Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase.
Iron-sulfur ([Fe-S]) clusters are common in electron transfer proteins, and their midpoint potentials (E(m) values) play a major role in defining the rate at which electrons are shuttled. The E(m) values of [Fe-S] clusters are largely dependent on the protein environment as well as solvent accessibility. The electron transfer subunit (DmsB) of Escherichia coli dimethylsulfoxide reductase contains four [4Fe-4S] clusters (FS1-FS4) with E(m) values between -50 and -330 mV. We have constructed an in silico model of DmsB and addressed the roles of a group of residues surrounding FS4 in electron transfer, menaquinol (MQH(2)) binding, and protein control of its E(m). Residues Pro80, Ser81, Cys102, and Tyr104 of DmsB are located at the DmsB-DmsC interface and are critical for the binding of the MQH(2) inhibitor analogue 2-n-heptyl-4-hydroxyquinoline N-oxide (HOQNO) and the transfer of electrons from MQH(2) to FS4. Because the EPR spectrum of FS4 is complicated by spectral overlap and spin-spin interactions with the other [4Fe-4S] clusters of DmsB, we evaluated mutant effects on FS4 in double mutants (with a DmsB-C102S mutation) in which FS4 is assembled as a [3Fe-4S] cluster (FS4([3Fe)(-)(4S])). The DmsB-C102S/Y104D and DmsB-C102S/Y104E mutants dramatically lower the E(m) of FS4([3Fe)(-)(4S]) from 275 to 150 mV and from 275 to 145 mV, respectively. Mutations of positively charged residues around FS4([3Fe)(-)(4S]) lower its E(m), but mutations of negatively charged residues have negligible effects. The E(m) of FS4([3Fe)(-)(4S]) in the DmsB-C102S mutant is insensitive to HOQNO as well as to changes in pH from 5 to 7. The FS4([3Fe)(-)(4S]) E(m) of the DmsB-C102S/Y104D mutant increases in the presence of HOQNO and decreasing pH. Analyses of the mutants suggest that the maximum achievable E(m) for FS4([3Fe)(-)(4S]) of DmsB is approximately 275 mV.
Cheng
Victor W T
VW
Membrane Protein Research Group, Department of Biochemistry, University of Alberta, 474 Medical Sciences Building, Edmonton, Alberta T6G 2H7, Canada.
Rothery
Richard A
RA
Bertero
Michela G
MG
Strynadka
Natalie C J
NC
Weiner
Joel H
JH
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D005560:Formate Dehydrogenases / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006912:Hydroxyquinolines / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D011199:Potentiometry; D011485:Protein Binding / Q000235:genetics; D013050:Spectrometry, Fluorescence; D040681:Structural Homology, Protein
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D006912:Hydroxyquinolines; D007506:Iron-Sulfur Proteins; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; D010088:Oxidoreductases; D005560:Formate Dehydrogenases; C045664:dimethyl sulfoxide reductase