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Tungsten-containing formate dehydrogenase from Methylorubrum extroquens AM1 (FoDH1)—a promising biocatalyst for the interconversion of carbon dioxide/formate and nicotine adenine dinucleotide (NAD+)/NADH redox couples—was investigated using structural biology and bioelectrochemistry. FoDH1 is reported to be an enzyme that can realize "direct electron transfer (DET)-type bioelectrocatalysis." However, its 3-D structure, electrode-active sites, and electron transfer (ET) pathways remain unclear. The ET pathways were investigated using structural information, electrostatic interactions between the electrode and the enzyme, and the differences in the substrates. Two electrode-active sites and multiple ET pathways in FoDH1 were discovered. [ABSTRACT FROM AUTHOR]

Research has been conducted on Paracoccus denitrificans quinohemoprotein amine dehydrogenase. The study of quinohemoprotein amine dehydrogenase's hemes c carried out via the use of mediator-assisted continuous-flow column electrolytic spectroelectrochemistry is presented.

Mediated biosensors consisting of an oxidase and peroxidase (POx) have attracted increasing attention because of their wide applicability. However, since most of oxidases utilize artificial electron acceptors in place of dioxygen, the competition between [O.sub.2] and the electron acceptor in the mediated sensors is anticipated. This has been evidenced with a glucose oxidase (GOx)- and POx-coentrapped and ferrocene-embedded carbon paste electrode, which exhibits peak-shaped current-time curves at increased concentrations of glucose and also gives a peak-shaped calibration curve. Digital simulation has been applied to clarify the cause of such unusual responses, by taking into account the ping-pong enzyme kinetics on two- and three-substrate models for POx and GOx, respectively. The simulation has well reproduced such unusual responses and has clearly revealed that the depletion of [O.sub.2] in the enzyme layer is the most important factor responsible for such unusual responses. To overcome such a drawback of oxidase/POx bienzyme sensors, it is expected to be essential to decrease the rate of the oxidase reaction. In contrast, increase in the POx activity is useful to improve the sensitivity. According to the simulation-based expectation, the GOx and POx concentrations in the bienzyme sensor are adjusted to exhibit normal behavior with high sensitivity.

Results suggest that the n-sigma type chloranil dianion interaction plays a role in the hydrogen bond formation in p-quinone dianion complexes with methyl alcohol as indicated by the blue-shift of the p-quinone dianion spectra.

Kinetic determination of protein redox potentials with a mediated continuous-flow column electrolytic spectro-electrochemical technique (CFCESET) is described. In this method, the redox state of the mediator is completely regulated by the continuous-flow column electrolysis, and the homogeneous redox reaction between the mediator and a protein sample in the column is monitored spectroscopically at the downstream of the column. The protein/mediator reaction is in the pseudo-first-order kinetics, and then the rate equation is analytically solved. The kinetic analysis provides the protein redox potential as well as the homogeneous rate constant. In the kinetic measurements, equilibration of the system within the column is not required, which allows the use of increased kinds of mediators. This method was successfully applied to quinoprotein methylamine dehydrogenase containing tryptophan tryptophylquinone (TFQ) as a prosthetic group. The kinetic aspect is also valuable for the thermodynamic analysis with the mediated CFCESET. The half-life time of the kinetics can be utilized to optimize the system for the attainment of the equilibrated state within the column and can provide the assurance that the system is in equilibrium.

A study was conducted to analyze a novel quinohemoprotein amine dehydrogenase from Paracoccus denitrificans IFO 12442. The redox potential of the quinone/semiquinone and semiquinone/quinol redox couples were evaluated through an analysis of the substrate titration curve. Steady-state kinetic assays were then carried out by measuring the amine-dependent reduction rate of Fe(CN)(sub 6)(super 3-). Results indicated the expression of at least two amine dehydrogenases.

An electrode modified with immobilized whole cells of Desulfovibrio vulgaris (Hildenborough) produces an S-shaped voltammogram with both cathodic- and anodic-catalytic-limiting currents in a methyl viologen-containing buffer saturated with [H.sub.2]. Methyl viologen penetrates into the bacterial cells to serve as an electron carrier in the reversible reaction of hydrogenase in the cells and functions as an electron-transfer mediator between the bacterial cells and the electrode, thus producing the catalytic currents for the evolution and consumption of [H.sub.2]. An equation for the catalytic current that takes into account the reversible hydrogenase reaction explains well the shape of the voltammogram. The potential at null current on the voltammogram agrees with the potential determined by potentiometry with the same electrode, which is equal to the redox potential of the [H.sup.-]/[H.sub.2] couple in the solution-the standard potential of a hydrogen electrode at the pH of the solution. When D. vulgaris cells are suspended in an argon-saturated buffer containing methyl viologen, the suspension produces a catalytic current at a bare glassy carbon electrode for the evolution of [H.sub.2]. Analysis of the current by a theory for a catalytic current for a unidirectional nonlinear enzyme catalysis allows us to determine the kinetic parameters of the reaction between methyl viologen and hydrogenase in intact D. vulgaris cells. Thus we obtain the apparent Michaelis constant for methyl viologen cation radical, [K[prime].sub.[MV.sup.+]] = 0.16 mM, and the apparent catalytic constant (that is, the turnover number per D. vulgaris cell), [zk.sub.cat,[H.sup.+]] = 1.2 x [10.sup.7] [s.sup.-1], for the [H.sub.2] evolution reaction at pH 5.5 and at 25 [degrees] C, z being the number of hydrogenases contained in a D. vulgaris cell. The bimolecular reaction rate constant, [k.sub.cat,[H.sup.+]]/[K[prime].sub.[MV.sup.+]], of the reaction between methyl viologen cation radical and oxidized hydrogenase in intact D. vulgaris cells is estimated as 4.2 x [10.sup.7] [M.sup.-1] [s.sup.-1]. Similarly, the bimolecular reaction rate constant, [k.sub.cat,[H.sub.2]]/[K[prime].sub.[MV.sup.2]], of the reaction between methyl viologen and reduced hydrogenase is estimated to be 1.2 x [10.sup.7] [M.sup.-1] [s.sup.-1] at pH 9.5 and 25 [degrees] C. Both rate constants are large enough for the reactions to be diffusion-limited processes.

Redox enzymes can work as efficient electrocatalysts. The coupling of redox enzymatic reactions with electrode reactions is called enzymatic bioelectrocatalysis, which imparts high reaction specificity to electrode reactions with nonspecific characteristics. The key factors required for bioelectrocatalysis are hydride ion/electron transfer characteristics and low specificity for either substrate in redox enzymes. Several theoretical features of steady-state responses are introduced to understand bioelectrocatalysis and to extend the performance of bioelectrocatalytic systems. Applications of the coupling concept to bioelectrochemical devices are also summarized with emphasis on the achievements recorded in the research group of the author. [ABSTRACT FROM AUTHOR]

A mediator-assisted continuous-flow column electrolytic spectroelectrochemical method was designed to study redox properties of proteins. This method is based on a flow-through column electrolytic control of a redox buffer containing relatively high concentrations of mediators and an accelerated reaction of a protein with the mediators to reach equilibrium within the electrolysis time. A small amount of a protein sample is introduced into the electrochemically regulated redox buffer in the mode of flow injection analysis (FIA). The equilibrated redox state of the protein is evaluated by a highly sensitive flow-through photodiode array detector. Reproducible and stable background spectra and the employment of the FIA mode allow precise background subtraction in spectral analysis. This method was successfully applied to determining the redox potentials of horseradish peroxidase (HRP) using potassium hexachloroiridate as a mediator. The absorbance versus the electrode potential curves for the redox reactions among the ferric form, compound II, and compound I of HRP were well interpreted by a Nernstian equation based on the two-step one-electron-transfer model. The advantage and the kinetic aspects of this method are discussed in detailed.

Ubiquinones act as mediators in electron transfer and proton translocation between redox enzymes within mitochondrial and bacterial respiratory systems. The knowledge of ubiquinone binding and redox properties is insufficient. A novel series of the ubiquinone-2 (Q2) analogues where just one structural factor of the diprenyl group as systematically modified, were synthesized, to identify structural features of the diprenyl tail that determine interactions with bovine complex I and cytochrome bo.

A study was conducted to analyze the synthesis and properties of dendritic tetranuclear ruthenium and osmium 2,2'-bipyridine complexes which were bridged by 2,2-bis(1-benzimidazo-2-yl)-4,4'-bipyridine and evaluate the effectiveness of the flow-through electrolysis method. The data obtained from the flow-through method was compared to the data obtained from traditional spectroelectrochemical methods. Results revealed that the flow-through approach can be more reliable in analyzing the synthesis and properties of metal complexes compared to other methods.

Separation of hydrophobic cations in capillary electrophoresis under the mixed micellar system composed of SDS and Tween 20 was investigated from the viewpoints of thermodynamics and practical application. Hydrophobic cations interact strongly with the anionic SDS micelle, and this often lead to predominant dissolution of the analytes into the micellar phase, resulting in poor resolution. The ionic interaction was evaluated to be close in strength to the hydrophobic interaction between benzenes and the SDS micelle. Tween 20 as a component to the mixed micelle was found to weaken the attractive ionic interactions between the cationic solutes and SDS. In addition, the polyether chain of Tween 20 serves also as a hydrogen acceptor to cause the attractive hydrogen-bonding interactions with hydrogen-donating analytes. The two different functions of Tween 20 improve the separation of hydrophobic cations remarkably. Separation patterns are well controlled by varying the mixing ratio of the two surfactants. Adrenaline and its six precursors were successfully separated with this mixed micellar system. The improved separation efficiency was not affected by the presence of bovine serum matrix, realizing the direct MEKC analysis of serum sample without deproteination, in which proteins were comigrated with the micelle.

The actions of uricase or urate oxidase (UOD) and glucose oxidase (GOD) in the oxidative polymerization of p-phenylenediamine (PPD) were compared using spectroscopic and electrochemical means. The results, which demonstrated the superiority of UOD as a colorant, revealed that GOD suppresses the polymerization due to its glucose dehydrogenase activity which reverts p-benzoquinonediimine, a two-electron oxidized form of PPD, to PPD.

The oxidation of D-glucose and nicotinic acid by intact cells of Gluconobacter industrius and Pseudomonus fluorescens, respectively, is successfully measured by an amperometric method using such compounds as Fe(CN)[6.sup.3-], p-benzoquinone, and dichlorophenolindophenol as electron acceptors. Analysis of the experimental results reveals that the intact cells behave like oxido-reductases whose kinetics follows a Michaelis-Menten-type equation. The catalytic behavior is explained by a model which treats the bacterial cells as bags of enzymes and assumes distribution equilibrium in the concentrations of both the substrate and the electron acceptor between the test solution and the medium within the cells. The catalytic activity can be characterized by three quantities: the maximum reaction rate ([v.sub.B]) and the ratios of the Michaelis constant to the distribution constant for the substrate ([K.sub.s,cell]/[K.sub.s,p]) and to that for the electron acceptor ([K.sub.M,cell]/[K.sub.M,p]). Advanced modification of the model to involve the membrane permeability reveals that the three quantities are effective for explaining the catalytic behavior even when the permeability effect is significant. Thus, the three quantities should be regarded as the parameters which can reflect the permeability effect.

Two new schemes enable the preparation of a new model compound of novel cofactor tryptophan tryptophylquinone (TTQ) of bacterial methylamine dehydrogenase. Spectral studies and physicochemical techniques helps characterize this compound, which shows it to be very similar to TTQ. EPR and electrochemical studies help analyze the stability and redox characteristics of semiquinone radical anion derived from an iminoquinone, prepared by a reaction between the model compound and ammonia.