Presence of HCN in Chlorella vulgaris and its possible role in controlling the reduction of nitrate

Abstract

NITRATE reductase of Chlorella vulgaris is present in crude extracts mainly in an inactive form^1–3 which may be converted to an active form by the addition of an artificial oxidant such as ferricyanide⁴. In crude extracts from which low-molecular weight substances have been removed by gel filtration, added NADH (or NADPH) causes a rapid conversion of the active form of nitrate reductase to the inactive form⁵. Losada and coworkers have also observed an inactivation of the nitrate reductase from Chlorella fusca⁶ and Chlamydomonas reinhardi⁷ by added NADH. A low-molecular weight fraction separated from crude extracts of Chlorella vulgaris also causes an inactivation of nitrate reductase when added back to the extract which had been treated with ferricyanide and passed through a Sephadex column to remove excess reagent⁵. In all cases the enzyme can be reactivated by the addition of the oxidant, ferricyanide^4–8. On purification of the enzyme, Solomonson⁹ found that two substances must be present simultaneously to cause the reversible inactivation: (1) a reducing substance, preferably NADH, and (2) a substance that behaves like HCN. The molar concentration of HCN needed to effect a rapid and reversible inactivation of the enzyme was of about the same magnitude as the enzyme concentration. Thus, activated, suitably purified nitrate reductase with added excess NADH, provides an exceedingly sensitive, though non-specific test for HCN, as shown in Fig. 1.