Kinetic studies of ferrous ion oxidation with crystalline human ferroxidase. II. Rate constants at various steps and formation of a possible enzyme-substrate complex.

The reaction between crystalline human ferroxidase (ferro:On oxido-reductase, EC 1.12.3) and the substrate, Fe(H), was studied by a stop-flow method. The rate constants for four separate steps-enzyme-substrate complex formation, separation of product from the enzyme, reoxidation by molecular oxygen, and a rate-determining step-were estimated to be kl = 1.2 x lo6 M-I set-I, kp = 13 set-*, ka = 570 x lo3 M-I set-l, and k’t = 1.1 set-I, respectively. The rate-determining process in catalytic action by ferroxidase is possibly a conformational change of the enzyme molecule before the reoxidation. Spectral evidence for the formation of an enzyme-substrate complex in the early stage of the reaction between ferroxidase and Fe(U) was obtained. The lifetime of the enzyme-substrate complex was found to be very short, approximately 40 msec. The sequence of the reaction and possible rate-determining step in ferroxidase activity is discussed, and the data obtained in the present experiment are compared with those reported elsewhere.