Artificial mediators

Particularly useful have been the artificial mediators that shuttle electrons between the FAD center and the surface by the following scheme ... 

The approaches that have been proposed to immobilize artificial mediators include the adsorption of the redox mediator (9), the immobilization in carbon paste (75), the covalent linkage on electroinactive (75) or conducting polymer backbone (10), the covalent attachement to the enzyme structures (3) and... 

Clostridium thermoaceticum contains the so-called AMAPOR (artificial-media-tor-accepting pyridine-nucleotide oxidoreductases), which are useful for electro-microbial regeneration of all four forms of pyridine nucleotides, too. An NADP(H) dependent AMAPOR from C. thermoaceticum has been purified and characterized [104]. It is able to react with rather different artificial mediators such as viologens or quinones, for example 1,4-benzoquinone, anthraquinone-2,6-disulfonate, or 2,6-dichloro-indophenol. 

As will be shown in Sections 5 and 6 methylviologen can be reduced at the cathode of an electrochemical cell to the mono cation radical MV which transfers electrons by the action of an enzyme type which we call AMAPOR for artificial mediator accepting pyridine nucleotide pxidoreductase. Finally the NADH is used for reducing the substrate by the catalytic action of an NADH dependent reductase. 

In the following reductions are described with cells which do not show the briefly mentioned problems of yeasts applied in the classical way. All the strictly anaerobically grown cells of anaerobic or facultative organisms are able to accept hydrogen gas, many accept formate and some carbon monoxide as electron donors and contain redox enzymes in often high activities which take up the electrons and deliver them to viologens and other artificial mediators which transfer them further (Section 1.2). The standard redox potential E° of the electron delivering reactions... 

Structure and standard potentials of some artificial mediators... 

SCHEME 2a Regeneration of reduced artificial mediators for reductions with nonpyridine nucleotide dependent or pyridine nucleotide dependent reductases. 

IfE3 is a pyridine nucleotide independent reductase accepting electrons from artificial mediators, only this enzyme is necessary, if the mediators are reduced electrochemically. If they are reduced enzymicly El has to be included. Reduced pyridine nucleotides are formed by E2. These artificial mediator accepting pyridine nucleotide oxidoreductases (AMAPOR) catalyse Reaction [8] and [8a] forming NAD(P)H. Further examples are given in the text. Artificial mediators are indicated by med . Y are natural mediators except pyridine nucleotides. They may assist the electron transfer, but they are not necessary. 

If El is a pyridine nucleotide independent dehydrogenase reducing medox to med d it can be reoxidized by an anode of an electrochemical cell or by an enzyme E3, which delivers the electrons to an acceptor such as dimethyl-sulphoxide or others. Pyridine nucleotide dependent enzymes El produce NADH or NADPH by the dehydrogenation of their substrates. The reduced pyridine nucleotides are reoxidized by the AMAPOR E2, which in turn reduces an artificial mediator medox to medred- Med d delivers electrons to E3 or an anode. For the reoxidation of E3 see text. 

Besides the artificial mediators mentioned so far safranine T in 0.75 mM concentration shows about 80 % of the activity observed with 1 mM methylviologen or 1 mM anthraquinone-2,6-disulphonate for the reduction of 2-methylfumarate. The absence of a mediator decreases the reduction rate to 3-4 %. C. formicoaceticum is also useful for stereoselective hydrations of fumarate and maleate derivatives (36). 

The oxidoreductase contains 1 molybdenum cofactor and 4 iron and 4 sulphur ions per subunit of 80 kDa (46). The natural electron mediator is not known yet. The enzyme reacts not only with viologens, but also with many other artificial mediators. Their redox potentials are in the range from -440 to +220 mV (Section 3.2, Table 14). 

In the presence of a catalytic concentration (0.5-2.0 mM) of an artificial mediator resting cells of both Proteus mirabilis or P. vulgaris reduce 2-oxo carboxylates to steri-cally pure (R)-2-hydroxy carboxylates at the expense of formate and/or hydrogen gas. The reductions can also be carried out in an electrochemical cell where the regeneration of the reduced mediator proceeds at the cathode (Section 6, Fig. 6). Under these condi-... 

The apparent Km values were determined with crude extracts of P. mirabilis at 38 °C and pH 8.5. The data were calculated according to Eadie and Hofstee (69). The substrates were tested in the presence of 6 mM anthraquinone-2.6-disulphonate (AQDS).Yield as isolated product. Kinetic data for the dimethyl-sulphoxide reductase were determined with 0.6 mM reduced anthraquinone-2.6-disulphonate. The Km values for the mediators were determined with 50 mM D-araWno-hex-2-ulosonate as substrate. mmol kg. (biocatalyst) h . The Km and Vm,x values of the 3 artificial mediators were determined with 50 mM D-ara6/ o-hex-2-ulosonate. 

USE OF ARTIFICIAL MEDIATOR ACCEPTING PYRIDINE NUCLEOTIDE OXIDOREDUCTASES (AMAPORs) FOR THE REGENERATION OF PYRIDINE NUCLEOTIDES... 

NADPH was regenerated under anaerobic conditions by crude extracts of C. thermoaceticum containing formate dehydrogenase (FDH) as well as artificial mediator accepting pyridine nucleotide oxidoreductases (AMAPORs) and formate as electron donor. For electromicrobial redox reactions see Section 6. 

Enoate reductase, 2-hydroxy carboxylate viologen oxidoreductase (HVOR) and AMAPORs (Section 5) are enzymes able to accept reversibly single electrons fi-om artificial mediators such as viologens and others. These mediators transfer electrons fi om or to electrodes. Therefore by the presence of the aforementioned enzyme activities biocata-lytic redox reactions can be carried out in electrochemical cells. As already mentioned in Section 1.2 AMAPORs catalysing Reactions [8] and/or [8a] are rather ubiquitous. Electromicrobial reductions can also be carried out with yeasts (Section 1.2). Since the potential of the working electrode can be chosen at will, reductions as well as... 

Under these conditions, the over all reaction rate in the electrochemical cell is often limited by the reduction of the artificial mediator. Sufficiently large electrodes and efficient stirring are helpful measures to avoid such limitations. 

The concentration of hydrogen peroxide can be measured directly using amperometric detection. A change in H2O2 concentration in the medium appears as a variarion in the output current. The quantified parameters are m nitude of the sensor response, response time, and current response. It is desirable to measure signals in conditions when the linear relationship exists between the current value and the analyte concentration. At that point, the reactions are considered to be in steady state when pseudoequilibrium occurs between the species close to the sensor and their consumption at the indicative electrode. One of the serious problems associated with measurement of complex analytes is the possible interference of the redox species present in the sample. Several methods have been reported which aimed at reducing level of interference. These methods include use of perm-selective coatii, use of artificial mediators, or selective electrocatalysis. The use of mediators or selective electrocatalysis helps to lower the detection potential to the level when the majority of interferii species are electroinactive. ... 

Most of these studies have not addressed the question of where the reaction between the artificial mediator and the electrode occurs. Although it is often claimed that the mediator is reoxidized on the polymer, direct evidence to support this assertion is sparse. In many cases results can be equally well explained by diffusion of the mediator species to, and reoxidation on, the underlying electrode. 

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