Intermediates in the catalytic action of lipoyl dehydrogenase (diaphorase)

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Abstract
The anaerobic addition of either reduced diphosphopyridine nucleotide or dihydrolipoic acid derivatives to diaphorase results in the production of a red colour, characterized by an increase in extinction in the 500-650 mu region of the spectrum, and changes in other regions of the visible spectrum. The effect of diphosphopyridine nucleotide, oxidized lipoic acids, potassium ferricyanide, oxygen and dithionite on the red form of the enzyme indicate that it represents an intermediate oxidation level of the enzyme-bound flavinadenine dinucleotide between the fully oxidized and fully reduced forms. By analogy with previous work, this form has been ascribed to the semiquinone or free radical state of the flavin (FADH). The effect of p-chloromercuriphenyl sulphonate on the red form is described; the results indicate that the semiquinone form is stablized by interaction with a protein sulphydryl group, possibly a sulphur radical. The rates of formation of the red intermediate and of its reoxidation under a variety of conditions have been studied by the stopped-flow method. Comparison of the rates obtained with catalytic rates estimated under the same conditions shows that the rate of formation of FADH by dihydrolipoic acids is the rate-determining step in the catalytic reduction of diphosphopyridine nucleotide by dihydrolipoic acids. Similarly, the rate of reoxidation of FADH by oxidized lipoic acids is the rate-determining step in the catalytic oxidation of reduced diphosphopyridine nucleotide by oxidized lipoic acids. The results of the rate experiments and titration experiments show that in the overall two-electron oxidation-reduction reaction catalysed by the enzyme the flavinadenine dinucleotide is reduced to FADH'' and reoxidized only once per catalytic cycle. The relation of this finding to the reaction mechanism of the enzyme is discussed.