Open AccessStopped‐flow studies of the binding of 2‐ n ‐heptyl‐4‐hydroxyquinoline‐ N ‐oxide to fumarate reductase of Escherichia coli
Author(s) -
Zhao Zhongwei,
Rothery Richard A.,
Weiner Joel H.
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00116.x
Subject(s) - chemistry , kinetics , dissociation constant , equilibrium constant , dimethyl sulfoxide , reaction rate constant , dissociation (chemistry) , stereochemistry , quenching (fluorescence) , fluorescence , organic chemistry , biochemistry , physics , receptor , quantum mechanics
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 ( k 1 and k 2 ) are determined to be k 1 = (1.1 ± 0.1) × 10 7 m −1 ·s −1 , and k 2 = 6.0 ± 0.6 s −1 , respectively. The dissociation constants of the first equilibrium ( K d1 = k 2 / k 1 ) is calculated to be about 550 n m . The overall dissociation constant for the two‐step equilibrium, K d overall = K d1 /[1 + (1/ K d2 )], is estimated to be ≤ 7 n m . 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.