Red-light effects sensitized by methylene blue on nitrate reductase from spinach (Spinacia Oleracea L.) leaves

Abstract

Nitrate reductase from spinach (Spinacia oleracea L.) leaves, which had been inactivated in vitro by incubation with N A D H and cyanide, was fully reactivated in minutes when irradiated in anaerobic conditions with red light in the presence o f m ethylene blue. Both the rate and the extent of reactivation increased with light intensity ( 6 to 100 W - m “ 2) and dye concentration (1 to 10 jiM). On the contrary, photoreactivation was com pletely abolished when N A D H or ethylenediaminetetra-acetic acid were present during irradiation. W e propose that methylene blue, when photo excited, exhibits a redox potential positive enough to reoxidise the CN~-reduced molybdenum complex settled in the inactive enzyme, thus causing its reactivation. On the other hand, prolonged irradiation o f nitrate reductase, under air and in the presence o f methylene blue, promoted an oxygen-dependent irreversible inactivation o f the two partial activities of the enzyme. This inactivation was markedly enhanced in 77% deuterated water and greatly prevented by azide, which indicates that singlet oxygen is the species primarily involved in the photooxidative inactivation o f the enzyme