Kinetic Studies of Ferrous Ion Oxidation with Crystalline Human Ferroxidase

Abstract

Abstract The reaction between crystalline human ferroxidase (ferro: O2 oxido-reductase, EC 1.12.3) and the substrate, Fe(II), 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 k1 = 1.2 x 106 m-1 sec-1, k2 ≈ 13 sec-1, k3 = 570 x 103 m-1 sec-1, and k'2 = 1.1 sec-1, 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(II) 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.