The Presence of Bound Cyanide in the Naturally Inactivated Form of Nitrate Reductase of Chlorella vulgaris

In the presence of NADH and cyanide, NADH-nitrate oxidoreductase (EC 1.6.6.1) from Chlorella vulgaris is converted to an inactive form which is readily reactivated by ferricyanide. Experiments with H14CN indicated that the inactivation process is associated with the firm binding to the protein of 0.066 nmole of cyanide per unit of enzyme inactivated. This cyanide binding was linearly proportional to the amount of inactivation. No firm cyanide binding and no inactivation occurred in the absence of NADH or an equivalent reductant. The bound 14C was released when the enzyme was reactivated by ferricyanide. After the cells had been treated with ammonia for several hours prior to disruption, the crude cell extracts contained the nitrate reductase primarily in the inactive form. Critical examination of the methods of cell disruption suggested that the inactivation of the enzyme had truly occurred in vivo. After 300-fold purification, activation of this in vivo inactivated enzyme resulted in the release of 0.066 nmole of HCN per unit of enzyme activated. We conclude that the inactivation of nitrate reductase in vivo involves the formation of a firmly bound complex of reduced enzyme and cyanide. The reaction between reduced enzyme and HCN may be written: Er + HCN (ka)/⇄/(kd) Er-HCN The measured value for ka was 1.25 x 106 m-1 min-1, for kd, 4.5 x 10-4 min-1, giving a dissociation constant, Kd = 3.6 x 10-10 m.