Nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) of spinach (Spinacia oleracea L.) leaves, inactivated in vitro by acetylene, was reactivated by irradiation with blue light. Red + infrared, green or white light of the same irradiance were less effective. The dehydrogenase activity of the nitrate reductase complex was not required for pliotoreactivation. Photoreactivation of cyanide‐inactivated nitrate reductase was greatly enhanced by the addition of 1 and 20 μ of either FMN or FAD; however, flavins showed a much smaller stimulatory effect on photoreactivation of acetylene‐inactivated enzyme. The effect of flavins was higher under anaerobic conditions. This might imply the direct ievolvement of excited flavins in the photoreactivation mechanism. Besides promoting photoreactivation, blue light irradiation led simultaneously to a gradual inactivation of the enzyme especially under air and 20 μ FMN, eventually abolishing the recovered activity of the enzyme.
All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase p h s catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.
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