In vivo and in vitro inactivation of spinach leaf nitrate reductase by cyanide photogenerated from carbonylcyanide m-chlorophenylhydrazone

Abstract

Illumination with white light (400 W · m−2) of spinach leaf discs infiltrated with 1 mM carbonylcyanide m-chlorophenylhydrazone (CCCP) promoted in vivo a fast inactivation of nitrate reductase (NR). This inactivation was stimulated by O2. Blue and red lights were much more effective than green light. From the two partial activities of NR, only the terminal activity (assayed with reduced flavin mononucleotide) was abolished. The in vivo inactivated enzyme could be readily reactivated by a short incubation with potassium ferricyanide. NR was also inactivated in vitro by illumination of the enzyme preparation in the presence of CCCP.Illumination of a CCCP solution with white light generated cyanide. Cyanide release was stimulated by O2 and only sustained by blue light. However, if chlorophylls were simultaneously present, red light was as effective as blue light. From these observations, and from the fact that NR inactivation exhibited striking similarities with the enzyme inactivation by cyanide, it is concluded that, both in vivo and in vitro, NR is inactivated by the cyanide generated from CCCP during illumination.