Investigation of the Environment Surrounding Iron−Sulfur Cluster 4 ofEscherichia coliDimethylsulfoxide Reductase

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.

The Iron-Sulfur Clusters in Escherichia coli Succinate Dehydrogenase Direct Electron Flow

“…Thus, the ET relay in E. coli fumarate reductase would favor electron transfer from FS3 to FS1. The effects of E m values in relation to electron transfer have also been previously examined in Me 2 SO reductase (7).…”

“…Phenylmethanesulfonyl fluoride was added to a final concentration of 0.2 mM, and cells were lysed using a French press. Membranes were prepared by differential centrifugation as previously described (7). Enriched membranes were prepared by centrifugation on a 55% sucrose cushion at 150,000 ϫ g for 90 min and were diluted to 25 ml.…”

“…Thus during catalysis, electrons must surmount the energy barrier imposed by the low potential cluster despite the overall downhill reaction between the reductant and oxidant. Controversy continues to surround the issue as to whether E m values of cofactors play a role in determining the rate of electron transfer through redox enzymes, especially that of the low potential cluster (5)(6)(7)(8)(9)(10). It would thus be of great interest to use a genetically modifiable model system to study the effects of E m modulation on observed catalytic rates of electron transfer.…”

Correct Assembly of Iron-Sulfur Cluster FS0 into Escherichia coli Dimethyl Sulfoxide Reductase (DmsABC) Is a Prerequisite for Molybdenum Cofactor Insertion

“…DSS301 was used for the growth experiments. TOPP2 was used for enzyme expression and spectroscopic studies, as we found that it was optimal for DmsABC overexpression and assembly (27). The plasmids (Table 1) used for cloning and expression included pBR322, pDMS160 (28), and pATZ (EcoRI-EcoRV fragment of pDMS160 (28) cloned into pTZ18R (Amp R lacZЈ (Pharmacia)) (laboratory collection)).…”

“…Cloning and Site-directed Mutagenesis-Construction of the site-directed mutants was carried out following the QuikChange method (Stratagene) (27) using pATZ as the template for mutagenesis. Mutants were verified by DNA sequencing carried out by the Department of Biochemistry DNA Core Facility at the University of Alberta.…”