Electrocatalysis and Amperometric Detection of the Reduced Form of Nicotinamide Adenine Dinucleotide at Toluidine Blue/Zinc Oxide Coated Electrodes

Thin toluidine blue (TBO) and zinc oxide (ZnO) hybrid films have been grown on glassy carbon electrode (GCE) and indium tin oxide coated (SnO₂) glass electrodes by using cyclic voltammetry (CV). Scanning electron microscopy (SEM) images revealed spherical and beads‐like shape of highly oriented TBO/ZnO hybrid films. Energy dispersive spectrometry (EDS) results declared that the films composed mainly of Zn and O. Moreover, TBO/ZnO hybrid films modified electrode is electrochemically active, dye molecules were not easily leached out from the ZnO matrix and the hybrid films can be considered for potential applications as sensor for amperometric determination of reduced nicotinamide adenine dinucleotide (NADH) at 0.0 V. A linear correlation between electrocatalytic current and NADH concentration was found to be in the range between 25 μM and 100 μM in phosphate buffer. In addition, we observed that dopamine, ascorbic acid and uric acid are not interference in amperometric detection of NADH in this proposed method. In addition, TBO/ZnO hybrid film modified electrode was highly stable and its response to the NADH also remained relentless.     

Electrochemical and catalytic investigation of carbon paste modified with Toluidine Blue O covalently immobilised on silica gel

Toluidine Blue O (TBO) was covalently bound on silica gel and mixed with graphite powder and paraffin oil to produce modified carbon paste electrodes. The formal potential (E°′) of the covalently bound TBO was found to be −100 mV versus Ag|AgCl (KCl sat.) at pH 7.0 and the E°′ varied less than anticipated for a 2-electron-proton type mediator with pH. The bound TBO was found to act as an efficient electron acceptor for NADH as well as electron donor for oxidised horseradish peroxidase (HRP). The kinetics and the mechanism of the reaction between NADH and TBO were investigated with cyclic voltammetry and using a rotating disc electrode. Further experiments were done in the flow injection mode injecting different concentrations of NADH. Similar studies were done in the presence of hydrogen peroxide when HRP was adsorbed onto the TBO modified silica gel carbon paste electrodes.     

Sequential electron-transfer and proton-transfer pathways in hydride-transfer reactions from dihydronicotinamide adenine dinucleotide analogues to non-heme oxoiron(IV) complexes and p-chloranil. Detection of radical cations of NADH analogues in acid-promoted hydride-transfer reactions

Hydride transfer from dihydronicotinamide adenine dinucleotide (NADH) analogues, such as 10-methyl-9,10-dihydroacridine (AcrH 2) and its derivatives, 1-benzyl-1,4-dihydronicotinamide (BNAH), and their deuterated compounds, to non-heme oxoiron(IV) complexes such as [(L)Fe (IV)(O)] (2+) (L = N4Py, Bn-TPEN, and TMC) occurs to yield the corresponding NAD (+) analogues and non-heme iron(II) complexes in acetonitrile. Hydride transfer from the NADH analogues to p-chloranil (Cl 4Q) also occurs to produce the corresponding NAD (+) analogues and the hydroquinone anion (Cl 4QH (-)). The logarithms of the observed second-order rate constants (log k H) of hydride transfer from NADH analogues to non-heme oxoiron(IV) complexes are linearly correlated with those of hydride transfer from the same series of NADH analogues to Cl 4Q, including similar kinetic deuterium isotope effects. The log k H values of hydride transfer from NADH analogues to non-heme oxoiron(IV) complexes are also linearly correlated with those of deprotonation of the radical cations of NADH analogues. Such linear correlations indicate that overall hydride-transfer reactions of NADH analogues to both non-heme oxoiron(IV) complexes and Cl 4Q occur via electron transfer from NADH analogues to the oxoiron(IV) complexes, followed by rate-limiting deprotonation from the radical cations of NADH analogues and subsequent rapid electron transfer from the deprotonated radicals to the Fe(III) complexes to yield the corresponding NAD (+) analogues and the Fe(II) complexes. The electron-transfer pathway was accelerated by the presence of perchloric acid, and the resulting radical cations of NADH analogues were detected by electron spin resonance spectroscopy and UV-vis spectrophotometry in the acid-promoted hydride-transfer reactions from NADH analogues to non-heme oxoiron(IV) complexes. This result provides the first direct evidence that a hydride transfer from NADH analogues to non-heme oxoiron(IV) complexes proceeds via an electron-transfer pathway.     

Photocatalytic hydrogen evolution under highly basic conditions by using Ru nanoparticles and 2-phenyl-4-(1-naphthyl)quinolinium ion

Photocatalytic hydrogen evolution with a ruthenium metal catalyst under basic conditions (pH 10) has been made possible for the first time by using 2-phenyl-4-(1-naphthyl)quinolinium ion (QuPh(+)-NA), dihydronicotinamide adenine dinucleotide (NADH), and Ru nanoparticles (RuNPs) as the photocatalyst, electron donor, and hydrogen-evolution catalyst, respectively. The catalytic reactivity of RuNPs was virtually the same as that of commercially available PtNPs. Nanosecond laser flash photolysis measurements were performed to examine the photodynamics of QuPh(+)-NA in the presence of NADH. Upon photoexcitation of QuPh(+)-NA, the electron-transfer state of QuPh(+)-NA (QuPh(?)-NA(?+)) is produced, followed by formation of the π-dimer radical cation with QuPh(+)-NA, [(QuPh(?)-NA(?+))(QuPh(+)-NA)]. Electron transfer from NADH to the π-dimer radical cation leads to the production of 2 equiv of QuPh(?)-NA via deprotonation of NADH(?+) and subsequent electron transfer from NAD(?) to QuPh(+)-NA. Electron transfer from the photogenerated QuPh(?)-NA to RuNPs results in hydrogen evolution even under basic conditions. The rate of electron transfer from QuPh(?)-NA to RuNPs is much higher than the rate of hydrogen evolution. The effect of the size of the RuNPs on the catalytic reactivity for hydrogen evolution was also examined by using size-controlled RuNPs. RuNPs with a size of 4.1 nm exhibited the highest hydrogen-evolution rate normalized by the weight of RuNPs.     

Amperometric determination of lactate dehydrogenase based on a carbon fiber microcylinder electrode modified covalently with Toluidine Blue O by acylation

A method has been developed for the modification of a carbon fiber microcylinder electrode with acylation. The stability and surface coverage of the Toluidine Blue O-modified microelectrode were studied by cyclic voltammetry. The modified electrode showed significant activity for the electrocatalytic oxidation of NADH in pH 6.8-7.8 solution. The catalytic current increased linearly with increasing concentration of NADH from 4.0 x 10(-5) to 1.5 x 10(-3) M. A simple amperometric determination based on electrochemical detection of NADH produced from the enzymatic reaction of lactate with NAD(+) under the catalysic effect of lactate dehydrogenase (LDH) is reported. The experimental factors which had primary influence on the analytical performance were studied. The sensor had a linear response over a range of LDH concentrations from 5.0 U l(-1) to 200 U l(-1) at -0.2 V vs. SCE under optimum conditions. A satisfactory result was obtained for the determination of LDH in clinical blood samples.     

Efficient catalytic interconversion between NADH and NAD+ accompanied by generation and consumption of hydrogen with a water-soluble iridium complex at ambient pressure and temperature

Regioselective hydrogenation of the oxidized form of β-nicotinamide adenine dinucleotide (NAD(+)) to the reduced form (NADH) with hydrogen (H(2)) has successfully been achieved in the presence of a catalytic amount of a [C,N] cyclometalated organoiridium complex [Ir(III)(Cp*)(4-(1H-pyrazol-1-yl-κN(2))benzoic acid-κC(3))(H(2)O)](2) SO(4) [1](2)·SO(4) under an atmospheric pressure of H(2) at room temperature in weakly basic water. The structure of the corresponding benzoate complex Ir(III)(Cp*)(4-(1H-pyrazol-1-yl-κN(2))-benzoate-κC(3))(H(2)O) 2 has been revealed by X-ray single-crystal structure analysis. The corresponding iridium hydride complex formed under an atmospheric pressure of H(2) undergoes the 1,4-selective hydrogenation of NAD(+) to form 1,4-NADH. On the other hand, in weakly acidic water the complex 1 was found to catalyze the hydrogen evolution from NADH to produce NAD(+) without photoirradiation at room temperature. NAD(+) exhibited an inhibitory behavior in both catalytic hydrogenation of NAD(+) with H(2) and H(2) evolution from NADH due to the binding of NAD(+) to the catalyst. The overall catalytic mechanism of interconversion between NADH and NAD(+) accompanied by generation and consumption of H(2) was revealed on the basis of the kinetic analysis and detection of the catalytic intermediates.     

Histochemical staining and quantification of dihydrolipoamide dehydrogenase diaphorase activity using blue native PAGE

Mammalian mitochondrial dihydrolipoamide dehydrogenase (DLDH, EC 1.8.1.4) catalyzes NAD(+)-dependent oxidation of dihydrolipoamide in vivo and can also act as a diaphorase catalyzing in vitro nicotinamide adenine dinucleotide (reduced form) (NADH)-dependent reduction of electron-accepting molecules such as ubiquinone and nitroblue tetrazolium (NBT). In this paper, we report a gel-based method for histochemical staining and quantification of DLDH diaphorase activity using blue native PAGE (BN-PAGE). Rat brain mitochondrial extracts, used as the source of DLDH, were resolved by nongradient BN-PAGE (9%), which was followed by diaphorase activity staining using NADH as the electron donor and NBT as the electron acceptor. It was shown that activity staining of DLDH diaphorase was both protein amount- and time-dependent. Moreover, this in-gel activity-staining method was demonstrated to be in good agreement with the conventional spectrophotometric method that measures DLDH dehydrogenase activity using dihydrolipoamide as the substrate. The method was applied to determine levels of DLDH diaphorase activity in several rat tissues other than the brain, and the results indicated a similar level of DLDH diaphorase activity for all the tissues examined. Finally, the effects of thiol-reactive reagents such as N-ethylmaleimide (NEM) and nitric oxide donors on DLDH diaphorase activity were evaluated, demonstrating that, with this method, DLDH diaphorase activity can be determined without having to remove these thiol-reactive reagents that may otherwise interfere with spectrophotometric measurement of DLDH dehydrogenase activity. The gel-based method can also be used as a means to isolate mitochondrial DLDH that is to be analyzed by mass spectral techniques in studying DLDH post-translational modifications.     

Low-potential nicotinamide adenine dinucleotide detection at a glassy carbon electrode modified with toluidine blue O functionalized multiwall carbon nanotubes.

The toluidine blue O (TBO) functionalized multiwall carbon nanotubes (MWNTs) nanomaterials (TBO-MWNTs) were prepared by assembling TBO onto the surface of a MWNTs modified glassy carbon (GC) electrode. Also TBO-MWNTs modified GC electrodes exhibiting a strong and stable electrocatalytic response toward beta-nicotinamide adenine dinucleotide (NADH) were described. Compared with a bare GC electrode, the TBO-MWNTs modified GC electrodes could decrease the oxidization overpotential of NADH by 730 mV, with a peak current at 0.0 V, since there was a positively synergistic electrocatalytic effect between the MWNTs and TBO toward NADH. Furthermore, the TBO-MWNTs modified GC electrodes had perfect performances, such as a low detection limit (down to 0.5 microM), being very stable (the current diminutions is lower than 6% in a period over 35 min), a fast response (within 3 s), and a wide linear range (from 2.0 microM to 3.5 mM). Such an ability of TBO-MWNTs to promote the NADH electron-transfer reaction suggests great promise for dehydrogenase-based amperometric biosensors.     

Gold nanoparticles: catalyst for the oxidation of NADH to NAD(+)

Nicotinamide adenine dinucleotide is an important coenzyme involved in the production of ATP, the fuel of energy, in every cell. It alternates between the oxidized form NAD(+) and the reduced form dihydronicotinamide adenine dinucleotide (NADH) and serves as a hydrogen and electron carrier in the cellular respiratory processes. In the present work, the catalytic effect of gold nanoparticles on the oxidization of NADH to NAD(+) was investigated. The addition of gold nanoparticles was found to quench the NADH fluorescence intensities but had no effect on the fluorescence lifetime. This suggested that the fluorescence quenching was not due to coupling with the excited state, but due to changing the ground state of NADH. The intensity of the 340 nm absorption band of NADH was found to decrease while that of the 260 nm band of NAD(+) was found to increase as the concentration of gold nanoparticles increased. This conversion reaction was further supported by nuclear magnetic resonance and mass spectroscopy. The effect of the addition of NADH was found to slightly red shift and increase the intensity of the surface plasmon absorption band of gold nanoparticles at 520 nm. This gives a strong support that the conversion of NADH to NAD(+) is occurring on the surface of the gold nanoparticles, i.e. NADH is surface catalyzed by the gold nanoparticles. The catalytic property of this important reaction might have important future applications in biological and medical fields.     

l- and d-Lactate assay in real milk samples with immobilized enzyme reactors and graphite electrode

A sensitive flow system for the determination of l- and d-lactate in milk samples is described. l- and d-Lactate dehydrogenase, LDH, were immobilized on aminopropyl-controlled pore glass beads. l- and d-Lactate are oxidized to pyruvate in the presence of NAD(+) and NADH is produced. The electrochemical determination of NADH allows the measurement of the substrate involved in the reaction. We used a graphite-based anode sensor without any mediator at +500 mV vs. Ag/AgCl. The analytes were measured, in standard solutions, in the concentration range from 1 x 10(-6) to 4 x 10(-4)M using 1 mM NAD(+) concentration and 0.1M Tris buffer pH 9. Experiments with real milk samples showed large values of currents probably due to electroactive substances usually contained in milk. To eliminate interfering compounds a microdialysis probe coupled with a pre-oxidizing cell was used. This method of pre-treatment removes the interfering substances, but leaves the analytes under study unaffected. The procedure allows the determination of l- and d-lactate in milk samples in the concentration range from 1 x 10(-5) to 5 x 10(-4)M. The assay was applied to monitor continuously the bacterial fermentation of Staphylococcus aureus in UHT milk as an example of possible contamination detection in the manufacturing process.     

Determination of lactate and lactic dehydrogenase by biamperometric monitoring of hexacyanoferrate(II) in a flowing stream

Summary Open tubular carbon electrodes were used to monitor the biamperometric current of hexacyanoferrate(II) ion produced by the LDH catalyzed reaction between hexacyanoferrate(III) ion and lactate, and that produced from the diaphorase catalyzed reaction between hexacyanoferrate(III) ion and NADH, product from the LDH catalyzed reaction between lactate and NAD. Reagents and samples were mixed in a flow stream. Reaction takes place between the mixing point and the electrodes, and measurements are taken after a constant time interval. Lactate and LDH standards in serum control solutions were measured using the NAD dependent reaction.

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Bestimmung von Lactat und Lactatdehydrogenase durch biamperometrische Messung von Hexacyanoferrat(II) im Durchfluß

Zusammenfassung Kohlenstoff-Röhrenelektroden wurden zur Messung des biamperometrischen Stromes des Hexacyanoferrat(II)-ions eingesetzt, das aus der LDH-katalysierten Reaktion zwischen Hexacyanoferrat(III) und Lactat bzw. der Diaphorase-katalysierten Reaktion zwischen Hexacyanoferrat(III) und NADH (Produkt der LDH-katalysierten Reaktion zwischen Lactat und NAD) stammte. Die Reagentien wurden mit den Proben im Durchfluß vermischt. Die Reaktion fand zwischen der Mischungsstelle und den Elektroden statt und die Messungen wurden nach einem Bestimmten Zeitintervall durchgeführt. Mit Hilfe der NAD-abhängigen Reaktion wurden Lactat- und LDH-Standards in Serumkontrollösungen bestimmt.

     

Enzyme monolayer- and bilayer-modified electrodes with diaphorase and dehydrogenases

Diaphorase was immobilized covalently as a monolayer on a tin(IV) oxide electrode, and the diaphorase electrode thus obtained responded to NADH amperometrically in the presence of ferricyanide or 2,6-dichloroindophenol as the electron mediator. The response was one to two orders of magnitude larger than that of a bare electrode. Further derivatization of the diaphorase electrode with a dehydrogenase (glucose, lactate or alcohol dehydrogenase), which reduces NAD to NADH by reaction with the substrate, yielded dehydrogenase/diaphorase heterobilayer-modified electrodes. These electrodes functioned as sensors for the respective substrate with NAD and ferricyanide as the mediators. Each bilayer electrode responded to the substrate only in the presence of added NAD; this provides evidence for the essential contribution of diaphorase to the sensor performance. As much as 60 to 80% of the electron mediator reduced by the enzymatic reaction was utilized in the amperometric response.     

Esterification of borate with NAD+ and NADH as studied by electrospray ionization mass spectrometry and 11B NMR spectroscopy

This paper describes for the first time the direct measurement of boric acid (B(OH)(3)) and borate (B(OH)(4) (-)) adduction to NAD(+) and NADH by electrospray ionization mass spectrometry (ESI-MS) and (11)B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD(+) forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD(+), and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD(+) were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD(+) and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 micro M for NAD(+) and boric acid (1 : 1) to detect NAD(+)-borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance.     

Platinum nanoparticles have an activity similar to mitochondrial NADH:ubiquinone oxidoreductase

This study was designed to examine if platinum nanoparticles have an activity similar to mitochondrial complex I, NADH:ubiquinone oxidoreductase. Platinum nanoparticles were prepared by a citrate reduction of H(2)PtCl(6) and protected by citrate itself and pectin (CP-Pt). Time- and dose-dependent decreases in NADH and a time-dependent increase in NAD(+) were observed in the presence of 50muM CP-Pt; these observations were made using a spectrophotometric method in which the maximum absorption spectra at 340 and 260nm were used for NADH and NAD(+), respectively. The required platinum concentration in CP-Pt to achieve a 50% oxidation of NADH for 3h was approximately 20muM, and this NADH oxidation did not require oxygen as an electron acceptor. We also verified NAD(+) formation using an NAD(+)/NADH quantification kit. The absorption peak shift from 278 to 284nm of 2,3-dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone (CoQ(1)) was observed by incubating CoQ(1) with CP-Pt in an aqueous buffer. A further analysis with HPLC revealed the reduction of CoQ(1) to CoQ(1)H(2) by CP-Pt. As a whole, platinum nanoparticles have an NADH:ubiquinone oxidoreductase-like activity. This suggests that platinum nanoparticles are a potential medicinal substance for oxidative stress diseases with suppressed mitochondrial complex I.     

示差脉冲伏安法测定乳酸脱氢酶活性及纳米微粒生物毒性检测新方法研究

采用示差脉冲伏安法，在乳酸脱氢酶(LDH)酶促体系“丙酮酸盐 + NADH +H⁺ (?) 乳酸盐 + NAD⁺”中，通过检测NAD⁺还原峰电流的变化，测定了不同条件下(不同酶用量、缓冲液pH值以及温度)LDH的活性、酶促体系的米氏常数K_m^NADH以及最大反应速率v_max。并且在最佳实验条件下，通过检测LDH活性的改变，实验考察了3种纳米物质(ZnS，TiO₂(R)和TiO₂(A))对乳酸脱氢酶酶促体系的影响。     

Multi-walled carbon nanotubes decrease lactate dehydrogenase activity in enzymatic reaction

The oxidation of 1, 4-nicotinamide adenine dinucleotide (NADH) to β-nicotinamide adenine dinucleotide (NAD(+)) coupled with converting of pyruvic acid (PA) to lactate catalyzed by lactate dehydrogenase (LDH), NADH+PA+H(+)?LDHNAD(+)+Lactate, was widely adopted to quantify the cell's death, membrane infiltration and proliferation induced by potential toxins. The differential pulse voltammetry (DPV) cathodic signal of NAD(+) at a hanging mercury drop electrode (HMDE) showed LDH activity decreased with the elevating dosages of and the pre-contact time (t(c)) with multi-walled carbon nanotubes (MWCNTs). Comparison of kinetic rate constant of above enzymatic reaction (ER) was able to sensitively assay the adverse influence of MWCNTs. Toxic concentration of altering relative LDH activity by 50% (TC(50)) of MWCNTs was derived to be 40mg/L. TC(50) values indicated a decrease toxicity order Al (III)>MWCNTs>nano-Al(13)>50nm-Al(2)O(3)≥1000nm-Al(2)O(3). The negatively charged surfaces of these nanoparticles (NPs) might be a main cause for the decrement of LDH activity. This decrement was capable to result in the underestimation of the toxicity of NPs in classic LDH assays. This observation would highlight to settle down contradictory medium dependent toxicity of MWCNTs among the literature.     

Photochemical Production of NADH Using Cobaloxime Catalysts and Visible-Light Energy

In this study, a visible-light-driven photocatalytic system for the generation of dihydronicotinamide adenine dinucleotide (NADH) from aqueous protons was examined using cobaloxime as a catalyst, eosin as a photosensitizer, and triethanolamine as a sacrificial electron donor. Irradiation of a reaction solution containing cobaloxime, eosin, and triethanolamine (TEOA) converted NAD(+) to NADH with a yield of 36% in a phosphate buffer. The reaction rates for the production of NADH were dependent on the concentrations of the catalyst, NAD(+), and TEOA. Introduction of an electron-donating or -withdrawing substituent in the para position of the pyridine changed the rate constant and affected the conversion efficiency. The rates obtained by the different substituents were linearly correlated with the Hammett coefficients of the introduced substituents. Last, reduction of CO(2) was carried out in the presence of formate dehydrogenase using NADH photochemically generated using the cobaloxime/eosin/TEOA system.     

Au nanoparticle-enhanced surface plasmon resonance sensing of biocatalytic transformations

N-(3-Aminopropyl)-N'-methyl-4,4'-bipyridinium is coupled to tiopronin-capped Au nanoparticles (diameter ca. 2 nm) to yield methyl(aminopropyl)viologen-functionalized Au nanoparticles (MPAV(2+)-Au nanoparticles). In situ electrochemical surface plasmon resonance (SPR) measurements are used to follow the electrochemical deposition of the bipyridinium radical cation modified Au nanoparticles on an Au-coated glass surface and the reoxidation and dissolution of the bipyridinium radical cation film. The MPAV(2+)-functionalized Au nanoparticles are also employed for the amplified SPR detection of NAD(+) and NADH cofactors. By SPR monitoring the partial biocatalyzed dissolution of the bipyridinium radical cation film in the presence of diaphorase (DP) NAD(+) is detected in the concentration range of 1x10(-4) M to 2x10(-3) M. Similarly, the diaphorase-mediated formation of the bipyridinium radical cation film on the Au-coated glass surface by the reduction of the MPAV(2+)-functionalized Au nanoparticles by NADH is used for the amplified SPR detection of NADH in the concentration range of 1x10(-4) M to 1x10(-3) M.     

Electrochemical sensing platform based on the carbon nanotubes/redox mediators-biopolymer system

A new electrochemical sensing platform was developed that relied on synergy between carbon nanotubes (CNT) and redox mediators that were co-immobilized in the biopolymer chitosan (CHIT). To demonstrate the concept, the redox mediator Toluidine Blue O (TBO) and CNT were integrated in CHIT and used for the determination of a reduced form of nicotinamide adenine dinucleotide (NADH). As compared to CHIT-TBO, the CHIT-TBO/CNT films displayed large amplification of a current due to the TBO-mediated oxidation of NADH at -0.10 V. This was discussed in terms of the TBO/CNT synergy that resulted in the improved charge propagation through the CHIT-TBO/CNT matrix.     

A modular system for regeneration of NAD cofactors using graphite particles modified with hydrogenase and diaphorase moieties

Pyrolytic graphite particles modified with hydrogenase and an NAD(+)/NADH cycling enzyme provide a modular heterogeneous catalyst system for regeneration of oxidised or reduced nicotinamide cofactors using H(2) and H(+) as electron source or sink. Particles can be tuned for cofactor supply under different conditions by appropriate choice of hydrogenase.