Viologen oxidoreductase

Kinetic parameters determined for reactions catalysed by hydroxy carboxylate viologen oxidoreductase and dimethyl-sulphoxide reductase in Proteus vulgaris. 

Relative initial rates of reductions of 2-oxo carboxylates to (R)-2-hydroxy carboxylates with partially purified 2-hydroxy carboxylate viologen oxidoreductase (HVOR) from Proteus vulgaris andR. mirabilis (58,59, unpublished). 

Dehydrogenations of aldonates and aldarates with resting cells of Proteus mirabilis. These cells contained 2-hydroxy carboxylate viologen oxidoreductase (HVOR) with a specific activity of 5.1 U mg protein for the dehydrogenation of (f )-lactate (61). 

Activities of hydroxy carboxylate viologen oxidoreductase (HVOR) and dimethyl-sulphoxide reductase in P. vulgaris grown on glucose or (R,S)-lactate with or without different electron acceptors (47). 

The specific enzyme activities (U mg protein) of the crude extract of P. vulgaris were tested with reduced benzylviologen for dimethyl-sulphoxide reductase and oxidized benzylviologen for (R)-2-hydroxy carboxylate viologen oxidoreductase dehydrogenating (R)-lactate. 

Enzyme activities of (7 )-2-hydroxy carboxylate viologen oxidoreductase (HVOR), dimethyl-sulphoxide reductase, formate reductase and hydro-genase in crude extracts of P. vulgaris and P. mirabilis (47). 

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... 

Strobl G, R feicht, H White, F Lottspeich, H Simon (1992) The tungsten-containing aldehyde oxidoreductase from Clostridium thermoaceticum and its complex with a viologen-accepting NADPH oxidoreductase. Biol Chem Hoppe-Seyler il i 123-132. 

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. 

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). 

The concentrated sample (150 ml) is applied to a column (5 x 12 cm) of Blue Sepharose (Pharmacia LKB) equilibrated with buffer B. The column is eluted with a linear gradient (1.4 liters) from 0 to 2.0 M NaCl in buffer B and 50 ml fractions are collected. Three peaks of BVNOR activity are separated by this step. The first peak contains 20% of the total activity, and the enzyme responsible has not been characterized. The second peak represents 40% of the activity and, in this case, the reduction of benzyl viologen is catalyzed by ferredoxin NADP oxidoreductase (FNOR). The third peak of activity contains about 40% of the total and corresponds to NROR. This starts to elute as 1.7 M NaCl is applied to the column. Those fractions containing NROR activity are combined (600 ml) and concentrated to 30 ml by ultrafiltration (PM30 membrane). 

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