Noriyuki Asakura

Oxygen concentration imaging in a single living cell using phosphorescence lifetime of Pt-porphyrin

An oxygen concentration imaging system inside a single living cell, based on the phosphorescence lifetime, under a microscope was developed. A fluorescence microscope equipped with a pulsed Nd:YAG laser (532 nm) and a CCD camera equipped with a gated imaging intensifier was used. When the cell was incubated with the phosphorescent compound, platinum tetra-(carboxyphenyl)-porphyrin (PtTCPP) was incorporated and localized in the cell. As the phosphorescence intensity depends not only on the concentration of a quencher such as oxygen but also on the concentration of phosphorescent molecules, the oxygen concentration was measured by the phosphorescence lifetime. To measure the oxygen concentration image, the time dependence of the phosphorescence intensity was observed under a microscope. The phosphorescence intensity decay obeyed first order kinetics and the oxygen concentration imaging in the single cell was obtained by the lifetime of a single exponential decay curve.

Photoinduced hydrogen evolution with lysine-linked viologen and hydrogenase

Lysine-linked viologen was prepared as a substrate for the hydrogenase. By using reduced lysine-linked viologen (LysV+•), the hydrogenase-LysV+• complex was formed efficiently, leading to effective hydrogen evolution compared with methyl viologen. Lysine-linked viologen as an electron carrier was applied for the photoinduced hydrogen evolution system containing hydrogenase, Tetrakis(4-carboxyphenyl)porphyrin (TCPP) as a photosensitizer, and a sacrificial electron donor for TCPP. In this system, effective photoinduced hydrogen evolution was observed.

Photoinduced hydrogen evolution with cytochrome c3-viologen-ruthenium(II) triad complex and hydrogenase

Abstract Cytochrome c 3 -viologen-ruthenium(II) triad complex, Ru-V-cyt. c 3 , was prepared and characterized by using spectroscopic techniques. Effective quenching of the photoexcited state of ruthenium complex moiety by the bound viologen was observed in Ru-V-cyt. c 3 . When the system containing Ru-V-cyt. c 3 and hydrogenase was irradiated by visible light, photoinduced hydrogen evolution was observed, showing the effective two-step electron transfer from the photoexcited state of ruthenium complex moiety to cytochrome c 3 via bound viologen.

Lysine-linked viologen for substrate of hydrogenase on hydrogen evolution

Lysine-linked viologen was synthesized and was applied for hydrogen evolution with hydrogenase. The lysine-linked viologen has high affinity for hydrogenase than methyl viologen, resulting the high hydrogen evolution rate.

Development of novel optical oxygen sensing system based on stationary T–T absorption

A novel oxygen sensor based on stationary triplet–triplet (T–T) absorption was developed and the stationary absorption change of zinc 5,10,15,20-tetrakis-(pentafluorophenyl)-porphyrin (ZnTFPP) depending on oxygen concentration was investigated. In this technique, a photochemical equilibrium made by two continuous lights, which are S–S excitation and T–T excitation, and the change of equilibrium was monitored as the change of stationary T–T absorption. In this study, ZnTFPP in polystyrene and ZnTFPP immobilizing aluminium oxide were used as a sensor device and the difference between the two devices was clarified by the stationary quenching measurement.

Incorporation of unnatural amino acids into cytochrome c3 and specific viologen binding to the unnatural amino acid.

Site-specific incorporation of unnatural amino acids into proteins is a useful technique for a site-specific reaction between proteins and chemicals such as chromophores, fluorescent dyes, biotin, and polymers. While some studies report unnatural amino acid incorporation, it has been unsuccessful in the hemoprotein-containing c-type heme. We report here that a system for the incorporation of an unnatural amino acid into cy ACHTUNGTRENNUNGtoACHTUNGTRENNUNGchrome c3 has been established. CyACHTUNGTRENNUNGto ACHTUNGTRENNUNGchrome c3 is an electron-transfer protein containing four c-type hemes, which have a role in a redox center. The 3D structure of cyACHTUNGTRENNUNGto ACHTUNGTRENNUNGchrome c3 is shown in Figure 1, however, mechanisms for interor intramo-

Direct monitoring of the electron pool effect of cytochrome c3 by highly sensitive EQCM measurements

Cytochrome c3 from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c3 with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c3 molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c3 is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c3 to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c3 and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c3 stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c3 facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c3 donates the accepted electrons to the viologen with difficulty.

The role of specific lysine in cytochrome c3 on the electron transfer with hydrogenase

Abstract Modification of specific lysine residue of cytochrome c 3 with 2,4,6-trinitorobenzenesulfonic acid sodium salt dehydrate (TNBS) was carried out. By comparing the hydrogen evolution rate with modified cytochrome c 3 and native cytochrome c 3 catalyzed by hydrogenase, the role of lysine residue of cytochrome c 3 was discussed. The modified lysine residue was responsible for the intermolecular electron transfer from cytochrome c 3 to hydrogenase.

Motion of redox molecules in solution monitored by the highly-sensitive EQCM technique

The behavior of redox molecules in solution that was not detected by electrochemical techniques was measured by a highly-sensitive electrochemical quartz crystal microbalance (EQCM) technique that has been improved in this study to obtain a high sensitivity of EQCM measurement in solution. The improved EQCM technique allowed to monitor the motion of a redox molecule, that is an access of the molecule to an electrode surface and repulsion from the surface during redox. An EQCM technique currently in use has measured adsorption of redox molecules on an electrode surface or polymerization on the surface caused by a chemical reaction following redox, which exhibits an enough large mass change response to detect with an EQCM measurement. However, access and repulsion of redox molecule, which is a slight motion of the molecule near on electrode surface, has not been detected and investigated by an EQCM technique, because the mass change response seems to be very small. In this study, the redox behavior of methyl viologen on a bare gold surface, pyridinethiol surface and methylpyridinethiol surface was investigated. Although the three electrodes give the same cyclic voltammogram of methyl viologen, the three are different in QCM response recorded at the same time as the voltammetry. Access/repulsion of methyl viologen within an electrical double layer was monitored by the highly-sensitive EQCM technique.

Role of positive charge of lysine residue on cytochrome c3 for electrostatic interaction with hydrogenase

Cytochrome c3 from Desulfovibrio vulgaris (Miyazaki) is an electron transfer protein containing four hemes per molecule. Its physiological electron transfer partner is the hydrogenase which catalyzes reversible oxidation of hydrogen. The complex formation between cytochrome c3 and hydrogenase is caused by electrostatic interaction, because cytochrome c3 is a basic protein and hydrogenase is an acidic protein. As cytochrome c3 has 20 lysine residues among 108 amino acids, the positive charges of some lysine residues may play an important role in the interaction with hydrogenase. To clarify the role of positive charge of lysine residue, the positive charge was changed to neutral or negative charge using chemical modification and site-directed mutagenesis. When the positive charges around heme IV were changed, the hydrogen evolution rate with hydrogenase decreased. The affinity between hydrogenase and mutated cytochrome c3 (K57Q, K57E, K72Q, K94Q, K94E) were not affected. On the other hand, the affinity of K72E cytochrome c3 for hydrogenase was very low. These results suggest that the positive charge around heme IV plays an important role in the electrostatic interaction with hydrogenase in hydrogen evolution.