Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.

Werth, Mark T.; Cecchini, Gary; Manodori, Anamaria; Ackrell, Brian A.C.; Schröder, Imke; Gunsalus, Robert P.; Johnson, Michael K.

doi:10.1073/pnas.87.22.8965

Cited by 86 publications

(117 citation statements)

References 34 publications

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“…SDHB mutants p.Cys101Tyr and p.Arg242His were both associated with significantly reduced SDH activity when compared with the WT. Using our in silico results we predicted loss of bonding to the 2Fe-2S centre at p.Cys101, which is more tolerant to amino acid substitution than the other two (4Fe-4S), (3Fe-4S) domains of SDHB (Werth et al 1990, Iverson et al 2012. Mutation at p.Arg242 was predicted to disrupt hydrogen bonds between SDH subunits, thereby reducing their biochemical efficiency and perhaps could also explain the loss of function in an otherwise fully compartmentalised SDH complex.…”

Section: Discussionmentioning

confidence: 99%

Structural and functional consequences of succinate dehydrogenase subunit B mutations

Kim¹,

Rath²,

Tsang³

et al. 2015

Endocrine-Related Cancer

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Mitochondrial dysfunction, due to mutations of the gene encoding succinate dehydrogenase (SDH), has been implicated in the development of adrenal phaeochromocytomas, sympathetic and parasympathetic paragangliomas, renal cell carcinomas, gastrointestinal stromal tumours and more recently pituitary tumours. Underlying mechanisms behind germline SDH subunit B (SDHB) mutations and their associated risk of disease are not clear. To investigate genotype-phenotype correlation of SDH subunit B (SDHB) variants, a homology model for human SDH was developed from a crystallographic structure. SDHB mutations were mapped, and biochemical effects of these mutations were predicted in silico. Results of structural modelling indicated that many mutations within SDHB are predicted to cause either failure of functional SDHB expression (p.Arg27*, p.Arg90*, c.88delC and c.311delAinsGG), or disruption of the electron path (p.Cys101Tyr, p.Pro197Arg and p.Arg242His). GFP-tagged WT SDHB and mutant SDHB constructs were transfected (HEK293) to determine biological outcomes of these mutants in vitro. According to in silico predictions, specific SDHB mutations resulted in impaired mitochondrial localisation and/or SDH enzymatic activity. These results indicated strong genotype-functional correlation for SDHB variants. This study reveals new insights into the effects of SDHB mutations and the power of structural modelling in predicting biological consequences. We predict that our functional assessment of SDHB mutations will serve to better define specific consequences for SDH activity as well as to provide a much needed assay to distinguish pathogenic mutations from benign variants.

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Section: Discussionmentioning

confidence: 99%

Structural and functional consequences of succinate dehydrogenase subunit B mutations

Kim¹,

Rath²,

Tsang³

et al. 2015

Endocrine-Related Cancer

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“…Site-directed mutagenesis was performed as described previously (Werth et al, 1990). Oligonucleotides for mutagenesis and sequencing were synthesized on a Biosearch model 8700 DNA synthesizer.…”

Section: ␤-Galactosidase Assaymentioning

confidence: 99%

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

Cotter

Melville

Albrecht

et al. 1997

Molecular Microbiology

125

120

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“…E. coli strain DW35 cells containing pH3 plasmids encoding wild-type or mutant forms of QFR were grown anaerobically on glucose/fumarate medium as described previously (31). Cells were harvested in the early stationary phase of growth, and the membrane fraction was prepared from a French pressure cell lysate as described previously (32). Redox titrations were performed at room temperature (25°C) in an airtight vessel flushed with oxygen-free argon and equipped with a magnetic stirring device, an Ag/AgCl-platinum combination electrode, and a pH electrode essentially as described (33).…”

Section: Methodsmentioning

confidence: 99%

An Escherichia coli Mutant Quinol:Fumarate Reductase Contains an EPR-detectable Semiquinone Stabilized at the Proximal Quinone-binding Site

Hägerhäll¹,

Magnitsky²,

Sled³

et al. 1999

Journal of Biological Chemistry

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The EPR and thermodynamic properties of semiquinone (SQ) species stabilized by mammalian succinate: quinone reductase (SQR) in situ in the mitochondrial membrane and in the isolated enzyme have been well documented. The equivalent semiquinones in bacterial membranes have not yet been characterized, either in SQR or quinol:fumarate reductase (QFR) in situ. In this work, we describe an EPR-detectable QFR semiquinone using Escherichia coli mutant QFR (FrdC E29L) and the wild-type enzyme. The SQ exhibits a g ‫؍‬ 2.005 signal with a peak-to-peak line width of ϳ1.1 milliteslas at 150 K, has a midpoint potential (E m(pH 7.2) ) of ؊56.6 mV, and has a stability constant of ϳ1.2 ؋ 10 ؊2 at pH 7.2. It shows extremely fast spin relaxation behavior with a P 1/2 value of > >500 milliwatts at 150 K, which closely resembles the previously described SQ species (SQ s ) in mitochondrial SQR. This SQ species seems to be present also in wildtype QFR, but its stability constant is much lower, and its signal intensity is near the EPR detection limit around neutral pH. In contrast to mammalian SQR, the membrane anchor of E. coli QFR lacks heme; thus, this prosthetic group can be excluded as a spin relaxation enhancer. The trinuclear iron-sulfur cluster FR3 in the [3Fe-4S] 1؉ state is suggested as the dominant spin relaxation enhancer of the SQ FR spins in this enzyme. E. coli QFR activity and the fast relaxing SQ species observed in the mutant enzyme are sensitive to the inhibitor 2-nheptyl-4-hydroxyquinoline N-oxide (HQNO). In wildtype E. coli QFR, HQNO causes EPR spectral line shape perturbations of the iron-sulfur cluster FR3. Similar spectral line shape changes of FR3 are caused by the FrdC E29L mutation, without addition of HQNO. This indicates that the SQ and the inhibitor-binding sites are located in close proximity to the trinuclear iron-sulfur cluster FR3. The data further suggest that this site corresponds to the proximal quinone-binding site in E. coli QFR.Succinate:quinone reductase (SQR) 1 and quinol:fumarate reductase (QFR) are structurally and functionally similar enzymes with an interesting evolution (1-3). 1ϩ,0 clusters are called S1 or FR1, S2 or FR2, and S3 or FR3 in SQR and QFR, respectively. The membrane anchor domain of the enzyme is more variable and may consist of one or two hydrophobic polypeptides (SdhC/FrdC and SdhD/ FrdD) and contain zero, one, or two b hemes depending on the enzyme species. When two hemes are present, they are denoted heme b H and heme b L . The primary sequence similarity is also much lower in this part of the enzyme. Nevertheless, accumulated evidence indicates that the membrane anchors have a conserved general structure (3, 4). One exception is a group of SQRs lacking the membrane domain and instead containing two different, more or less hydrophilic subunits (5).The membrane-bound enzymes catalyze the oxidation of succinate or the reduction of fumarate in the bacterial cytoplasm or mitochondrial matrix and the reduction or oxidation of quinone/quinol in the membrane. It should be emphasi...

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Site-directed mutagenesis of conserved cysteine residues in Escherichia coli fumarate reductase: modification of the spectroscopic and electrochemical properties of the [2Fe-2S] cluster.

Cited by 86 publications

References 34 publications

Structural and functional consequences of succinate dehydrogenase subunit B mutations

Structural and functional consequences of succinate dehydrogenase subunit B mutations

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

An Escherichia coli Mutant Quinol:Fumarate Reductase Contains an EPR-detectable Semiquinone Stabilized at the Proximal Quinone-binding Site

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