tailieunhanh - Báo cáo khoa học: Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis, fluorescence quench titrations and EPR spectroscopy

We have used fluorescence quench titrations, EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB, FrdC and FrdD on the proximal menaquinol (MQH2) binding site (QP)of Escherichia colifumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH2 analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indi-cate that the QPsite is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparentKdof nm, and the following mutations significantly increase this value: FrdB-T205H (Kd¼39 nm);. | ềFEBS Journal Defining the QP-site of Escherichia coli fumarate reductase by site-directed mutagenesis fluorescence quench titrations and EPR spectroscopy Richard A. Rothery1 Andrea M. Seime1 . Caroline Spiers1 Elena Maklashina2 3 Imke Schroder4 Robert P. Gunsalus4 Gary Cecchini2 3 and Joel H. Weiner1 1 CIHR Membrane Protein Research Group Department of Biochemistry University of Alberta Edmonton Alberta Canada 2 Molecular Biology Division Veterans Affairs MedicalCenter San Francisco CA USA 3 Department of Biochemistry and Biophysics University of California San Francisco CA USA 4 Department of Microbiology Immunology and Molecular Genetics University of California Los Angeles CA USA Keywords fumate reductase Q-site iron-sulfur menaquinol Correspondence R. A. Rothery Department of Biochemistry 474 MedicalSciences Building University of Alberta Edmonton Alberta T6G 2H7 Fax 1 780 492 0886 Tel 1 780 492 2229 E-mail Received 13 September 2004 revised 22 October 2004 accepted 1 November 2004 doi We have used fluorescence quench titrations EPR spectroscopy and steady-state kinetics to study the effects of site-directed mutants of FrdB FrdC and FrdD on the proximal menaquinol MQH2 binding site QP of Escherichia coli fumarate reductase FrdABCD in cytoplasmic membrane preparations. Fluorescence quench FQ titrations with the fluorophore and MQH2 analog 2-n-heptyl-4-hydroxyquinoline-N-oxide HOQNO indicate that the QP site is defined by residues from FrdB FrdC and FrdD. In FQ titrations wild-type FrdABCD binds HOQNO with an apparent Kd of nM and the following mutations significantly increase this value FrdB-T205H Kd 39 nM FrdB-V207C Kd 20 nM FrdC-E29L Kd 25 nM FrdC-W86R no detectable binding and FrdD-H80K Kd 20 nM . In all titrations performed data were fitted to a monophasic binding equation indicating that no additional high-affinity HOQNO binding sites exist in FrdABCD. In all cases where HOQNO binding is .