Interaction of an engineered [3Fe-4S] cluster with a menaquinol binding site of Escherichia coli DMSO reductase.
We have characterized by EPR the interaction of the Em,7 = -50 mV [4Fe-4S] cluster of Escherichia coli DMSO reductase (DmsABC) with a menaquinol (MQH2) binding site. Potentiometric titrations indicate that in DmsAB(C102S)C, the Em,7 = -50 mV [4Fe-4S] cluster is replaced by an Em,7 = +260 mV [3Fe-4S] cluster. The Q-pool coupling assay in combination with the MQH2 analog HOQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) was used to examine the effect of the DmsB(Cl02S) mutation on physiological electron transfer through DmsABC. Forward electron transfer through the mutant (MQH2 to DmsA) is blocked in the Q-pool coupling assay, but reverse electron transfer (DmsA to MQ) is not. HOQNO elicits a significant change in the EPR line shape of the oxidized DmsAB(Cl02S)C [3Fe-4S] cluster but has no effect on the line shape of the reduced [4Fe-4S] clusters. We have identified a residue in DmsC involved in MQH2 oxidation. DmsC(H65), and in a double mutant, DmsAB(C102S)C(H65R), the DmsC mutation blocks the HOQNO effect on the [3Fe-4S] EPR line shape, suggesting, that the DmsC(H65R) mutation either blocks HOQNO binding or blocks a conformational link between a HOQNO binding site and the DmsB(C102S) [3Fe-4S] cluster. These results suggest that the MQH2 binding site of DmsC is conformationally and functionally linked to the Em,7 = -50 mV [4Fe-4S] cluster of DmsB.
Rothery
R A
RA
MRC Group in the molecular Biology of Membranes, Department of Biochemistry, Edmonton, Alberta, Canada.
Weiner
J H
JH
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006912:Hydroxyquinolines / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011199:Potentiometry; D015202:Protein Engineering; D013455:Sulfur / Q000378:metabolism; D014812:Vitamin K / Q000031:analogs & derivatives* / Q000378:metabolism*
D006912:Hydroxyquinolines; D007506:Iron-Sulfur Proteins; D008565:Membrane Proteins; D014812:Vitamin K; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D013455:Sulfur; D007501:Iron; D010088:Oxidoreductases; C045664:dimethyl sulfoxide reductase