PMID:15924426

Citation	Cheng, VW, Rothery, RA, Bertero, MG, Strynadka, NC and Weiner, JH (2005) Investigation of the environment surrounding iron-sulfur cluster 4 of Escherichia coli dimethylsulfoxide reductase. Biochemistry 44:8068-77
Abstract	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.
Links	PubMed Online version:10.1021/bi050362p
Keywords	Amino Acid Sequence; Amino Acid Substitution/genetics; Binding Sites/genetics; Electron Spin Resonance Spectroscopy; Electron Transport/genetics; Enzyme Inhibitors/chemistry; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/chemistry; Escherichia coli Proteins/genetics; Formate Dehydrogenases/chemistry; Hydrogen-Ion Concentration; Hydroxyquinolines/chemistry; Iron-Sulfur Proteins/antagonists & inhibitors; Iron-Sulfur Proteins/chemistry; Iron-Sulfur Proteins/genetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Oxidation-Reduction; Oxidoreductases/antagonists & inhibitors; Oxidoreductases/chemistry; Oxidoreductases/genetics; Potentiometry; Protein Binding/genetics; Spectrometry, Fluorescence; Structural Homology, Protein
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