Artificial electron mediators

Finally, in order to ascertain if our PSI-particles were also useful for enzyme activation by potochemically reduced TRX, the purc/1. cylindrica PRuK was incubated with PSI-enriched particles, TRX and Fd-TRX reductase purified from /l. cylindrica (Serra et al., 1987) and a number of natural and artificial electron mediators as described under Materials and Methods. As shown in Fig. 5, PRuK was activated by photoreduced TRX and the degree of activation achieved depended on the time of pieincubation (compare curves b-d). No activation was observed when the enzyme was preincubated in the dark with the complete Fd/TRX system, or when the Fd-TRX reductase was omitted. 

In the last few years the term generation has mainly been used to differentiate between the modes of signal transfer between a redox enzyme and an electrode, i.e., via the natural secondary substrates and products of the enzyme catalysed reaction first generation), artificial electron mediators instead of the natural cosubstrates second generation) or in direct electronic... 

The enzyme enoate reductase has been isolated and described76,11. It is a conjugated iron sulphur flavoprotein. For preparative purposes purified enoate reductase can be applied in electrochemical cells7S. In this system methyl viologen (or another artificial electron mediator for the electron transfer) is continuously regenerated by an electrode and serves as an electron donor for the reductive reaction as shown in the following diagram. 

In the presence of catalytic concentrations of artificial electron mediators (Section... 

The application of enzymes detected by us in recent years together with the above indicated electron donors in the presence of catalytic concentrations of artificial electron mediators will be described. 

According to our experience the productivity numbers obtained with the aforementioned microorganisms can be enhanced by the use of catalytic concentrations (0.5-2 mM) of artificial electron mediators. Their properties and aspects of their use have been described in a review article (6). Table 1 lists some mediators with their redox potentials. 

The cells or crude cell extracts which will be described proceed by ways revealed by Schemes 2a and 2b, respectively. Electrons supplied by hydrogen gas, formate, carbon monoxide or the cathode of an electrochemical cell are channelled via an artificial electron mediator such as a reduced viologen (V ) or others to the enzymes reducing... 

It was of interest to purify and characterize the (R)-2-hydroxy carboxylate oxidore-ductase fi om Proteus vulgaris since it turned out that it does not react with NAD(H) or NADP(H) (46). Enzymes catalysing reversible reductions of carbonyl groups have been characterized fi om archaea, eubacteria and eukaryotes. The enzymes fi om eubacterial or eukaryotic sources depend on pyridine nucleotides the enzymes fi om archaea also use pyridine nucleotides or deazaflavin F42o- This coenzyme has a structure related to that of NAD(P)". Since viologens are suitable artificial electron mediators for this previously unknown enzyme we named it (/ )-2- droxy carboxylate viologen pxidoreductase (HVOR). In the purified form the membrane bound enzyme shows a specific activity of 1800 U mg protein for the reduction of phenylpyruvate (46). (One U (unit) reduces 1 pmol 2-0X0 carboxylate per minute). 

For the AMAPORs from C. thermoaceticum and C. formicoaceticum the values for the artificial electron mediators and those for pyridine nucleotides are in a reasonable range for preparative transformations. Table 24 shows some values for enzymes present in C. thermoaceticum. 

Biocatalyzed photosynthetic transformations using artificial electron mediators... 

The electron-transfer rate between large redox protein and electrode surface is usually prohibitively slow, which is the major barricade of the electrochemical system. The way to achieve efficient electrical communication between redox protein and electrode has been among the most challenging objects in the field of bioelectrochemistry. In summary, two ways have been proposed. One is based on the so-called electrochemical mediators, both natural enzyme substrates and products, and artificial redox mediators, mostly dye molecules and conducted polymers. The other approach is based on the direct electron transfer of protein. With its inherited simplicity in either theoretical calculations or practical applications, the latter has received far greater interest despite its limited applications at the present stage. 

The acceleration mechanism of redox mediators are presumed by van der Zee [15]. Redox mediators as reductase or coenzymes catalyze reactions by lowering the activation energy of the total reaction. Redox mediators, for example, artificial redox mediators such as AQDS, can accelerate both direct enzymatic reduction and mediated/indirect biological azo dye reduction (Fig. 3). In the case of direct enzymatic azo dye reduction, the accelerating effect of redox mediator will be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the azo dye. Possibly, both reactions will be catalyzed by the same nonspecific periplasmic enzymes. In the case of azo dye reduction by reduced enzyme cofactors, the accelerating effect of redox mediator will either be due to an electron shuttle between the reduced enzyme cofactor and redox mediator or be due to redox mediator enzymatic reduction in addition to enzymatic reduction of the coenzymes. In the latter case, the addition of redox mediator simply increases the pool of electron carriers. 

Attempts to reduce interference and minimize the effect of variations in oxygen tension have resulted in the development of biosensors with improved linear ranges which operate at lower electrode potentials. They incorporate artificial electron acceptors, called mediators, to transfer electrons from the flavoenzyme (e.g. glucose oxidase) to the electrode and thus are not dependent on oxygen. Ferrocene (bis(i75-cyclopentadienyl)iron) and its derivatives are examples of redox mediators for flavoenzymes. The reaction now becomes... 

Biocatalyzed Photosynthetic Systems Mediated by Artificial Electron... 

Artificial electron carriers are recognizable by the active sites of different redox enzymes and specifically biocatalysts containing Fe of Mo sulfur clusters as active sites. Bipyridinium radical cations, i.e. methyl viologen radical, MV+, exhibit proper electrical and size properties to penetrate into protein structures and to mediate reduction processes at the enzymes active sites. 

Fig. 40. Biocatalyzed photosynthetic systems mediated by an artificial electron carrier, JV,iV -dimethyl-4,4 -bipyridinium radical cation, MV+. Photogenerated MV+ mediates H2-evolution, C02 reduction to formate and sequential reduction of NO3 to ammonia, using Hyd, ForDH and a mixture of NitraR and NitriR, respectively...

Figure 6.5 Three generations of amperometric enzyme electrodes based on the use of natural secondary substrate (a), artificial redox mediators (b), or direct electron transfer between the enzyme and the electrode (c).

Figure 32. Photosensitized regeneration of NAD(P)H cofactor involving enzymes (FDR or LipDH) and the artificial electron-transfer mediator MV +, and using (A) Ru(bpy)j-" or (B) Zn(II) mMo-(A-tetramethylpyridinium)porphyrin, Zn-TMPyP +, as a photosensitizer.

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