluble anode is possible due to membrane structures (cytochromes, pili, etc.). Association of microorganisms, and thus the biochemical mechanisms of electron transfer depend on the origin of the inoculum, substrate composition, mass transfer, conditions of aeration, potentials and location of electrodes and others, that are defined by technological and design parameters.

Accurate calculations of geometries and singlet-triplet energy differences for active-site models of [NiFe] hydrogenase.

We have studied the geometry and singlet-triplet energy difference of two mono-nuclear Ni(2+) models related to the active site in [NiFe] hydrogenase. Multiconfigurational second-order perturbation theory based on a complete active-space wavefunction with an active space of 12 electrons in 12 orbitals, CASPT2(12,12), reproduces experimental bond lengths to within 1 pm. Calculated singlet-triplet energy differences agree with those obtained from coupled-cluster calculations with single, double and (perturbatively treated) triple excitations (CCSD(T)) to within 12 kJ mol(-1). For a bimetallic model of the active site of [NiFe] hydrogenase, the CASPT2(12,12) results were compared with the results obtained with an extended active space of 22 electrons in 22 orbitals. This is so large that we need to use restricted active-space theory (RASPT2). The calculations predict that the singlet state is 48-57 kJ mol(-1) more stable than the triplet state for this model of the Ni-SIa state. However, in the [NiFe] hydrogenase protein, the structure around the Ni ion is far from the square-planar structure preferred by the singlet state. This destabilises the singlet state so that it is only ∼24 kJ mol(-1) more stable than the triplet state. Finally, we have studied how various density functional theory methods compare to the experimental, CCSD(T), CASPT2, and RASPT2 results. Semi-local functionals predict the best singlet-triplet energy differences, with BP86, TPSS, and PBE giving mean unsigned errors of 12-13 kJ mol(-1) (maximum errors of 25-31 kJ mol(-1)) compared to CCSD(T). For bond lengths, several methods give good results, e.g. TPSS, BP86, and M06, with mean unsigned errors of 2 pm for the bond lengths if relativistic effects are considered.

Kinetic properties of adenosine triphosphate sulfurylase of intestinal sulfate-reducing bacteria.

The investigation of specific activity of ATP sulfurylase and kinetic properties of the enzyme in cell-free extracts of intestinal bacterial strains Desulfovibrio piger Vib-7 and Desulfomicrobium sp. Rod-9 is presented. The microbiological, biochemical, biophysical and statistical methods were used in the work. The optimal temperature (35°C) and pH 8.0-8.5 for enzyme reaction were determined. An analysis of kinetic properties of ATP sulfurylase has been carried out. Initial (instantaneous) reaction velocity (V0), maximum amount of the product of reaction (Pmax), the reaction time (half saturation period, τ) and maximum velocity of the ATP sulfurylase reaction (Vmax) have been defined. Michaelis constants (Km(Sulfate), Km(ATP), Km(APS), and Km(Pyrophosphate)) of the enzyme reaction were demonstrated for both D. piger Vib-7 and Desulfomicrobium sp. Rod-9 intestinal bacterial strains.

Arsenic removal in a sulfidogenic fixed-bed column bioreactor.

In the present study, the bioremoval of arsenic from synthetic acidic wastewater containing arsenate (As(5+)) (0.5-20mg/L), ferrous iron (Fe(2+)) (100-200mg/L) and sulfate (2,000 mg/L) was investigated in an ethanol fed (780-1,560 mg/L chemical oxygen demand (COD)) anaerobic up-flow fixed bed column bioreactor at constant hydraulic retention time (HRT) of 9.6h. Arsenic removal efficiency was low and averaged 8% in case iron was not supplemented to the synthetic wastewater. Neutral to slightly alkaline pH and high sulfide concentration in the bioreactor retarded the precipitation of arsenic. Addition of 100mg/L Fe(2+) increased arsenic removal efficiency to 63%. Further increase of influent Fe(2+) concentration to 200mg/L improved arsenic removal to 85%. Decrease of influent COD concentration to its half, 780 mg/L, resulted in further increase of As removal to 96% when Fe(2+) and As(5+) concentrations remained at 200mg/L and 20mg/L, respectively. As a result of the sulfidogenic activity in the bioreactor the effluent pH and alkalinity concentration averaged 7.4 ± 0.2 and 1,736 ± 239 mg CaCO3/L respectively. Electron flow from ethanol to sulfate averaged 72 ± 10%. X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analyses were carried out to identify the nature of the precipitate generated by sulfate reducing bacteria (SRB) activity. Precipitation of arsenic in the form of As2S3 (orpiment) and co-precipitation with ferrous sulfide (FeS), pyrite (FeS2) or arsenopyrite (FeAsS) were the main arsenic removal mechanisms.

Estudo da interação de Desulfovibrio indonesiensis com células epiteliais intestinais: influência sobre as junções celulares e função imunológica / Interaction between desulfovibrio indonesiensis and intestinal epithelial cells - influence on celular junctions and immunological function

A imunopatologia das doenças inflamatórias intestinais (DIIs) está associada a níveis aumentados de citocinas pró-inflamatórias, alterações na microbiota local e perda da integridade da barreira epitelial. De fato, a relação parasita-hospedeiro, definida aqui pela interação da microbiota com o microambiente da mucosa, tem sido relatada como fator central na imunopatogênese das DIIs. Neste contexto, o presente trabalho teve como objetivo avaliar o efeito da interação de Desulfovibrio indonesiensis, bactéria redutora de sulfato (BRS), com a linhagem de célula epitelial intestinal Caco-2, sobre a integridade das junções oclusivas e seu papel na modulação da produção de IL-8. Células Caco-2 foram incubadas (3h a 37ºC) com D. indonesiensis (proporção 10:1 bactéria–célula hospedeira), e a interação foi interrompida após 24h e 48h. Microscopia correlativa foi empregada para avaliar a integridade das junções celulares, sendo as culturas controle e as que interagiram com D. indonesiensis processadas para imunofluorescência indireta e microscopia eletrônica de varredura (MEV). Análises da expressão de ocludina, proteína de junção oclusiva, e permeabilidade paracelular foram realizadas por Western blot e resistência elétrica transepitelial (RET), respectivamente. Microscopia de contraste interferencial diferencial (DIC) e microscopia de fluorescência das culturas de células Caco-2 revelaram intensa coesão entre células da monocamada celular, com junções oclusivas visualizadas pela detecção de ocludina e ZO-1. A presença da BRS não acarretou mudanças nas junções celulares e na distribuição espacial de ocludina e ZO-1...

The immunopathology of inflammatory bowel diseases (IBD) is associated withincreased levels of pro-inflammatory cytokines, changes in local microbiota and loss of epithelial barrier integrity. In fact, the host-parasite relationship, here defined by the interaction of microbes with the mucosal microenvironment, has been reported as a central factor in IBD immunopathogenesis. Herein, we aimed to evaluate the interaction of Desulfovibrio indonesiensis, a sulfate reducing bacteria (SRB), with anintestinal epithelial cell strain, Caco-2, and its effect on the integrity of tight junctions and on IL- 8 production. Caco-2 cells were incubated (3h at 37°C) with D. indonesiensis (10:1 bacteria-host cell ratio), and the interaction stopped after 24h and 48h. Correlative microscopy was used to assess the integrity of cell junctions, and both control and D. indonesiensis interaction cultures were processed for indirectimmunofluorescence and scanning electron microscopy (SEM). Expression analysis of occludin, a tight junction protein, and paracellular permeability were performed by Western blot and transepithelial electrical resistance (TEER), respectively. Differential interference contrast (DIC) and fluorescence microscopy analyses ofCaco-2 monolayer cultures revealed intense cell cohesion, with tight junctions visualized by occludin and ZO-1 labeling. Cell junctions and spatial distribution of occludin and ZO-1 were not altered in the presence of BRS. Ultrastructural analysisshowed isolated bacteria and biofilm structures bound to Caco-2 cells without anyalteration in intercellular association. This inaltered junctional integrity was also seenby TEER assays, and Western blot results revealed no changes in occludinexpression...

Crystal structure, exogenous ligand binding, and redox properties of an engineered diiron active site in a bacterial hemerythrin.

A nonheme diiron active site in a 13 kDa hemerythrin-like domain of the bacterial chemotaxis protein DcrH-Hr contains an oxo bridge, two bridging carboxylate groups from Glu and Asp residues, and five terminally ligated His residues. We created a unique diiron coordination sphere containing five His and three Glu/Asp residues by replacing an Ile residue with Glu in DcrH-Hr. Direct coordination of the carboxylate group of E119 to Fe2 of the diiron site in the I119E variant was confirmed by X-ray crystallography. The substituted Glu is adjacent to an exogenous ligand-accessible tunnel. UV-vis absorption spectra indicate that the additional coordination of E119 inhibits the binding of the exogenous ligands azide and phenol to the diiron site. The extent of azide binding to the diiron site increases at pH ≤ 6, which is ascribed to protonation of the carboxylate ligand of E119. The diferrous state (deoxy form) of the engineered diiron site with the extra Glu residue is found to react more slowly than wild type with O2 to yield the diferric state (met form). The additional coordination of E119 to the diiron site also slows the rate of reduction from the met form. All these processes were found to be pH-dependent, which can be attributed to protonation state and coordination status of the E119 carboxylate. These results demonstrate that modifications of the endogenous coordination sphere can produce significant changes in the ligand binding and redox properties in a prototypical nonheme diiron-carboxylate protein active site.

Hydrogen activation by biomimetic [NiFe]-hydrogenase model containing protected cyanide cofactors.

Described are experiments demonstrating incorporation of cyanide cofactors and hydride substrate into [NiFe]-hydrogenase (H2ase) active site models. Complexes of the type (CO)2(CN)2Fe(pdt)Ni(dxpe) (dxpe = dppe, 1; dxpe = dcpe, 2) bind the Lewis acid B(C6F5)3 (BAr(F)3) to give the adducts (CO)2(CNBAr(F)3)2Fe(pdt)Ni(dxpe), (1(BAr(F)3)2, 2(BAr(F)3)2). Upon decarbonylation using amine oxides, these adducts react with H2 to give hydrido derivatives [(CO)(CNBAr(F)3)2Fe(H)(pdt)Ni(dxpe)](-) (dxpe = dppe, [H3(BAr(F)3)2](-); dxpe = dcpe, [H4(BAr(F)3)2](-)). Crystallographic analysis shows that Et4N[H3(BAr(F)3)2] generally resembles the active site of the enzyme in the reduced, hydride-containing states (Ni-C/R). The Fe-H···Ni center is unsymmetrical with r(Fe-H) = 1.51(3) Å and r(Ni-H) = 1.71(3) Å. Both crystallographic and (19)F NMR analyses show that the CNBAr(F)3(-) ligands occupy basal and apical sites. Unlike cationic Ni-Fe hydrides, [H3(BAr(F)3)2](-) and [H4(BAr(F)3)2](-) oxidize at mild potentials, near the Fc(+/0) couple. Electrochemical measurements indicate that in the presence of base, [H3(BAr(F)3)2](-) catalyzes the oxidation of H2. NMR evidence indicates dihydrogen bonding between these anionic hydrides and R3NH(+) salts, which is relevant to the mechanism of hydrogenogenesis. In the case of Et4N[H3(BAr(F)3)2], strong acids such as HCl induce H2 release to give the chloride Et4N[(CO)(CNBAr(F)3)2Fe(Cl)(pdt)Ni(dppe)].

Membrane protein complex of APS reductase and Qmo is present in Desulfovibrio vulgaris and Desulfovibrio alaskensis.

Due to their adjacent location in the genomes of Desulfovibrio species and their potential for formation of an electron transfer pathway in sulfate-reducing prokaryotes, adenosyl phosphosulfate (APS) reductase (Apr) and quinone-interacting membrane-bound oxidoreductase (Qmo) have been thought to interact together during the reduction of APS. This interaction was recently verified in Desulfovibrio desulfuricans. Membrane proteins of Desulfovibrio vulgaris Hildenborough ΔqmoABCD JW9021, a deletion mutant, were compared to the parent strain using blue-native PAGE to determine whether Qmo formed a complex with Apr or other proteins. In the parent strain of D. vulgaris, a unique band was observed that contained all four Qmo subunits, and another band contained three subunits of Qmo, as well as subunits of AprA and AprB. Similar results were observed with bands excised from membrane preparations of Desulfovibrio alaskensis strain G20. These results are in support of the formation of a physical complex between the two proteins; a result that was further confirmed by the co-purification of QmoA/B and AprA/B from affinity-tagged D. vulgaris Hildenborough strains (AprA, QmoA and QmoB) regardless of which subunit had been tagged. This provides clear evidence for the presence of a Qmo-Apr complex that is at least partially stable in protein extracts of D. vulgaris and D. alaskensis.

Understanding and tuning the catalytic bias of hydrogenase.

When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.

Kinetics and thermodynamics of gas diffusion in a NiFe hydrogenase.

We have investigated O2 and H2 transport across a NiFe hydrogenase at the atomic scale by means of computational methods. The Wild Type protein has been compared with the V74Q mutant. Two distinct methodologies have been applied to study the gas access to the active site. Temperature locally enhanced sampling simulations have emphasized the importance of protein dynamics on gas diffusion. The O2 diffusion free energy profiles, obtained by umbrella sampling, are in agreement with the known kinetic data and show that in the V74Q mutant, the inhibition process is lowered from both a kinetic and a thermodynamic point of view.

Structural redox control in a 7Fe ferredoxin isolated from Desulfovibrio alaskensis.

The redox behaviour of a ferredoxin (Fd) from Desulfovibrio alaskensis was characterized by electrochemistry. The protein was isolated and purified, and showed to be a tetramer containing one [3Fe-4S] and one [4Fe-4S] centre. This ferredoxin has high homology with FdI from Desulfovibrio vulgaris Miyazaki and Hildenborough and FdIII from Desulfovibrio africanus. From differential pulse voltammetry the following signals were identified: [3Fe-4S](+1/0) (E(0')=-158±5mV); [4Fe-4S](+2/+1) (E(0')=-474±5mV) and [3Fe-4S](0/-2) (E(0')=-660±5mV). The effect of pH on these signals showed that the reduced [3Fe-4S](0) cluster has a pK'(red)(')=5.1±0.1, the [4Fe-4S](+2/+1) centre is pH independent, and the [3Fe-4S](0/-2) reduction is accompanied by the binding of two protons. The ability of the [3Fe-4S](0) cluster to be converted into a new [4Fe-4S] cluster was proven. The redox potential of the original [4Fe-4S] centre showed to be dependent on the formation of the new [4Fe-4S] centre, which results in a positive shift (ca. 70mV) of the redox potential of the original centre. Being most [Fe-S] proteins involved in electron transport processes, the electrochemical characterization of their clusters is essential to understand their biological function. Complementary EPR studies were performed.