Isao Taniguchi

A post genomic characterization of Arabidopsis ferredoxins

In higher plant plastids, ferredoxin (Fd) is the unique soluble electron carrier protein located in the stroma. Consequently, a wide variety of essential metabolic and signaling processes depend upon reduction by Fd. The currently available plant genomes of Arabidopsis and rice (Oryza sativa) contain several genes encoding putative Fds, although little is known about the proteins themselves. To establish whether this variety represents redundancy or specialized function, we have recombinantly expressed and purified the four conventional [2Fe-2S] Fd proteins encoded in the Arabidopsis genome and analyzed their physical and functional properties. Two proteins are leaf type Fds, having relatively low redox potentials and supporting a higher photosynthetic activity. One protein is a root type Fd, being more efficiently reduced under nonphotosynthetic conditions and supporting a higher activity of sulfite reduction. A further Fd has a remarkably positive redox potential and so, although redox active, is limited in redox partners to which it can donate electrons. Immunological analysis indicates that all four proteins are expressed in mature leaves. This holistic view demonstrates how varied and essential soluble electron transfer functions in higher plants are fulfilled through a diversity of Fd proteins.

Comparison of the Electrostatic Binding Sites on the Surface of Ferredoxin for Two Ferredoxin-dependent Enzymes, Ferredoxin-NADP+ Reductase and Sulfite Reductase

Plant-type ferredoxin (Fd), a [2Fe-2S] iron-sulfur protein, functions as an one-electron donor to Fd-NADP+ reductase (FNR) or sulfite reductase (SiR), interacting electrostatically with them. In order to understand the protein-protein interaction between Fd and these two different enzymes, 10 acidic surface residues in maize Fd (isoform III), Asp-27, Glu-30, Asp-58, Asp-61, Asp-66/Asp-67, Glu-71/Glu-72, Asp-85, and Glu-93, were substituted with the corresponding amide residues by site-directed mutagenesis. The redox potentials of the mutated Fds were not markedly changed, except for E93Q, the redox potential of which was more positive by 67 mV than that of the wild type. Kinetic experiments showed that the mutations at Asp-66/Asp-67 and Glu-93 significantly affected electron transfer to the two enzymes. Interestingly, D66N/D67N was less efficient in the reaction with FNR than E93Q, whereas this relationship was reversed in the reaction with SiR. The static interaction of the mutant Fds with each the two enzymes was analyzed by gel filtration of a mixture of Fd and each enzyme, and by affinity chromatography on Fd-immobilized resins. The contributions of Asp-66/Asp-67 and Glu-93 were found to be most important for the binding to FNR and SiR, respectively, in accordance with the kinetic data. These results allowed us to map the acidic regions of Fd required for electron transfer and for binding to FNR and SiR and demonstrate that the interaction sites for the two enzymes are at least partly distinct.

Voltammetric and in situ STM studies on self-assembled monolayers of 4-mercaptopyridine, 2-mercaptopyridine and thiophenol on Au(111) electrodes

Voltammetric and in situ STM studies were carried out for self-assembled monolayers of 4-mercaptopyridine (4-PySH), 2-mercaptopyridine (2-PySH) and thiophenol (PhSH) on well-defined single-crystal Au(111) electrodes in aqueous solutions. A reversible voltammetric response for cytochrome c was clearly observed only at the 4-PyS/Au(111) electrode, showing that only the 4-pyridinethiolate monolayer promotes facial electron transfer reaction between the Au(111) and cytochrome c. On the basis of reductive desorption, the surface coverages of the three aromatic thiolate monolayers were found to be similar to each other; 4.6×10−10 mol/cm2 for 4-PyS/Au(111), 4.7×10−10 mol/cm2 for 2-PyS/Au(111), and 4.4×10−10 mol/cm2 for PhS/Au(111). High-resolution STM images in perchloric acid solutions revealed p(5×√3R-30°) and p(4×√7R-40.9°) structures for the 4- and 2-pyridinethiolate monolayers on Au(111), respectively. No structure order was observed for the PhSH monolayers. While the pyridine units of both 4- and 2-pyridinethiolate monolayers were found to be oriented normal to the surface, 2-pyridinethiolates adsorbed through not only sulfur but also nitrogen atom of the pyridine ring. From these STM images, the orientation of the N atom of the pyridine moiety must face to the bulk solution, as in the case of 4-PyS/Au(111), in order to obtain a facile electrochemical reaction for cytochrome c.

The Direct Electron Transfer Reactions of Cytochrome c at Electrode Surfaces

Abstract Examples of facile electron transfer between electrode surfaces and electron transfer proteins were first reported nearly twenty years ago. Substantial progress has been achieved in understanding the fundamental requirements that must be met in order to observe such reactions. Despite the large body of work that has now been published on a host of examples of such reactions, there continues to be a lack of understanding over just what conditions must be met in order to use direct electrochemical methods to characterize the electron transfer thermodynamics and kinetics of electron transfer proteins. Part of the problem lies in the inherent difficulties associated with using solid electrodes. Equally as important is the wide range of attention that has been paid to the purity of the electron transfer protein solutions being studied. The goal here is to bring together those aspects of this problem that enjoy some degree of agreement among scientists in this field and to reflect upon those issues tha...

722—The effect of pH on the temperature dependence of the redox potential of horse heart cytochrome c at a bis(4-pyridyl)disulfide-modified gold electrode

Abstract The thermodynamic parameters of the cytochrome c electron-transfer reaction have been estimated electrochemically using a bis(4-pyridyl)disulfide-modified gold electrode in phosphate buffer solutions (pHs 6–8) containing 0.1 M NaC104. The temperature dependence of the formal redox potential, U°′, obtained in the temperature range of 0–55°C showed biphasic behavior in an alkaline solution with an intersection point at ca. 40°C, which would be attributable to a structural change in the protein moiety of cytochrome c, while in acidic and neutral solutions a monotonous relationship between U°′ and temperature was observed. For the electron-transfer reaction entropies (ΔS°rc = S°red - S°ox) of −12.7 ± 1.6, −11.8 ± 1.1, and −10.3 ± 1.5 (below 40°C) and −41.1 ± 7.5 (above 40°C) were obtained at pHs 6, 7, and 8, respectively.

Catalytic oxidation of hydrogen peroxide at Ni-cyclam modified electrodes and its application to the preparation of an amperometric glucose sensor

In the present study, we demonstrate, for the first time, that a Ni-cyclam derivative modified electrode is an excellent catalyst for H 2 O 2 oxidation in a neutral solution and also that this electrode is applicable to the preparation of an interesting amperometric glucose sensor

Reversible electrochemical reduction and oxidation of cytochrome c at a bis(4-pyridyl) disulphide-modified gold electrode

At a gold electrode on which bis(4-pyridyl) disulphide had been pre-adsorbed, horse heart ferricytochrome c exhibited a well developed quasi-reversible redox wave by cyclic voltammetry in the absence of any promoter in the solution.

Simple methods for preparation of a well-defined 4-pyridinethiol modified surface on Au(111) electrodes for cytochrome c electrochemistry

Abstract A very small amount of sulfide impurity in 4-pyridinethiol (4-PySH) modifier solution was found to interfere with the proper formation of the 4-PySH modified surface for cytochrome c electrochemistry on an Au(111) electrode. When the modification was conducted in an alkaline (e.g. 0.1 M KOH) solution, in aqueous solutions under applying a potential more positive than 0.3 V vs. Ag/AgCl, or at a low modifier concentration (e.g. 20 μM), the proper 4-PySH modified surface was obtained even using 4-PySH as received, which contained a small amount of sulfide. The selective adsorption of 4-PySH in the presence of a small amount of sulfide under these conditions was due to the rapid formation of proper 4-PySH modified surface, which prevented the sulfide from reacting with the electrode surface.

Electrochemical and Photoelectrochemical Reduction of Carbon Dioxide

The reduction of carbon dioxide has been a subject of active interest for more than a century.1 Especially in recent years, electrochemical and photoelectrochemical reduction of carbon dioxide has been extensively studied.2-4 This is because this reaction has several attractive features. In view of the increasing possibility of unavailability of oil and other fossil fuels in the near future,5,6 alternative fuels have to be produced from abundant resources such as carbon dioxide and water. Carbon dioxide reduction is also an important branch of C1 chemistry. In addition, the effect of recent excessive production of carbon dioxide on the future climate of the Earth is being seriously discussed,7 and carbon dioxide reduction to organic raw materials or fuels would help to reduce this type of atmospheric pollution as well. Carbon dioxide reduction can be used as a suitable reaction for energy storage, as is required, for instance, in the conversion of solar to storable chemical energy.8,9 Moreover, formic acid, which is one of the reduction products of carbon dioxide, has been proposed as a convenient means of hydrogen storage.10

Electrochemical study on competitive adsorption of pyridinethiol with sulfide onto Au(111) surfaces

Abstract Using electrochemical reductive desorption of surface modifiers the structure of the modified surface of a single crystal electrode was studied. A very small amount (e.g. 1 mol %) of sulfide impurity in a 4-pyridinethiol (4-PySH) modifier solution was found to adsorb on an electrode in competing with 4-PySH and eventually adsorbed 4-PySH (or bis(4-pyridyl)disulfide, 4,4′-PySSPy) molecules were replaced completely by sulfide. In an ethanolic solution the sulfide impurity affected the modified surface structure of an Au(111) electrode more significantly than in an aqueous solution. The purified 4-PySH and 4,4′-PySSPy samples reproducibly gave a preferable surface for cytochrome c electrochemistry. The reductive desorption peak potential of a thiol adsorbed on the electrode surface was suggested to be a measure for predicting the structure of the modified surface. When the desorption peak potential of thiol becomes more positive, faster replacement of thiol by sulfide occurs. On the same lines, it was explained why an Au(111) single crystal surface is more sensitive to the sulfide impurity than are the Au(100) and Au(110) surfaces.