Interaction of a menaquinol binding site with the [3Fe-4S] cluster of Escherichia coli fumarate reductase.
We have used EPR to study the interaction between the [3Fe-4S] FR3 cluster and a menaquinol (MKH2) binding site of Escherichia coli fumarate reductase (FrdABCD). The MKH2 analog 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) binds to FrdABCD and elicits a significant change in the EPR lineshape of the FR3 cluster of FrdB. In a mutant of FrdABCD in which His82 of FrdC is changed to Arg, this HOQNO effect is eliminated. The HOQNO effect can also be eliminated by reaction of membranes with the imidazole-reactive reagent ethoxyformic anhydride. Potentiometric titrations indicate that neither HOQNO nor [Arg82]FrdC have a detectable effect on the midpoint potential (Em.7) of FR3. Quinol:fumarate oxidoreductase activity assays confirm that HOQNO and [Arg82]FrdC both block MKH2 oxidation by FrdABCD. Fluorescence spectroscopy was used to quantitate HOQNO binding to FrdABCD; approximately 1.30 binding sites were detected/FR3 cluster. Overall, the data support a quinol-binding model for FrdABCD in which one site has a close functional relationship with the FR3 cluster.
Department of Biochemistry and the Medical Research Council Group in the Molecular Biology of Membranes, University of Alberta, Edmonton, Canada.
R A
RA
Rothery
J H
JH
Weiner
D019943:Amino Acid Substitution; D001665:Binding Sites; D002384:Catalysis; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism*
D007506:Iron-Sulfur Proteins; D009284:Naphthols; D013729:Terpenes; C066500:menaquinol 6; D013385:Succinate Dehydrogenase; D004047:Diethyl Pyrocarbonate