Electron paramagnetic resonance spectroscopic characterization of dimethyl sulfoxide reductase of Escherichia coli

The electron transfer centers in dimethyl sulfoxide reductase were examined by EPR spectroscopy in membranes of the overproducing Escherichia coli strain HB101/pDMS159, and in purified enzyme. Iron-sulfur clusters of the [4Fe-4S] type and a molybdenum center were detected in the protein, which comprises three different subunits: DmsA, -B, and -C. The intensity of the reduced iron-sulfur clusters corresponded to 3.82 +/- 0.5 spins per molecule. The dithionite-reduced clusters were reoxidized by DMSO or TMAO. The enzyme, as prepared, showed a spectrum of Mo(V), which resembles the high-pH form of E. coli nitrate reductase. The Mo(V) detected by EPR was absent from a mutant which does not assemble the molybdenum cofactor. In these cases, the levels of EPR-detectable iron-sulfur clusters in the cells were increased. Extracts from HB101/pDMS159 enriched in DmsA showed more Mo(V) signals and considerably less iron-sulfur. These results are in agreement with predictions from amino acid sequence comparisons, that the molybdenum center is located in DmsA, while four iron-sulfur clusters are in DmsB. The midpoint potentials of the molybdenum and iron-sulfur clusters in the various preparations were determined by mediator titrations. The iron-sulfur signals could be best fitted by four clusters, with midpoint potentials spread between -50 and -330 mV. The midpoint potentials of the iron-sulfur clusters and Mo(V) species were pH dependent. In addition, all potentials became less negative in the presence of the detergent Triton X-100. Observation of relaxation enhancement of the Mo(V) species by the reduced [4Fe-4S] clusters indicated that the centers are in proximity within the protein.     

Alteration of the iron-sulfur cluster composition of Escherichia coli dimethyl sulfoxide reductase by site-directed mutagenesis.

We have used site-directed mutagenesis to alter the [Fe-S] cluster composition of Escherichia coli dimethyl sulfoxide (DMSO) reductase (DmsABC). The electron-transfer subunit (DmsB) of this enzyme contains 16 Cys residues arranged in 4 groups (I-IV) which provide ligands to 4 [4Fe-4S] clusters [Cammack, R., & Weiner, J. H. (1990) Biochemistry 29, 8410-8416]. Strong homologies exist between these Cys groups and the four Cys groups of the electron-transfer subunit (NarH) of E. coli nitrate reductase (NarGHJI), which contains a [3Fe-4S] cluster in addition to multiple [4Fe-4S] clusters. The Cys group primarily involved in providing ligands to the [3Fe-4S] cluster of NarH has a Trp residue at a position equivalent to Cys102 of DmsB. We have mutated Cys102 to Trp, Ser, Tyr, and Phe and have investigated the altered enzymes in terms of their enzymatic activities and EPR properties. The mutant enzymes do not support electron transfer from menaquinol to DMSO, although they retain high rates of electron transport from reduced benzyl viologen to DMSO. The mutations cause major changes in the EPR properties of the enzyme in the fully reduced and oxidized states. In the oxidized state, new species are observed in all the mutants; these have spectral features comprising a peak at g = 2.03 (gz) and a peak-trough at g = 2.00 (gxy). The temperature dependencies, microwave power dependencies, and spin quantitations of these species are consistent with the Trp102, Ser102, Phe102, and Tyr102 mutations causing conversion of one of the [4Fe-4S] clusters present in the wild-type enzyme into [3Fe-4S] clusters in the mutant enzymes.     

Multiple roles for the twin arginine leader sequence of dimethyl sulfoxide reductase of Escherichia coli

Dimethyl sulfoxide (Me(2)SO) reductase of Escherichia coli is a terminal electron transport chain enzyme that is expressed under anaerobic growth conditions and is required for anaerobic growth with Me(2)SO as the terminal electron acceptor. The trimeric enzyme is composed of a membrane extrinsic catalytic dimer (DmsAB) and a membrane intrinsic anchor (DmsC). The amino terminus of DmsA has a leader sequence with a twin arginine motif that targets DmsAB to the membrane via a novel Sec-independent mechanism termed MTT for membrane targeting and translocation. We demonstrate that the Met-1 present upstream of the twin arginine motif serves as the correct translational start site. The leader is essential for the expression of DmsA, stability of the DmsAB dimer, and membrane targeting of the reductase holoenzyme. Mutation of arginine 17 to aspartate abolished membrane targeting. The reductase was labile in the leader sequence mutants. These mutants failed to support growth on glycerol-Me(2)SO minimal medium. Replacing the DmsA leader with the TorA leader of trimethylamine N-oxide reductase produced a membrane-bound DmsABC with greatly reduced enzyme activity and inefficient anaerobic respiration indicating that the twin arginine leaders may play specific roles in the assembly of redox enzymes.     

Investigation of Escherichia coli dimethyl sulfoxide reductase assembly and processing in strains defective for the sec-independent protein translocation system membrane targeting and translocation

Dimethyl sulfoxide reductase is a heterotrimeric enzyme (DmsABC) localized to the cytoplasmic surface of the inner membrane. Targeting of the DmsA and DmsB catalytic subunits to the membrane requires the membrane targeting and translocation (Mtt) system. The DmsAB dimer is a member of a family of extrinsic, cytoplasmic facing membrane subunits that require Mtt in order to assemble on the membrane. We show that the MttA(2), MttB, and presumably MttA(1) but not the MttC proteins are required for targeting DmsAB to the membrane. Unlike other Mtt substrates such as trimethylamine N-oxide reductase, the soluble cytoplasmic DmsAB dimer that accumulates in the mtt deletions is very labile. Deletion of the mttA(2) or mttB genes also prevents anaerobic growth on fumarate even though fumarate reductase does not require Mtt for assembly. This was due to the lethality of membrane insertion of DmsC in the absence of the DmsAB subunits. In the absence of DmsC, DmsAB accumulates in the cytoplasm. A 45-amino acid leader on DmsA is removed during assembly. Processing does not require DmsC but does require Mtt. Translocation of DmsAB to the periplasm is not required for processing. The leader may be cleaved by a novel leader peptidase, or the long DmsA leader may traverse the membrane through the Mtt system resulting in cleavage by the periplasmic leader peptidase I followed by release of DmsA into the cytoplasm.     

Purification and properties of Escherichia coli dimethyl sulfoxide reductase, an iron-sulfur molybdoenzyme with broad substrate specificity.

Dimethyl sulfoxide reductase, a terminal electron transfer enzyme, was purified from anaerobically grown Escherichia coli harboring a plasmid which codes for dimethyl sulfoxide reductase. The enzyme was purified to greater than 90% homogeneity from cell envelopes by a three-step purification procedure involving extraction with the detergent Triton X-100, chromatofocusing, and DEAE ion-exchange chromatography. The purified enzyme was composed of three subunits with molecular weights of 82,600, 23,600, and 22,700 as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular weight was determined by gel electrophoresis to be 155,000. The purified enzyme contained 7.5 atoms of iron and 0.34 atom of molybdenum per mol of enzyme. The presence of molybdopterin cofactor in dimethyl sulfoxide reductase was identified by reconstitution of cofactor-deficient NADPH nitrate reductase activity from Neurospora crassa nit-I mutant and by UV absorption and fluorescence emission spectra. The enzyme displayed a very broad substrate specificity, reducing various N-oxide and sulfoxide compounds as well as chlorate and hydroxylamine.     

H+/e- stoichiometry for NADH dehydrogenase I and dimethyl sulfoxide reductase in anaerobically grown Escherichia coli cells.

Anaerobically grown Escherichia coli cells were shown to acidify the reaction medium in response to oxygen or dimethyl sulfoxide (DMSO) pulses, with the H+/e- stoichiometry being close to 2.5 and 1.5, respectively. In the presence of the NADH dehydrogenase I (NDH-I) inhibitor 8-methyl-N-vanillyl-6-nonenamide (capsaicin) or in mutants lacking NDH-I, this ratio decreased to 1 for O2 and to 0 for DMSO. These data suggest that (i) the H+/e- stoichiometry for E. coli NDH-I is at least 1.5 and (ii) the DMSO reductase does not generate a proton motive force.     

Effects of dimethyl sulfoxide on catalysis in Escherichia coli F1-ATPase.

(1) Dimethyl sulfoxide (DMSO) markedly inhibited the Vmax of multisite ATPase activity in Escherichia coli F1-ATPase at concentrations greater than 30% (v/v). Vmax/KM was reduced by 2 orders of magnitude in 40% (v/v) DMSO at pH 7.5, primarily due to reduction of Vmax. The inhibition was rapidly reversed on dilution into aqueous buffer. (2) KdATP at the first, high-affinity catalytic site was increased 1500-fold from 2.3 x 10(-10) to 3.4 x 10(-7) M in 40% DMSO at pH 7.5, whereas KdADP was increased 3.2-fold from 8.8 to 28 microM. This suggests that the high-affinity catalytic site presents a hydrophobic environment for ATP binding in native enzyme, that there is a significant difference between the conformation for ADP binding as opposed to ATP binding, and that the ADP-binding conformation is more hydrophilic. (3) Rate constants for hydrolysis and resynthesis of bound ATP in unisite catalysis were slowed approximately 10-fold by 40% DMSO; however, the equilibrium between bound Pi/bound ATP was little changed. The reduction in catalysis rates may well be related to the large increase in KdATP (less constrained site). (4) Significant Pi binding to E. coli F1 could not be detected either in 40% DMSO or in aqueous buffer using a centrifuge column procedure. (5) We infer, on the basis of the measured constants KaATP, K2 (hydrolysis/resynthesis of ATP), k+3 (Pi release), and KdADP and from estimates of k-3 (Pi binding) that delta G for ATP hydrolysis in 40% DMSO-containing pH 7.5 buffer is between -9.2 and -16.8 kJ/mol.     

Amperometric dimethyl sulfoxide sensor using dimethyl sulfoxide reductase from Rhodobacter sphaeroides

An amperometric dimethyl sulfoxide (DMSO) sensor was constructed based on DMSO reductase (DMSO-R). DMSO-R from Rhodobacter sphaeroides f. sp. denitrificans was immobilized by BSA-glutaraldehyde cross-linking at the surface of a glassy carbon electrode. Mediators were added to the sample solution in a free form. Several mediators (methyl viologen (MV), benzyl viologen (BV), neutral red (NR), safranin T (ST), FMN, phenazine methosulfate (PMS)), which can donate electrons to DMSO-R, were examined with the DMSO-R immobilized electrode. Among them MV was selected as a model mediator because of its wide linear response range and fast response time. The response current was effected by the measurement temperature but hardly effected by the pH of the sample solution. The response current was increased with the measurement temperature up to 50 degrees C. A response current was observed at 1 microM DMSO and the response time was 20 s under the optimum conditions. The response was observed for approximately 2 weeks. By the reduction of Schiff base in the cross-linking layer the response range became narrower but most of the response current was retained at 300 microM of DMSO for more than 5 weeks.     

The Escherichia coli ynfEFGHI operon encodes polypeptides which are paralogues of dimethyl sulfoxide reductase (DmsABC)

The ynfEFGHI operon is a paralogue of the Escherichia coli dmsABC operon. ynfE and ynfF are paralogues of dmsA. ynfG and ynfH are paralogues of dmsB and dmsC, respectively. YnfI (dmsD) has no dms paralogue. YnfE/F and YnfG could be detected by immunoblotting with anti-DmsAB antibodies when expressed under the control of a tac or dms promoter. Cells harbouring ynfFGH on a multicopy plasmid supported anaerobic growth with dimethyl sulfoxide (DMSO) as respiratory oxidant in a dmsABC deletion, suggesting that YnfFGH forms a heterotimeric enzyme complex similar to DmsABC. Exchange of DmsC by YnfH (DmsAB-YnfH) resulted in membrane localization, anaerobic growth on DMSO, and binding of 2-n-heptyl 4-hydroxyquinoline-N-oxide, indicating that YnfH was a competent anchor. YnfG can also replace DmsB as the electron transfer subunit and assembled [Fe-S] clusters as judged by electron paramagnetic resonance spectroscopy. YnfE and/or YnfF could not form a functional complex with DmsBC and expression of YnfE prevented the accumulation of YnfFGH.     

A methionine sulfoxide reductase in Escherichia coli that reduces the R enantiomer of methionine sulfoxide

It is known that Escherichia coli methionine mutants can grow on both enantiomers of methionine sulfoxide (met(o)), i.e., met-R-(o) or met-S-(o), indicating the presence of enzymes in E. coli that can reduce each of these enantiomers to methionine (met). Previous studies have identified two members of the methionine sulfoxide reductase (Msr) family of enzymes, MsrA and fSMsr, that could reduce free met-S-(o), but the reduction of free met-R-(o) to met has not been elucidated. One possible candidate is MsrB which is known to reduce met-R-(o) in proteins to met. However, free met-R-(o) is a very poor substrate for MsrB and the level of MsrB activity in E. coli extracts is very low. A new member of the Msr family (fRMsr) has been identified in E. coli extracts that reduces free met-R-(o) to met. Partial purification of FRMsr has been obtained using extracts from an MsrA/MsrB double mutant of E. coli.     

Association of molybdopterin guanine dinucleotide with Escherichia coli dimethyl sulfoxide reductase: effect of tungstate and a mob mutation.

We have identified the organic component of the molybdenum cofactor in Escherichia coli dimethyl sulfoxide reductase (DmsABC) to be molybdopterin (MPT) guanine dinucleotide (MGD) and have studied the effects of tungstate and a mob mutation on cofactor (Mo-MGD) insertion. Tungstate severely inhibits anaerobic growth of E. coli on a glycerol-dimethyl sulfoxide minimal medium, and this inhibition is partially overcome by overexpression of DmsABC. Isolation and characterization of an oxidized derivative of MGD (form A) from DmsABC overexpressed in cells grown in the presence of molybdate or tungstate indicate that tungstate inhibits insertion of Mo-MGD. No electron paramagnetic resonance evidence for the assembly of tungsten into DmsABC was found between Eh = -450 mV and Eh = +200 mV. The E. coli mob locus is responsible for the addition of a guanine nucleotide to molybdo-MPT (Mo-MPT) to form Mo-MGD. DmsABC does not bind Mo-MPT or Mo-MGD in a mob mutant, indicating that nucleotide addition must precede cofactor insertion. No electron paramagnetic resonance evidence for the assembly of molybdenum into DmsABC in a mob mutant was found between Eh = -450 mV and Eh = +200 mV. These data support a model for Mo-MGD biosynthesis and assembly into DmsABC in which both metal chelation and nucleotide addition to MPT precede cofactor insertion.     

Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101.

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.     

Stepwise selection of defective nitrogen-fixing phenotypes in Escherichia coli K-12 by dimethyl sulfoxide.

Storage in dimethyl sulfoxide of Escherichia coli K-12 hybrids carrying Klebsiella pneumoniae nif+ genes can result in selection of a defective nitrogen-fixing phenotype. Dimethyl sulfoxide appears to affect particular inner membrane proteins associated with energy metabolism in E. coli K-12.     

High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli.

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.     

Anaerobic induction of trimethylamine N-oxide reductase and cytochromes by dimethyl sulfoxide inEscherichia coli

Reduction of trimethylamine N-oxide is catalyzed by at least two enzymes inEscherichia coli: trimethylamine N-oxide reductase, which is anaerobically induced by trimethylamine N-oxide, and the constitutive enzyme dimethyl sulfoxide reductase. In this study, an increase in the specific activity of trimethylamine N-oxide reduction was observed in the anaerobic culture with dimethyl sulfoxide, but the specific activity of dimethyl sulfoxide reduction was not changed. The inducible enzyme trimethylamine N-oxide reductase was found in this culture. A marked expression of the structural genetorA for trimethylamine N-oxide reductase was also observed in atorA-lacZ gene fusion strain under anaerobic conditions with either trimethylamine N-oxide or dimethyl sulfoxide.l-Methionine sulfoxide and the N-oxides of adenosine, picolines, and nicotinamide slightly repressed expression of the gene. Membrane-boundb- andc-type cytochromes involved in the trimethylamine N-oxide reduction were also produced in a wild-type strain grown anaerobically with dimethyl sulfoxide. But thec-type cytochrome was not produced in thetorA-lacZ strain grown anaerobically with trimethylamine N-oxide or dimethyl sulfoxide; this suggests that there is a correlation between the expression oftorA and the synthesis of the cytochrome.     

Proton translocation coupled to dimethyl sulfoxide reduction in anaerobically grown Escherichia coli HB101.

Proton translocation coupled to dimethyl sulfoxide (DMSO) reduction was examined in Escherichia coli HB101 grown anaerobically on glycerol and DMSO. Rapid acidification of the medium was observed when an anaerobic suspension of cells, preincubated with glycerol, was pulsed with DMSO, methionine sulfoxide, nitrate, or trimethylamine N-oxide. The DMSO-induced acidification was sensitive to the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (60 microM) and was inhibited by the quinone analog 2-n-heptyl-4-hydroxy-quinoline-N-oxide (5.6 microM). Neither sodium azide nor potassium cyanide inhibited the DMSO response. An apparent----H+/2e- ratio of 2.9 was obtained for DMSO reduction with glycerol as the reductant. Formate and H2(g), but not lactate, could serve as alternate electron donors for DMSO reduction. Cells grown anaerobically on glycerol and fumarate displayed a similar response to pulses of DMSO, methionine sulfoxide, nitrate, and trimethylamine N-oxide with either glycerol or H2(g) as the electron donor. However, fumarate pulses did not result in acidification of the suspension medium. Proton translocation coupled to DMSO reduction was also demonstrated in membrane vesicles by fluorescence quenching. The addition of DMSO to hydrogen-saturated everted membrane vesicles resulted in a carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone-sensitive fluorescence quenching of quinacrine dihydrochloride. The data indicate that reduction of DMSO by E. coli is catalyzed by an anaerobic electron transport chain, resulting in the formation of a proton motive force.     

Methionine sulfoxide reduction and assimilation in Escherichia coli: new role for the biotin sulfoxide reductase BisC

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine-S-sulfoxide (Met-S-SO) and methionine-R-sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli. BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.     

Membrane potential before and after deep freezing of Escherichia coli in the presence of dimethyl sulfoxide and diethyl sulfoxide

It was shown by the method of penetrating tetraphenylphosphonium cations that low-temperature freezing (-196 degrees C) of Escherichia coli leads to a sharp decrease (from 198 to 85 mV) in membrane potential. Incubation of bacteria in a medium containing dimethyl sulfoxide and diethyl sulfoxide as cryoprotectors results in a reduction of the potential by 16 and 27 mV, respectively. It was also shown that diethyl sulfoxide is more effective in maintaining the membrane potential after freezing--thawing than dimethyl sulfoxide.     

Structure of fumarate reductase on the cytoplasmic membrane of Escherichia coli.

The terminal electron transfer enzyme fumarate reductase has been shown to be composed of a membrane-extrinsic catalytic dimer of 69- and 27-kilodalton (kd) subunits and a membrane-intrinsic anchor portion of 15- and 13-kd subunits. We prepared inverted membrane vesicles from a strain carrying the frd operon on a multicopy plasmid. When grown anaerobically on fumarate-containing medium, the membranes of this strain are highly enriched in fumarate reductase. When negatively stained preparations of these vesicles were examined with an electron microscope, they appeared to be covered with knob-like structures about 4 nm in diameter attached to the membrane by short stalks. Treatment of the membranes with chymotrypsin destroyed the 69-kd subunit, leaving the 27-, 15-, and 13-kd subunits bound to the membrane; these membranes appeared to retain remnants of the structure. Treatment of the membranes with 6 M urea removed the 69- and 27-kd subunits, leaving the anchor polypeptides intact. These vesicles appeared smooth and structureless. A functional four-subunit enzyme and the knob-like structure could be reconstituted by the addition of soluble catalytic subunits to the urea-stripped membranes. In addition to the vesicular structures, we observed unusual tubular structures which were covered with a helical array of fumarate reductase knobs.