Electrochemical Data Research Articles

Design and Validation of an Experimental Setup for Evaluation of Gas Permeation in Ceramic Membranes

An experimental setup for the evaluation of permeation of gaseous species with the possibility of simultaneously collecting electrochemical impedance spectroscopy data in disk-shaped ceramic membranes was designed and assembled. It consists of an alumina sample holder with thermocouple tips and platinum electrodes located close to both sides of the sample. Water-cooled inlet and outlet gas connections allowed for the insertion of the sample chamber into a programmable split tubular furnace. Gas permeation through a ceramic membrane can be monitored with mass flow controllers, a mass spectrometer, and an electrochemical impedance analyzer. For testing and data validation, ceramic composite membranes were prepared with the infiltration of molten eutectic compositions of alkali salts (lithium, sodium, and potassium carbonates) into porous gadolinia-doped ceria. Values of the alkali salt melting points and the permeation rates of carbon dioxide, in agreement with reported data, were successfully collected.

3-Substituted Blatter Radicals: Cyclization of N-Arylguanidines and N-Arylamidines to Benzo[e][1,2,4]triazines and PhLi Addition.

A series of 3-amino- and 3-alkyl-substituted 1-phenyl-1,4-dihydrobenzo[e][1,2,4]triazin-4-yls was prepared in four steps involving N-arylation, cyclization of N-arylguanidines and N-arylamidines, reduction of the resulting N-oxides to benzo[e][1,2,4]triazines, and subsequent addition of PhLi followed by aerial oxidation. The resulting seven C(3)-substituted benzo[e][1,2,4]triazin-4-yls were analyzed by spectroscopic and electrochemical methods augmented with density functional theory (DFT) methods. Electrochemical data were compared to DFT results and correlated with substituent parameters.

Derivatives of cyclam-1,8-diacetic acid: Synthesis and complexes with divalent transition metal ions

1,4,8,11-tetraazacyclotetradecane-1,8-diacetatic acid, H2te2a, and series of its derivative bearing two substituted N-benzyl groups were synthesized and their complexes were studied. Series of CoII and ZnII complexes was prepared in the solid-state and X-ray diffraction analysis showed that the metal ions are hexacoordinated by four nitrogen atoms of the macrocycle and two oxygen atoms of the carboxylate pendants. The two oxygen atoms were in cis and trans mutual position in the CoII and ZnII complexes, respectively. Potentiometric study of CoII, ZnII, CuII and NiII-H2te2a systems showed that the complexes are highly thermodynamically stable and the ligand is selective for CuII ions (logK = 27.5) over the other ions (logK = 21–22). UV-VIS and electrochemical data for the CoII-H2te2a system showed that complexation was rather slow (several hours at pH 4.5, 25 °C) and the complex undergoes slow oxidation with air oxygen.

High performance direct liquid fuel cells powered by xylose or glucose

In this work, we report the first abiotic, direct liquid fuel cells powered by the monosaccharide xylose using both a fully alkaline fuel cell (with anion exchange membrane) and a split pH fuel cell (with cation exchange membrane). We also report that the same fuel cells can be used with the monosaccharide glucose to produce much higher maximum power density than previously reported for abiotic, direct glucose fuel cells. This first alkaline direct xylose fuel cell (DXFC) produces a maximum power density of 57 mW cm−2 at optimum conditions, while the split pH DXFC produces a maximum power density of 160 mW cm−2. Our significantly improved alkaline direct glucose fuel cell (DGFC) produces 90 mW cm−2 at optimum conditions, while the split pH DGFC produces 189 mW cm−2. In addition to being high performing, these sugar molecules are naturally abundant, renewable, and known to convert to valuable products such as gluconic acid, glucaric acid, and xylonic acid during electrochemical oxidation. Other fuel cell and electrochemical cell data is also reported herein to understand the role of pH and fuel concentrations on behavior toward the electrochemical oxidation of these sugar molecules in alkaline media.

Quantitative analysis of cyclic aging of lithium-ion batteries using synchrotron tomography and electrochemical impedance spectroscopy

The highly complex aging mechanisms of lithium-ion batteries are still not sufficiently well understood. In the present paper, we characterize cyclically aged cells by means of a statistical 3D microstructure analysis and electrochemical properties. More precisely, synchrotron tomography is used to capture the morphology of lithium-ion battery cathodes with a state of health of 100%, 90%, 80%, 60% and 40%, cyclically aged with two different C-rates (0.5C and 1C). The three phases, namely active material, pores and the carbon-binder domain, have been reconstructed from 3D grayscale images by a k-means clustering approach. Moreover, individual active particles are segmented by combining the concept of morphological reconstruction with the watershed algorithm. The processed image data allows to quantitatively characterize the 3D microstructure by phase-based and particle-based characteristics, where a special focus is put on local heterogeneity, which will be described by locally computed descriptors. In addition, electrochemical data and electrochemical impedance spectroscopy are used to characterize the cyclically aged cells. Furthermore, we correlate these results with the detected structural changes. To the best of our knowledge, this is the most extensive data set of cyclically aged lithium-ion cells including electrochemical data as well as 3D structures.

Employing Long-Range Inductive Effects to Modulate Metal-to-Ligand Charge Transfer Photoluminescence in Homoleptic Cu(I) Complexes

Four Cu(I) bis(phenanthroline) photosensitizers formulated from a new ligand structural motif (Cu1-Cu4) coded according to their 2,9-substituents were synthesized, structurally characterized, and fully evaluated using steady-state and time-resolved absorption and photoluminescence (PL) measurements as well as electrochemistry. The 2,9-disubstituted-3,4,7,8-tetramethyl-1,10-phenanthroline ligands feature the following six-membered ring systems prepared through photochemical synthesis: 4,4-dimethylcyclohexyl (1), tetrahydro-2H-pyran-4-yl (2), tetrahydro-2H-thiopyran-4-yl (3), and 4,4-difluorocyclohexyl (4). Universally, these Cu(I) metal-to-ligand charge transfer (MLCT) chromophores display excited-state lifetimes on the microsecond time scale at room temperature, including the three longest-lived homoleptic cuprous phenanthroline excited states measured to date in de-aerated CH2Cl2, τ = 2.5-4.3 μs. This series of molecules also feature high PL quantum efficiencies (ΦPL = 5.3-12% in CH2Cl2). Temperature-dependent PL lifetime experiments confirmed that all these molecules exhibit reverse intersystem crossing and display thermally activated delayed PL from a 1MLCT excited state lying slightly above the 3MLCT state, 1050-1490 cm-1. Ultrafast and conventional transient absorption measurements confirmed that the PL originates from the MLCT excited state, which remains sterically arrested, preventing an excessive flattening distortion even when dissolved in Lewis basic CH3CN. Combined PL and electrochemical data provided evidence that Cu1-Cu4 are highly potent photoreductants (Eox* = -1.73 to -1.62 V vs Fc+/0 in CH3CN), whose potentials are altered solely based on which heteroatoms or substituents are resident on the 2,9-appended ring derivatives. It is proposed that long-range electronic inductive effects are responsible for the systematic modulation observed in the PL spectra, excited-state lifetimes, and the ground state absorption spectra and redox potentials. Cu1-Cu4 quantitatively follow the energy gap law, correlating well with structurally related cuprous phenanthrolines and are also shown to triplet photosensitize the excited states of 9,10-diphenylanthracene with bimolecular rate constants ranging from 1.61 to 2.82 × 108 M-1 s-1. The ability to tailor both photophysical and electrochemical properties using long-range inductive effects imposed by the 2,9-ring platforms advocates new directions for future MLCT chromophore discovery.

Hydrogen Diffusion and Trapping in Low-Alloy Tempered Martensitic Steels

Structural characterization of ten low-alloy tempered martensitic steels of varied composition (C, Cr, Mo, Mn, and V contents) and tempering temperature was performed to question the impact of microstructural features on hydrogen state. Thermal desorption spectroscopy and electrochemical permeation data for each alloy were acquired and interpreted in view of hydrogen diffusion/trapping models. This large database provided precise information regarding solubility, diffusion coefficient, activation energies for diffusion and trapping, hydrogen distribution into lattice, and reversible and irreversible trap sites. The results reveal a tendency for the apparent diffusion coefficient to decrease with increasing yield strength, mainly related to the density of trap sites rather than lattice diffusion. Estimates of trapping at dislocation core could explain the irreversible trapping in the six steels with sub-surface hydrogen concentration smaller than 1.5 wppm. For the four steels with higher solubility, it was calculated the superabundant vacancies concentration necessary to justify the amount of trapping sites. The steel with the highest Mo and V contents presented superior solubility of trapped hydrogen which was related to its precipitation of few nanometers in size. It was considered irreversible trapping at carbon vacancies as well as reversible trapping at elastic strain fields around the detected MC carbides.

Performance-Determining Factors for Si–Graphite Electrode Evaluation: The Role of Mass Loading and Amount of Electrolyte Additive

The mass loading of Si–graphite electrodes is often considered as a parameter of secondary importance when testing their electrochemical performance. However, if a sacrificial additive is present in the electrolyte to improve the electrochemical performance, the electrode loading becomes the battery cycle-life-determining factor. The correlation between mass-loading, electrolyte additive, and binder type was investigated by analyzing the cycling behavior of Si–graphite electrodes, prepared with water-based binders, with mass loading ranging from 3 to 9.5 mg cm−2 and cycled with FEC electrolyte additive, while keeping electrolyte amount constant. A lower loading was obtained by keeping slurry preparation steps unchanged from binder to binder and resulted in a longer lifetime for some of the binders. When the final loading was kept constant instead, the performance became independent of the binder used. Since such results can lead to the misinterpretation of the influence of electrode components on the cycling stability (and to a preference of one binder over another in our case), we propose that a comparison of long-term electrochemical performance data of Si–graphite electrodes needs to be always collected by using the same mass-loading with the constant electrolyte and additive.

Integrating machine learning and electrochemistry to develop a glucose biosensor assembled with Ganoderma applanatum lectin

Fungal lectins have enormous biotechnological potential, but limited knowledge about their biochemical and biophysical features prevents their proper use. Herein, we report an innovative alternative to use Ganoderma applanatum lectin (GAL) as a glucose biorecognition element, after identifying the ideal electroanalytical conditions by machine learning studies performed with a homologous agglutinin from the same macrofungus. The research revealed that GAL has moderate resistance to pH (4–8) and temperature (20–60 °C) variations, but its hemagglutinating activity (376.5 HU mg−1 GAL at 20 °C) was better conserved under physiological conditions. Integrating electrochemical data and semi-empirical molecular modeling, biocompatible and electrostatically favorable conditions were found to immobilize the lectin on Prussian blue-modified glassy carbon electrode, after thermal activation of the metal-complex film. The glucose dose–response relationship obtained with the developed biosensor, defined as GAL/ta-PB/GCE, showed a typical Hill equation correlation, suggesting electrodic interactions represented by a sigmoidal mathematical function. GAL/ta-PB/GCE achieved remarkable electroanalytical performance, with emphasis on the detection limit (10.2 pM) and sensitivity (0.012 µA µM−1cm−2). The biosensor was successfully used to quantify glucose in pharmaceutical formulations, reiterating that the association of theoretical and experimental information drives important advances in bioelectrochemical studies.

Reproducible polarization test methods and fair evaluation of polarization data by using interconversion factors in a single chamber cubic microbial fuel cell with a brush anode

MFC research has been conducted enormously in the last decade, and their electrochemical data has been accumulated enormously. Although reproducible measurement method and fair evaluation are essential elements in research, these elements are missing in MFC research. Herein, using single-chamber cubic MFCs, diverse polarization methods were conducted along with other electrochemical tests. 3-hour optimum resistance operation followed by 2-h open circuit time prevented power overshoots. In a faster polarization method, data reproducibility increased drastically, power and current were larger, and polarization resistance was smaller. In ERC-30 min, LSV-0.1, LSV-1, LSV-10 mV/s and, maximum power density was 1242, 1518, 2287 and 3417 mW/m2, and internal resistance 76, 63, 48 and 35 Ω, respectively. For fair evaluation of different polarization data, the interconversion factors were developed and applied. During the optimum resistance operation, average power and current densities were 994 mW/m2 and 3767 mA/m2, respectively. As similarly in the polarization tests, cathodic impedances (24.1 Ω) were 2.4 times larger than the anodic impedances (10.2 Ω). Charge transfer impedance (32.4 Ω) was the major component (>95%) in the electrodes and the whole cell (34.3 Ω). Ohmic impedance was detected only in the anode (0.2 Ω), and diffusion impedance was detected only in the cathodes (1.7 Ω). Cathodic capacitance (1.62 F) was 2.5 times larger than the anodic capacitance (0.64 F). Cyclic voltammogram cathodic capacitance (7.78 F) was 1.6 times larger than the anodic capacitance (4.76 F). Longer operation, lower internal resistance and higher anode performance increased coulombic and energy efficiencies. This study contains very important information on experimental accuracy and reproducibility, essential elements in research, and will greatly contribute to the firm foundation and the meaningful growth of the MFC research.

Insight into anti-corrosion mechanism of Dalbergia odorifera leaves extract as a biodegradable inhibitor for X70 steel in sulfuric acid medium

Dalbergia odorifera leaves extract (DOLE) was obtained by soaking, filtering, and freeze-drying with 0.5 M H2SO4 solution as solvent. The corrosion inhibition performance of X70 steel by DOLE was systematically studied by electrochemical research methods, surface morphology test, infrared spectrum test and theoretical calculation. The electrochemical test data show that the addition of DOLE in sulfuric acid medium can vastly reduce the electric double layer capacitance (Cdl) value of the steel/acid interface, improve the charge transfer resistance, and simultaneously retard the cathodic and anodic reactions of X70 steel. The DOLE reaches 400 mg/L, the anti-corrosion efficiency (η) is 96 %. Fourier transform infrared spectroscopy (FTIR) test data show that DOLE contains a large number of hydroxyl groups, carbonyl groups and unsaturated functional groups, and a large number of lone electron pairs can coordinate with the empty d orbital of Fe, which leads to the formation of chemical adsorption of DOLE at X70 steel. The morphology test showed that the corrosion level of X70 steel decreased memorably after adding DOLE. The adsorption model study manifests that the adhere of DOLE at the interface of X70 steel comply with the Langmuir adsorption isotherm. Theoretical calculation demonstrate that DOLE can exhibit high-level inhibition property.

Vinylene carbonate, toluene and diethyl ether as electrolyte additives for a wide-temperature range operating of EDLCs

This work presents the development of an electrolyte solution based on 1.2 M TEMA BF4 AN:EA (3:1) electrolyte mixture modified with diethyl ether (5–20 vol%), or toluene (5–20 vol%), or vinylene carbonate (0.5–5 vol%) ensuring stable operation of the supercapacitors (SC) in a wide temperature range from −68 °C to +60 °C. Supercapacitor cells with electrolytes of various volume ratios of components were investigated using cyclic voltammetry, galvanostatic charge-discharge, and impedance spectroscopy (EIS) methods. Specific conductivity values have been measured and compared with electrochemical impedance data. It was shown that the additives to the base electrolyte blend expand the operating temperature range of the SC cells by at least 7–8°, enabling its exploitation at lower temperatures (−68 °C). It has been established that the addition of 10 vol% of diethyl ether or toluene stabilizes the operation of the SC at −65 °C, demonstrating stability during long-term exposure. Vinylene carbonate additive most effectively stabilizes the work of the SC at high temperatures (+60 °C). Diethyl ether as an additive effectively promotes the operation of SC only at low temperatures (−68 °C), while at high temperatures, it leads to the degradation of SC.

Impedance investigation of the surface film formed on aluminum alloy exposed to nuclear reactor emergency core coolant

A method was proposed for in-situ evaluating the thickness and resistivity of the oxide/hydroxide film formed on the surface of aluminum alloy exposed to sump water formed in the containment after a loss-of-coolant accident. The evaluation entailed fitting a model for the film impedance, which has film thickness and other variables describing the resistivity profile of the film along its thickness direction as fitting parameters, to the practically measured electrochemical impedance data. The obtained resistivity profiles implied that the films formed at pHs25°C 7, 8, 9, 10, and 11 all had a duplex structure; compared to the outer layer in contact with the solution, the inner layer of the film had a much higher resistivity and was inferred to be denser and provide most of the protectiveness of the film. Both the thickness and the total resistance of the film decreased with the increasing solution pH25°C, suggesting that the films formed in more alkaline solutions had less protectiveness against corrosion, consistent with the increasing aluminum alloy corrosion rates previously identified.

A novel method for identifying corrosion types and transitions based on Adaboost and electrochemical noise

PurposeThe purpose of this paper is to identify corrosion types and corrosion transitions by a novel electrochemical noise analysis method based on Adaboost.Design/methodology/approachThe corrosion behavior of Q235 steel was investigated in typical passivation, uniform corrosion and pitting solution by electrochemical noise. Nine feature parameters were extracted from the electrochemical noise data based on statistical analysis and shot noise theory. The feature parameters were analysis by Adaboost to train model and identify corrosion types. The trained Adaboost model was used to identify corrosion type transitions.FindingsAdaboost algorithm can accurately identify the corrosion type, and the accuracy rate is 99.25%. The identification results of Adaboost for the corrosion type are consistent with corroded morphology analysis. Compared with other machine learning, Adaboost can identify corrosion types more accurately. For corrosion type transition, Adaboost can effectively identify the transition from passivation to uniform corrosion and from passivation to pitting corrosion consistent with corroded morphology analysis.Originality/valueAdaboost is a suitable method for prediction of corrosion type and transitions. Adaboost can establish the classification model of metal corrosion, which can more conveniently and accurately explore the corrosion types. Adaboost provides important reference for corrosion prediction and protection.

Zinc Electrode Cycling in Deep Eutectic Solvent Electrolytes: An Electrochemical Study.

Among post-lithium ion battery technologies, rechargeable chemistries with Zn anodes bear notable technological promise owing to their high theoretical energy density, lower manufacturing cost, availability of raw materials and inherent safety. However, Zn anodes, when employed in aqueous electrolytes, suffer from hydrogen evolution, passivation, and shape changes. Alternative electrolytes can help tackle these issues, preserving the green and safe characteristics of aqueous-based ones. Deep eutectic solvents (DESs) are promising green and low-cost non-aqueous solvents for battery electrolytes. Specifically, the cycling of Zn anodes in DESs is expected to be reversible, chiefly owing to their dendrite-suppression capability. Nevertheless, apart from a few studies on Zn plating, insight into the cathodic-anodic electrochemistry of Zn in DESs is still very limited. In view of developing DES-based battery electrolytes, it is crucial to consider that a potential drawback might be their low ionic conductivity. Water molecules can be added to the eutectic mixtures by up to 40% to increase the diffusion coefficient of the electroactive species and lower the electrolyte viscosity without destroying the eutectic nature. In this study, we address the electrochemistry of Zn in two different hydrated DESs (ChU and ChEG with ~30% H2O). Fundamental electrokinetic and electrocrystallization studies based on cyclic voltammetry and chronoamperometry at different cathodic substrates are completed with a galvanostatic cycling test of Zn|Zn symmetric CR2032 coin cells, SEM imaging of electrodes and in situ SERS spectroscopy. This investigation concludes with the proposal of a specific DES/H2O/ZnSO4-based electrolyte that exhibits optimal functional performance, rationalized on the basis of fundamental electrochemical data, morphology evaluation and modeling of the cycling response.

Folding-Induced Promotion of Proton-Coupled Electron Transfers via Proximal Base for Light-Driven Water Oxidation.

Proton-coupled electron-transfer (PCET) processes play a key role in biocatalytic energy conversion and storage, for example, photosynthesis or nitrogen fixation. Here, we report a series of bipyridine-containing di- to tetranuclear Ru(bda) macrocycles 2 C-4 C (bda: 2,2'-bipyridine-6,6'-dicarboxylate) to promote O-O bond formation. In photocatalytic water oxidation under neutral conditions, all complexes 2 C-4 C prevail in a folded conformation that support the water nucleophilic attack (WNA) pathway with remarkable turnover frequencies of up to 15.5 s-1 per Ru unit respectively. Single-crystal X-ray analysis revealed an increased tendency for intramolecular π-π stacking and preorganization of the proximal bases close to the active centers for the larger macrocycles. H/D kinetic isotope effect studies and electrochemical data demonstrate the key role of the proximal bipyridines as proton acceptors in lowering the activation barrier for the crucial nucleophilic attack of H2 O in the WNA mechanism.

Folding‐Induced Promotion of Proton‐Coupled Electron Transfers via Proximal Base for Light‐Driven Water Oxidation

AbstractProton‐coupled electron‐transfer (PCET) processes play a key role in biocatalytic energy conversion and storage, for example, photosynthesis or nitrogen fixation. Here, we report a series of bipyridine‐containing di‐ to tetranuclear Ru(bda) macrocycles 2 C–4 C (bda: 2,2′‐bipyridine‐6,6′‐dicarboxylate) to promote O−O bond formation. In photocatalytic water oxidation under neutral conditions, all complexes 2 C–4 C prevail in a folded conformation that support the water nucleophilic attack (WNA) pathway with remarkable turnover frequencies of up to 15.5 s−1 per Ru unit respectively. Single‐crystal X‐ray analysis revealed an increased tendency for intramolecular π‐π stacking and preorganization of the proximal bases close to the active centers for the larger macrocycles. H/D kinetic isotope effect studies and electrochemical data demonstrate the key role of the proximal bipyridines as proton acceptors in lowering the activation barrier for the crucial nucleophilic attack of H2O in the WNA mechanism.

Comparison of the Electrochemical Response of Carbon-Fiber-Reinforced Plastic (CFRP), Glassy Carbon, and Highly Ordered Pyrolytic Graphite (HOPG) in Near-Neutral Aqueous Chloride Media

Carbon-fiber-reinforced polymers (CFRP), being conductive, are capable of supporting cathodic oxygen reduction reactions (ORR) and thus promote galvanic corrosion when coupled to many metallic materials. Hence, understanding cathodic processes at carbon surfaces is critical to developing new strategies for the corrosion protection of multi-material assemblies. In the present work, the electrochemical responses of CFRP, glassy carbon, and HOPG (Highly Ordered Pyrolytic Graphite) have been evaluated in a quiescent 50 mM NaCl solution, and their respective activities towards ORR have been ranked. Employing the averages of the specific charges (CFRP, 129.52 mC cm−2; glassy carbon, 89.95 mC cm−2; HOPG, 60.77 mC cm−2) passed during 1 h polarization of each of the 3 carbon surfaces at −1000 mVSCE in the test media as a ranking criterion, the propensities of the 3 carbon surfaces (CFRP, GC, and HOPG) to support cathodic activities that can lead to anodic metal dissolution on galvanic coupling to metallic materials are ranked thusly; CFRP > GC > HOPG. This ranking is consistent with the trend of capacitance values obtained in this work: CFRP (19.5 to 34.5 μF cm−2), glassy carbon (13.6 to 85.5 μF cm−2), and HOPG (1.4 to 1.8 μF cm−2). A comparison of electrochemical data at potentials relevant to galvanic coupling to metals indicated that at these cathodic potential(s) the CFRP surface is the most electrochemically active of the studied carbon surfaces. On the basis of the values and trends of the electrochemical parameters evaluated, it is postulated that the observed differences in the electrochemical responses of these 3 carbon-rich surfaces to ORR are significantly due to differences in the proportions of edge sites present on each carbon surface. These results could provide valuable insights on plausible strategies for designing carbon surfaces and carbon fiber composites with reduced activity toward ORR for corrosion protection applications or enhanced activity towards ORR for energy applications.

Electrochemical data on the corrosion inhibition performance of admixed Citrus paradisi and Zingiber officinale oil extracts in 0.5 M H2SO4 solution

Data output on the protection performance of the combined admixture of Citrus paradisi and Zingiber officinale oil extracts (CPZO) on the corrosion inhibition of mild steel in 0.5 M H2SO4 solution are presented and described. CPZO effectively suppressed the electrochemical reactions on the steel surface with final inhibition values ranging between 95.16 mm/y at 1 % CPZO and 96.61 % at 3.5 % CPZO concentration. CPZO reduced the corrosion rate of the steel from the value of 184.48 % at 0 % CPZO concentration to values between 3.45 mm/y and 8.94 mm/y in the presence of CPZO extract. CPZO performance remained marginally steady with respect to its concentration. However, variation with time was significant till 240 h. Effective inhibition performance was attained at 48 h of exposure. Calculated data from ANOVA analysis shows exposure was the only statistically relevant parameter at 98.63 % compared to 0.75 % for CPZO concentration. Adsorption of CPZO molecules on the steel aligns with the Langmuir and Frumkin isotherm models with correlation coefficient values of 0.9997 and 0.7292. Thermodynamic calculations depict chemisorption adsorption mechanism with ΔG values ranging between −46.13 and −43.23 KJ/mol. Optical images of the inhibited and non-inhibited steel significantly contrast each other.

Stepwise conversion of the Cys6[4Fe-3S] to a Cys4[4Fe-4S] cluster and its impact on the oxygen tolerance of [NiFe]-hydrogenase.

The membrane-bound [NiFe]-hydrogenase of Cupriavidus necator is a rare example of a truly O2-tolerant hydrogenase. It catalyzes the oxidation of H2 into 2e- and 2H+ in the presence of high O2 concentrations. This characteristic trait is intimately linked to the unique Cys6[4Fe-3S] cluster located in the proximal position to the catalytic center and coordinated by six cysteine residues. Two of these cysteines play an essential role in redox-dependent cluster plasticity, which bestows the cofactor with the capacity to mediate two redox transitions at physiological potentials. Here, we investigated the individual roles of the two additional cysteines by replacing them individually as well as simultaneously with glycine. The crystal structures of the corresponding MBH variants revealed the presence of Cys5[4Fe-4S] or Cys4[4Fe-4S] clusters of different architecture. The protein X-ray crystallography results were correlated with accompanying biochemical, spectroscopic and electrochemical data. The exchanges resulted in a diminished O2 tolerance of all MBH variants, which was attributed to the fact that the modified proximal clusters mediated only one redox transition. The previously proposed O2 protection mechanism that detoxifies O2 to H2O using four protons and four electrons supplied by the cofactor infrastructure, is extended by our results, which suggest efficient shutdown of enzyme function by formation of a hydroxy ligand in the active site that protects the enzyme from O2 binding under electron-deficient conditions.