Benzylidenemalononitrile Derivatives as Substrates and Inhibitors of a New NAD(P)H Dehydrogenase of Erythrocytes. Purification and Crystallisation of Two Forms of the Enzyme

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Abstract
Using the powerful lachrymator (2-chlorobenzylidene)malononitrile as electron acceptor, 2 types of NAD(P)H dehydrogenases were isolated from human blood. Crystallization of the homogenous enzymes was performed in 50% polyethylene glycol solution. The enzymes (average MW 18,000) are composed of only 1 polypeptide chain and have a very similar amino acid composition. B-side stereospecificity was determined regarding the cofactor by gas chromatography-mass spectrometry for the reductase. Besides (2-chlorobenzylidene)malononitrile, 2,6-dichloroindophenol, methylene blue, 4-benzoquinone, FMN and FAD are also reduced using NADH or NADPH as hydrogen donor with the rates decreasing in the given order. Reduction of methemoglobin is observed only upon addition of methylene blue, FMN or FAD as carriers. (2-chlorobenzylidene)malononitrile reduction is inhibited by most of the compounds known to be decouplers of oxidative phosphorylation.