Defining the Q-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 (MQH(2)) binding site (Q(P)) of Escherichia coli fumarate reductase (FrdABCD) in cytoplasmic membrane preparations. Fluorescence quench (FQ) titrations with the fluorophore and MQH(2) analog 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) indicate that the Q(P) site is defined by residues from FrdB, FrdC and FrdD. In FQ titrations, wild-type FrdABCD binds HOQNO with an apparent K(d) of 2.5 nM, and the following mutations significantly increase this value: FrdB-T205H (K(d) = 39 nM); FrdB-V207C (K(d) = 20 nM); FrdC-E29L (K(d) = 25 nM); FrdC-W86R (no detectable binding); and FrdD-H80K (K(d) = 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 detectable by FQ titration, it can also be observed by EPR spectroscopy. Steady-state kinetic studies of fumarate-dependent quinol oxidation indicate that there is a correlation between effects on HOQNO binding and effects on the observed K(m) and k(cat) values, except in the FrdC-E29L mutant, in which HOQNO binding is observed, but no enzyme turnover is detected. In this case, EPR studies indicate that the lack of activity arises because the enzyme can only remove one electron from reduced MQH(2), resulting in it being trapped in a form with a bound menasemiquinone radical anion. Overall, the data support a model for FrdABCD in which there is a single redox-active and dissociable Q-site.
Rothery
Richard A
RA
Department of Biochemistry, CIHR Membrane Protein Research Group, 474 Medical Sciences Building, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Richard.Rothery@UAlberta.ca
Seime
Andrea M
AM
Spiers
A-M Caroline
AM
Maklashina
Elena
E
Schröder
Imke
I
Gunsalus
Robert P
RP
Cecchini
Gary
G
Weiner
Joel H
JH
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005453:Fluorescence; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009284:Naphthols / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000737:chemistry*; D013729:Terpenes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009284:Naphthols; D013729:Terpenes; C066500:menaquinol 6; D013385:Succinate Dehydrogenase