Viologen-diaphorase

Often the electron transfer between the reduced nicotinamide coenzyme and the mediator is rather slow because of kinetic limitations. In many of these cases electron transfer catalyzed by diaphorase (E. C. 1.6.99.x) results in a drastic enhancement of the reaction rate (Fig. 16.2-5 C). Diaphorase-catalyzed NAD(P)+ regeneration was reported for example with methylene blue1321, PQQ1331 (under aerobic conditions), ferrocene(28), N -methyl-p-aminophenol(34 , N, N-dimethylindoaniline, 2,6-di-chlorophenol indophenol (DCIP), [Fe(CN)6]2 [351, viologenes or several quinoid structures [36l... 

Artificial donors include the viologen dyes and hydrosulfite. Methyl viologen can replace clostridial Fd or Fid. Oxidation of the monomer of poly-)S-hydroxybutyrate, a major component of soybean nodule bac-teroids, has been coupled to N2ase to give very low activity via a complex system composed of a dehydrogenase, NAD a diaphorase, and a viologen dye 14). Hydrosulfite is the most useful donor for in vitro N2ase assays since it can be used in substrate amounts in contrast to all other natural and artificial donors (22). 

Diaphorase activity was measured at 600 nm by the reduction of DCPIP in the presence of NADPH. Light-saturated NADP reduction was measured at 340 nm with thylakoids supplemented with 5 juM ferredoxin. PSI dependent activities were measured with 20 jjM DCMU, 100 jjM DCPIP, 10 mM ascorbate euid 5 pM nigericin either in the presence of 200 jiil NADP or 100 pM methyl viologen (MV) as terminal electron acceptors. 

NAD (P)+-dependent enzymes, electrically contacted with electrode surfaces, can provide efhcient bioelectrocatalysis for the NAD(P)H oxidation. For example, diaphorase (DI) was applied to oxidize NADFl, using a variety of quinone compounds, several kinds of flavins, or viologens as mediators between the enzyme and electrode [217, 218]. The bimolecular reaction rate constants between the enzyme and mediators whose redox potentials are more positive than -0.28 V at pH 8.5 can be as high as 10 s , suggesting that the reac-... 

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