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

Rothery, Richard A.; Weiner, Joël H.

doi:10.1021/bi00098a003

Cited by 96 publications

(116 citation statements)

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“…We believe that the decreased activities are the result of altering the E m value of FS3, which is adjacent to and may exchange electrons directly with the Q site. Experiments done on the Saccharomyces cerevisiae bc 1 complex, E. coli Me 2 SO reductase, and E. coli fumarate reductase have shown that alteration of the E m value of the [Fe-S] cluster adjacent to the Q site can cause major changes in enzyme activity (9,26,40).…”

Section: Discussionmentioning

confidence: 99%

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

Cheng¹,

Zhao

et al. 2006

Journal of Biological Chemistry

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Succinate dehydrogenase is an indispensable enzyme involved in the Krebs cycle as well as energy coupling in the mitochondria and certain prokaryotes. During catalysis, succinate oxidation is coupled to ubiquinone reduction by an electron transfer relay comprising a flavin adenine dinucleotide cofactor, three iron-sulfur clusters, and possibly a heme b 556 . At the heart of the electron transport chain is a [4Fe-4S] cluster with a low midpoint potential that acts as an energy barrier against electron transfer. Hydrophobic residues around the [4Fe-4S] cluster were mutated to determine their effects on the midpoint potential of the cluster as well as electron transfer rates. SdhB-I150E and SdhB-I150H mutants lowered the midpoint potential of this cluster; surprisingly, the His variant had a lower midpoint potential than the Glu mutant. Mutation of SdhB-Leu-220 to Ser did not alter the redox behavior of the cluster but instead lowered the midpoint potential of the [3Fe-4S] cluster. To correlate the midpoint potential changes in these mutants to enzyme function, we monitored aerobic growth in succinate minimal medium, anaerobic growth in glycerol-fumarate minimal medium, non-physiological and physiological enzyme activities, and heme reduction. It was discovered that a decrease in midpoint potential of either the [4Fe-4S] cluster or the [3Fe-4S] cluster is accompanied by a decrease in the rate of enzyme turnover. We hypothesize that this occurs because the midpoint potentials of the [Fe-S] clusters in the native enzyme are poised such that direction of electron transfer from succinate to ubiquinone is favored. Electron transport (ET)3 chains are ubiquitous and play a key role in energy conservation in both aerobic and anaerobic respiration. Cofactors such as iron-sulfur ([Fe-S]) clusters, hemes, and flavins comprise the ET relays of respiratory chain enzymes and mediate electron transfer from a powerful reductant with a relatively low midpoint potential (E m ) to a final oxidant with a relatively high E m . The ET chain usually involves cofactors from multiple enzymes and the membrane-soluble ubiquinone or menaquinone pool, and the energetics of individual ET steps are not always downhill. In many redox enzymes, the exception often occurs in the form of an [Fe-S] cluster with an unusually low E m located at an intermediate position in the ET relay (1-4). 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 (5-10). 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.Succinate dehydrogenase (Sdh, Complex II in eukaryotes) is an indispensable enzyme involved in the Krebs cycle and...

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Section: Discussionmentioning

confidence: 99%

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

Cheng¹,

Zhao

et al. 2006

Journal of Biological Chemistry

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“…Preparations-Crude membrane vesicles were prepared from E. coli cells by French pressure cell lysis and differential centrifugation (12). Enriched inner membrane vesicles were isolated from these crude membranes by sucrose step centrifugation as described previously (6).…”

Section: Isolation Of Membrane Fractions and Purifiedmentioning

confidence: 99%

Structural and Biochemical Characterization of a Quinol Binding Site of Escherichia coli Nitrate Reductase A

Bertero

Rothery

Boroumand

et al. 2005

Journal of Biological Chemistry

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117

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The crystal structure of Escherichia coli nitrate reductase A (NarGHI) in complex with pentachlorophenol has been determined to 2.0 Å of resolution. We have shown that pentachlorophenol is a potent inhibitor of quinol:nitrate oxidoreductase activity and that it also perturbs the EPR spectrum of one of the hemes located in the membrane anchoring subunit (NarI). This new structural information together with site-directed mutagenesis data, biochemical analyses, and molecular modeling provide the first molecular characterization of a quinol binding and oxidation site (Q-site) in NarGHI. A possible proton conduction pathway linked to electron transfer reactions has also been defined, providing fundamental atomic details of ubiquinol oxidation by NarGHI at the bacterial membrane.Escherichia coli, when grown anaerobically in the presence of nitrate, synthesizes the membrane-bound quinol:nitrate oxidoreductase NarGHI 1 (1, 2). This enzyme has been the subject of intense biochemical, biophysical, and structural studies. The protein complex contains three subunits with characteristic redox prosthetic groups: NarG (140 kDa), the catalytic subunit with a molybdo-bis(molybdopterin guanine dinucleotide) cofactor and an [Fe-S] cluster (FS0); NarH (58 kDa), the electron transfer subunit with four [Fe-S] clusters (FS1, FS2, FS3, and FS4); NarI (26 kDa), the integral membrane subunit with two b-type hemes, termed b P and b D to indicate their proximal (b P ) and distal (b D ) positions to the catalytic site. NarG and NarH form a soluble cytoplasmically localized catalytic domain anchored to the membrane by NarI. NarGHI catalyzes electron transfer from a quinol binding site located in NarI through the redox cofactors aligned as an "electric wire" through the complex (b D 3 b P 3 FS4 3 FS3 3 FS2 3 FS1 3 FS0) to the molybdo-bis(molybdopterin guanine dinucleotide cofactor in NarG, where nitrate is reduced to nitrite.NarGHI often forms a respiratory chain with the formate dehydrogenase FdnGHI via the lipid soluble quinol pool. Electron transfer from formate to nitrate is coupled to proton translocation across the cytoplasmic membrane generating proton motive force by a redox loop mechanism (3). In the redox loop mechanism proton translocation is the net result of the topographically segregated reduction of quinone and oxidation of quinol on opposite sites of the membrane. The high resolution structures of both respiratory complexes, FdnGHI and NarGHI, have been recently solved (1, 4). Crystallographic analysis of FdnGHI has shown the presence of a quinone reduction site oriented toward the cytoplasm (4). Existing biochemical and biophysical evidence indicates that the quinol binding and oxidation functionality of NarGHI is provided by the NarI subunit (5-7), but no high resolution structural information has been made available to date.We have solved the crystal structure at 2.0 Å of resolution of NarGHI in complex with the quinol binding inhibitor pentachlorophenol (PCP), which is structurally related to the physiological quinol subs...

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“…This constitutive membrane-bound enzyme is composed of three subunits : a catalytic subunit containing molybdenum molybdopterin guanine dinucleotide cofactor (DmsA), an electron transfer subunit containing four [4Fe-4S] clusters (DmsB) and a membrane anchor subunit (DmsC) (Bilous e t al., 1988;Cammack & Weiner, 1990;Rothery & Weiner, 1991). Characterization of the molybdenum cofactor and [Fe-S] centres has proceeded using EPR (Cammack 8 z Weiner, 1990;Rothery & Weiner, 1991, 1993 and fluorescence spectroscopy (Rothery e t al., 1995), and an understanding of the electron transfer pathway from menaquinol to the molybdenum cofactor is currently being developed (Trieber e t al., 1994).…”

Section: Introductionmentioning

confidence: 99%

“…E. coli strains used in this study were: HBlOl [s~pE44 ksdS20 Z(r;m;) recA73 ara-74 proAZ ZacYI galK2 rpsL20 xjl-5 mtl-I ] (Boyer & Roulland-Dussoix, 1969) ; MC4100 [F-ara D139 (lad POZYA--argF)U169 rpsL150 relAI fEb-5307 deoCl PtsF25 rbsR] (Casadaban, 1976); LCB128 [as MC4100; torA : : Mud1 (Ap' -lrsc)] (M. Chippaux, Marseilles, France); DSS401 (as MC4100; Adms Km') (Sambasivarao & Weiner, 1991a); and DSS501 (as LCB128; Mms Km") (Sambasivarao & Weiner, 1991a). For overexpression of DmsABC, bacteria were transformed with pDMSl6O (Rothery & Weiner, 1991). All manipulations of plasmids and strains were carried out essentially as described by Sambrook et al (1989).…”

Section: Introductionmentioning

confidence: 99%

“…HBlOl(pDMS160) was grown in 19 1 carboys and harvested using a Pellicon membranes system as indicated previously (Weiner e t d., 1988). The cell paste was resuspended in buffer (Rothery & Weiner, 1991), and everted envelopes were prepared as indicated by Bilous & Weiner (1985a). The membrane fraction was extracted with Triton X-100 as indicated by Cammack & Weiner (1990).…”

Section: Introductionmentioning

confidence: 99%

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Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase

Simala‐Grant

Weiner

1996

Microbiology

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~~ ~~We have characterized the substrate specificity of dimethyl sulfoxide reductase (DmsABC) of Escherichia coli by determining Km and kut values for 22 different substrates. The enzyme has a very broad substrate specificity. The Km values varied 470-fold, while k,, values varied only 20-fold, implicating K, as the major determinant of kJK, values. Sulfoxides and pyridine N-oxide exhibited the lowest K, values, followed by aliphatic N-oxides. The k,, values for these compounds also followed the same pattern. Substitution at the 2 or 3 position of the pyridine N-oxide ring had little effect on Kw while substitution at the 4 position had a greater effect, and increased Km. Negatively charged substrates were poorly accepted. A few compounds that are not S-or N-oxides were also reduced by the enzyme. Most compounds reduced by DmsABC were not toxic to E. coli under anaerobic growth conditions, and Em coli was able to use many of these compounds anaerobically as terminal electron acceptors in the presence of glycerol. Anaerobic growth on sulfoxides is solely due to DmsABC expression. However, there appears to be another as yet unidentified terminal reductase capable of using pyridine N-oxides as terminal electron acceptors.

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

Cited by 96 publications

References 38 publications

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

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

Structural and Biochemical Characterization of a Quinol Binding Site of Escherichia coli Nitrate Reductase A

Kinetic analysis and substrate specificity of Escherichia coli dimethyl sulfoxide reductase

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