The reactivation of nitrate reductase from spinach (Spinacea oleracea L.) inactivated by NADH and cyanide: effects of peroxidase and associated systems

Nitrate reductase of spinach (Spinacea oleracea L.) leaves which had been inactivated in vitro by treatment with NADH and cyanide, was reactivated by incubation with oxidant systems and measured as FMNH₂-dependent activity. Ferricyanide, a purely chemical oxidant, produced rapid maximal reactivation (100%) which was 90% complete in less than 3 min. Reactivation occurred slowly and less completely (30–75% in 30 or 60 min) when the enzyme was incubated with pure horseradish peroxidase alone, depending on using one or 20 units and time. Addition of glucose and glucose oxidase to generate hydrogen peroxide increased reactivation slightly (10–15%) with 20 units of peroxidase but more (30–50%) with one unit and to 75–90% of ferricyanide values. Adding catalase decreased reactivation by more than half either with or without glucose oxidase. Glucose and glucose oxidase alone did not cause reactivation. Addition of superoxide dismutase increased reactivation from 50–75% of ferricyanide values with one unit of peroxidase alone but had no effect on greater reactivation obtained in the presence of glucose oxidase. The addition of p-cresol and manganese together increased reactivation with one unit of peroxidase and in the presence of glucose oxidase by about double, but omission of manganese had no effect. However, as shown previously, although trivalent manganese was formed, the residual presence of manganous ions inhibited reactivation. Nevertheless, peroxidase systems either alone or with additionally generated hydrogen peroxide can induce substantial reactivation of nitrate reductase in physiologically relevant conditions.Abbreviations EDTA ethylenediaminetetraacetic acid - FMN flavine mononucleotide