Oriented immobilization of a membrane-bound hydrogenase onto an electrode for direct electron transfer

The interaction of redox enzymes with electrodes is of great interest for studying the catalytic mechanisms of redox enzymes and for bioelectronic applications. Efficient electron transport between the biocatalysts and the electrodes has achieved more success with soluble enzymes than with membrane enzymes because of the higher structural complexity and instability of the latter proteins. In this work, we report a strategy for immobilizing a membrane-bound enzyme onto gold electrodes with a controlled orientation in its fully active conformation. The immobilized redox enzyme is the Ni-Fe-Se hydrogenase from Desulfovibrio vulgaris Hildenborough, which catalyzes H(2)-oxidation reversibly and is associated with the cytoplasmic membrane by a lipidic tail. Gold surfaces modified with this enzyme and phospholipids have been studied by atomic force microscopy (AFM) and electrochemical methods. The combined study indicates that by a two-step immobilization procedure the hydrogenase can be inserted via its lipidic tail onto a phospholipidic bilayer formed over the gold surface, allowing only mediated electron transfer between the enzyme and electrode. However, a one-step immobilization procedure favors the formation of a hydrogenase monolayer over the gold surface with its lipidic tail inserted into a phospholipid bilayer formed on top of the hydrogenase molecules. This latter method has allowed for the first time efficient electron transfer between a membrane-bound enzyme in its native conformation and an electrode.     

Electrocatalysis of the hydrogen production by [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough

Hydrogenases are enzymes which catalyze hydrogen production/consumption reactions. In this paper, the [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough is shown to catalyze the direct hydrogen production from protons, in the absence of any promoter, at a basal pyrolytic graphite electrode using cyclic voltammetry techniques. The effect of several parameters upon catalytic current is investigated: pH, hydrogenase concentration, ionic strength, competition with another protein (bovine serum albumin), cycling repetition, mode of electrode polishing. The extent and efficiency of the hydrogenase electroactivity are examined in the presence of either an artificial electron carrier (methylviologen) or a physiological partner cytochrome c₃ isolated from the same bacterial strain. Results suggest that the electron-transfer process is essentially controlled by a complex between both hydrogenase and cytochrome c₃. Electrocatalysis appears to be largely governed by the adsorption of hydrogenase on the electrode surface very likely involving hydrophobic interactions.     

FeFe]-hydrogenase-catalyzed H2 production in a photoelectrochemical biofuel cell

The Clostridium acetobutylicum [FeFe]-hydrogenase HydA has been investigated as a hydrogen production catalyst in a photoelectrochemical biofuel cell. Hydrogenase was adsorbed to pyrolytic graphite edge and carbon felt electrodes. Cyclic voltammograms of the immobilized hydrogenase films reveal cathodic proton reduction and anodic hydrogen oxidation, with a catalytic bias toward hydrogen evolution. When corrected for the electrochemically active surface area, the cathodic current densities are similar for both carbon electrodes, and approximately 40% of those obtained with a platinum electrode. The high surface area carbon felt/hydrogenase electrode was subsequently used as the cathode in a photoelectrochemical biofuel cell. Under illumination, this device is able to oxidize a biofuel substrate and reduce protons to hydrogen. Similar photocurrents and hydrogen production rates were observed in the photoelectrochemical biofuel cell using either hydrogenase or platinum cathodes.     

M?ssbauer characterization of the iron-sulfur clusters in Desulfovibrio vulgaris hydrogenase.

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S](H)) and a binuclear Fe cluster ([2Fe](H)), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2), indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field M?ssbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the CO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters, and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S]-[2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S](H) cluster from the 2+ to the 1+ state, followed by transfer of the reducing equivalent from the [4Fe-4S](H) subcluster to the binuclear [2Fe](H) subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S](H) and the [2Fe](H) subclusters. Implication of such a CO binding effect is discussed.     

High-performance hydrogen production and oxidation electrodes with hydrogenase supported on metallic single-wall carbon nanotube networks

We studied the electrocatalytic activity of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaH2ase) immobilized on single-wall carbon nanotube (SWNT) networks. SWNT networks were prepared on carbon cloth by ultrasonic spraying of suspensions with predetermined ratios of metallic and semiconducting nanotubes. Current densities for both proton reduction and hydrogen oxidation electrocatalytic activities were at least 1 order of magnitude higher when hydrogenase was immobilized onto SWNT networks with high metallic tube (m-SWNT) content in comparison to hydrogenase supported on networks with low metallic tube content or when SWNTs were absent. We conclude that the increase in electrocatalytic activities in the presence of SWNTs was mainly due to the m-SWNT fraction and can be attributed to (i) substantial increases in the active electrode surface area, and (ii) improved electronic coupling between CaH2ase redox-active sites and the electrode surface.     

Photoelectrochemical H2 Evolution with a Hydrogenase Immobilized on a TiO2‐Protected Silicon Electrode

The combination of enzymes with semiconductors enables the photoelectrochemical characterization of electron‐transfer processes at highly active and well‐defined catalytic sites on a light‐harvesting electrode surface. Herein, we report the integration of a hydrogenase on a TiO₂‐coated p‐Si photocathode for the photo‐reduction of protons to H₂. The immobilized hydrogenase exhibits activity on Si attributable to a bifunctional TiO₂ layer, which protects the Si electrode from oxidation and acts as a biocompatible support layer for the productive adsorption of the enzyme. The p‐Si|TiO₂|hydrogenase photocathode displays visible‐light driven production of H₂ at an energy‐storing, positive electrochemical potential and an essentially quantitative faradaic efficiency. We have thus established a widely applicable platform to wire redox enzymes in an active configuration on a p‐type semiconductor photocathode through the engineering of the enzyme–materials interface.     

Photoinduced hydrogen production by direct electron transfer from photosystem I cross-linked with cytochrome c3 to [NiFe]-hydrogenase

The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.     

硫酸溶液中苯酚在Ti/PbO2电极上的循环伏安研究

用循环伏安法研究了Ti/PbO₂电极在苯酚硫酸溶液中的电催化作用. 结果表明, 在硫酸溶液中, Ti/PbO₂阳极对苯酚具有电催化氧化作用. 如果苯酚浓度较低, 产生的吸附态羟基自由基可以将苯酚氧化, 直至完全矿化. 当苯酚浓度较高或产生的羟基自由基量相对较小时, 苯酚或中间产物可吸附在电极表面, 降低电极的真实表面积, 减少电极的活性点, 阻止反应物接近电极表面, 抑制苯酚的进一步氧化. 随着电解时间的延长, 这些吸附物由于逐渐被氧化, 电极活性恢复.     

Catalytic turnover of [FeFe]-hydrogenase based on single-molecule imaging

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ? H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.     

Infrared Spectroscopy During Electrocatalytic Turnover Reveals the Ni‐L Active Site State During H2 Oxidation by a NiFe Hydrogenase

A novel in situ IR spectroscopic approach is demonstrated for the characterization of hydrogenase during catalytic turnover. E. coli hydrogenase 1 (Hyd‐1) is adsorbed on a high surface‐area carbon electrode and subjected to the same electrochemical control and efficient supply of substrate as in protein film electrochemistry during spectral acquisition. The spectra reveal that the active site state known as Ni‐L, observed in other NiFe hydrogenases only under illumination or at cryogenic temperatures, can be generated reversibly in the dark at ambient temperature under both turnover and non‐turnover conditions. The observation that Ni‐L is present at all potentials during turnover under H₂ suggests that the final steps in the catalytic cycle of H₂ oxidation by Hyd‐1 involve sequential proton and electron transfer via Ni‐L. A broadly applicable IR spectroscopic technique is presented for addressing electrode‐adsorbed redox enzymes under fast catalytic turnover.     

Catalysts for hydrogen evolution from the [NiFe] hydrogenase to the Ni2P(001) surface: the importance of ensemble effect

Density functional theory (DFT) was employed to investigate the behavior of a series of catalysts used in the hydrogen evolution reaction (HER, 2H(+) + 2e(-) --> H(2)). The kinetics of the HER was studied on the [NiFe] hydrogenase, the [Ni(PS3*)(CO)](1)(-) and [Ni(PNP)(2)](2+) complexes, and surfaces such as Ni(111), Pt(111), or Ni(2)P(001). Our results show that the [NiFe] hydrogenase exhibits the highest activity toward the HER, followed by [Ni(PNP)(2)](2+) > Ni(2)P > [Ni(PS3*)(CO)](1)(-) > Pt > Ni in a decreasing sequence. The slow kinetics of the HER on the surfaces is due to the fact that the metal hollow sites bond hydrogen too strongly to allow the facile removal of H(2). In fact, the strong H-Ni interaction on Ni(2)P(001) can lead to poisoning of the highly active sites of the surface, which enhances the rate of the HER and makes it comparable to that of the [NiFe] hydrogenase. In contrast, the promotional effect of H-poisoning on the HER on Pt and Ni surfaces is relatively small. Our calculations suggest that among all of the systems investigated, Ni(2)P should be the best practical catalyst for the HER, combining the high thermostability of the surfaces and high catalytic activity of the [NiFe] hydrogenase. The good behavior of Ni(2)P(001) toward the HER is found to be associated with an ensemble effect, where the number of active Ni sites is decreased due to presence of P, which leads to moderate bonding of the intermediates and products with the surface. In addition, the P sites are not simple spectators and directly participate in the HER.     

4-氨基苯硫酚在金电极上电化学转变过程的表面增强拉曼光谱及其理论研究

采用循环伏安法和表面增强拉曼散射现场研究了酸性溶液中4-氨基苯硫酚(PATP)在金电极表面的电化学转变过程, 并结合密度泛函理论(DFT)对光谱进行了指认, 由此确定电极表面的最终产物. 研究结果表明, PATP分子在电极表面首先氧化为阳离子自由基, 该自由基与邻近的分子通过头尾相接生成二聚体4'-巯基-N-苯基苯醌二亚胺, 随后发生水解反应生成4'-巯基-N-苯基苯醌单亚胺. 用DFT在B3LYP/6-311＋G** (C, N, S, H)/ LANL2DZ (Au)水平上计算模拟4'-巯基-N-苯基苯醌二亚胺在金表面的拉曼光谱, 结果与所获得的表面拉曼光谱较好吻合, 平均相对偏差约为2.1%.     

次亚磷酸根离子在铂电极上氧化的机理研究(英文)

用SNIFTIRS和循环伏安法研究次亚磷酸根离子在多晶铂电极上的电氧化机理 .分析了0 .5mol/LH2 SO4 + 0 .1mol/LNaH2 PO2 溶液中原位红外反射谱图与Pt电极电位的关系 ,发现次亚磷酸根离子在Pt上发生解离吸附 ,其氧化产物是H3 PO4 ,不同于在Ni上的氧化产物H2 PO- 3 ,据此提出了酸性溶液中次亚磷酸根离子在Pt上氧化机理的新看法     

纳米铂电极的制备及其电化学行为

以镍铬合金为基体,采用电化学方法在其表面沉积制备了纳米Pt电极,用SEM对电极的形貌进行了表征,以亚铁氰化钾为分子探针考察了Pt电极的电化学性能。结果表明该电极对亚铁氰化钾具有稳定、灵敏的电化学响应,并对H2O2的电化学还原具有良好的催化活性。H2O2峰电流与其浓度在0.03-1.0 mmol/L范围内呈良好的线性关系,检出限（S/N=3）为7×10^-6mol/L。相同实验条件下,该纳米Pt电极的催化活性远高于纯Pt电极。     

Pt电极上乙醛吸附及氧化的现场FTIR反射吸收光谱电化学研究

本文用现场FTIR反射吸收光谱法和循环伏安法研究了Pt电极上HCl, H2SO4水溶液中乙醛的吸附及氧化过程。实验结果表明, 0.3至1.0V(vs. SCE)电势范围内乙醛主要以式I吸附于电极表面上, 并发生了生成乙酸的电化学反应, 产物乙酸可能以式II吸附。对于H2SO4水溶液中, 上述电势范围内电极表面上还检测出硫酸根离子与乙醛和乙酸的竞争吸附。     

Effect of dissolved CO2 on the potential stability of all-solid-state ion-selective electrodes.

The influence of dissolved CO2 on the potentiometric responses of all-solid-state ion-selective electrodes (ISEs) was systematically examined with four different types of electrodes fabricated by pairing pH-sensitive and pH-insensitive metal electrodes (Pt and Ag/AgCl, respectively) with pH-sensitive and pH-insensitive ion-selective membranes (H+-selective membrane based on tridodecylamine and Na+-selective membrane based on tetraethyl calix[4]arenetetraacetate, respectively). The experimental results clearly showed that the carbonic acid formed by the diffused CO2 and water vapor at the membrane/metal electrode interface varies the phase boundary potentials both at the inner side of the H+-selective membrane (deltaE(in)mem) and at the metal electrode surface (deltaEelec). The potential changes, deltaE(in)mem and deltaEelec, occurring at the facing boundaries, are opposite in their sign and result in a canceling effect if both the membrane and metal surface are pH-sensitive. Consequently, the H+-selective membrane coated on a pH-sensitive electrode (Pt) tends to exhibit a smaller CO2 interference than that on a pH-insensitive electrode (Ag/AgCl). When the all-solid-state Na+ and K+ ISEs were fabricated with both pH-insensitive metal electrode and ion-selective membrane, they did not suffer from CO2 interference. It was also confirmed that plasticization of the PVC leads to increased CO2 permeation. Various types of intermediate layers were examined to reduce the CO2 interference problem in the fabrication of H+-selective all-solid-state ISEs. The results indicated that the H+-selective electrode needs an intermediate layer that maintains a constant pH unless the carbonic acid formation at the interfacial area is effectively quenched.     

H2‐Fueled ATP Synthesis on an Electrode: Mimicking Cellular Respiration

ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F₁F₀‐ATPase in a lipid membrane can couple the electrochemical oxidation of H₂ to the synthesis of ATP. This electrode‐assisted conversion of H₂ gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.     

化学修饰电极的研究XVIII,四苯基钴卟啉化学修饰玻碳电极的热处理及其对氧的催化还原

本文研究四苯基钴卟啉化学修饰玻碳电极的热处理,经热处理的这种电极[(PCo/GC)h]具有对氧催化还原的异常高的稳定性和活性.在纯O2饱和的0.05mol.L^-^1H2SO4溶液中经循环伏安(CV)扫描3000次(100mV/s),其催化活性未见明显降低.研究了热处理温度(500-1000℃)对(PCo/GC)h电极电催化性能的影响.用紫外可见光谱对热处理产物的结构进行了分析.用CV法及旋转圆盘电极研究了O2在(PCo/GC)h电极上电催化反应动力学,测定了速率常数.在该电极上O2的还原反应为二电子还原成H2O2的不可逆过程.     

In Situ FTIR Spectroscopic Study of Electrochemical Adsorption of Phosphate Ions on Gold

Infrared reflection-absorption spectra for primary, secondary and tertiary orthophosphate anions on a gold electrode in aqueous solution were studied by in situ FTIR spectroscopy. The spectra show that H2PO4- , HPO ions do not interact with the electrode surface as strong as PO do. According to the surface selection rule, we deduce the modes of adsorption of these anions on the electrode from these spectra. The experiment also confirms the affection to adsorption of ion on the electrode due to ion-migration into thin-layer cavity.     

钯纳米粒子在电极表面的制备及其对氧的催化还原

纳米微粒的体积效应使其成为表面纳米工程及功能化纳米结构材料制备的理想研究对象 [1～ 3] .纳米粒子具有独特的电子、催化及光学特性[4 ] ,近年来关于纳米粒子的制备及其在材料科学领域中的应用受到研究者的极大关注 .而贵金属纳米粒子由于其在催化领域中的广泛应用而成为最重要的研究对象之一[5,6 ] .电催化氧还原是一直为化学家瞩目的研究领域[7～ 9] .研究主要目的之一是寻找合适的氧电极反应催化剂 ,并使之能够应用于燃料电池中 .其中催化氧电极材料研究得最多的是贵金属 Pt[10 ,11] .贵金属 Pd对氧催化还原的研究工作很少 .我们首次…