title: Stopped-flow studies of the binding of 2-n-heptyl-4-hydroxyquinoline-N-oxide to fumarate reductase of Escherichia coli.
abstract: We have studied the kinetics of binding of the menaquinol analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) by fumarate reductase (FrdABCD) using the stopped-flow method. The results show that the fluorescence of HOQNO is quenched when HOQNO binds to FrdABCD. The observed quenching of HOQNO fluorescence has two phases and it can be best fitted to a double exponential equation. A two-step equilibrium model is applied to describe the binding process in which HOQNO associates with FrdABCD by a fast bimolecular step to form a loosely bound complex; this is subsequently converted into a tightly bound complex by a slow unimolecular step. The rates of the forward and the reverse reactions for the first equilibrium (k1 and k2) are determined to be k1 = (1.1 +/- 0.1) x 10(7) M-1.s-1, and k2 = 6.0 +/- 0.6 s-1, respectively. The dissociation constants of the first equilibrium (Kd1 = k2/k1) is calculated to be about 550 nM. The overall dissociation constant for the two-step equilibrium, Kd overall = Kd1/[1+ (1/Kd2)], is estimated to be < or = 7 nM. Comparison of the kinetic parameters of HOQNO binding by FrdABCD and by dimethyl sulfoxide reductase provides important information on menaquinol binding by these two enzymes.
authors: Department of Biochemistry, University of Alberta, Edmonton, Canada.
authors: Z
authors: Z
authors: Zhao
authors: R A
authors: RA
authors: Rothery
authors: J H
authors: JH
authors: Weiner
mesh_terms: D004926:Escherichia coli / Q000201:enzymology*; D005470:Fluorometry; D006912:Hydroxyquinolines / Q000378:metabolism*; D007506:Iron-Sulfur Proteins; D007700:Kinetics; D010088:Oxidoreductases / Q000378:metabolism; D011485:Protein Binding; D013385:Succinate Dehydrogenase / Q000378:metabolism*
chemical_list: D006912:Hydroxyquinolines; D007506:Iron-Sulfur Proteins; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D010088:Oxidoreductases; D013385:Succinate Dehydrogenase; C045664:dimethyl sulfoxide reductase