Interaction of a menaquinol binding site with the [3Fe‐4S] cluster of <i>Escherichia coli</i> fumarate reductase

Rothery, Richard A.; Weiner, Joël H.

doi:10.1046/j.1432-1327.1998.2540588.x

Cited by 24 publications

(52 citation statements)

References 12 publications

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“…No further FR3 line shape changes occurred after HQNO addition to FrdC E29L mutant QFR (data not shown). These observations provide evidence that FR3 is in close proximity to the Q-proximal binding site and agree with the observations of Rothery and Weiner (20). Furthermore, the location of the observed semiquinone in the vicinity of FR3 is consistent with the position of Glu-29 of FrdC in the structural model ( Fig.…”

Section: ϫ2supporting

confidence: 91%

“…trum (Fig. 5B) as described (20). EPR line shape perturbation of the FR3 spectrum in the E29L mutant is similar to that seen when the wild-type QFR FR3 spectrum is perturbed by HQNO (Fig.…”

Section: ϫ2supporting

confidence: 73%

“…that it corresponds to Q-distal. It has also been shown that HQNO elicits a significant change in the EPR line shape of E. coli FR3, indicating the close proximity of the quinone-binding site and iron-sulfur cluster (20). Such EPR line shape changes were not detected in the B. subtilis enzyme (21,22) in the presence of HQNO.…”

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confidence: 95%

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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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Section: ϫ2supporting

confidence: 91%

“…trum (Fig. 5B) as described (20). EPR line shape perturbation of the FR3 spectrum in the E29L mutant is similar to that seen when the wild-type QFR FR3 spectrum is perturbed by HQNO (Fig.…”

Section: ϫ2supporting

confidence: 73%

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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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“…FQ Titrations with 2-n-Heptyl-4-hydroxyquinoline-N-oxide (HOQNO) and Estimation of NarGHI Content in Membrane Samples-The affinity of NarGHI for HOQNO was determined by performing FQ titrations using a PerkinElmer Life Sciences LS-50B luminescence spectrometer (6,17,18). Fluorescence intensities were measured using an excitation wavelength of 341 nm and an emission wavelength of 479 nm.…”

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

Self Cite

118

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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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“…Acetate concentration changes shown represent the total acetate consumption including dissimilation (54%) and assimilation (46%) Membranes of G. sulfurreducens reduced fumarate with NADPH at a rate higher than with NADH [190 vs 70 nmol min -1 (mg protein) -1 , respectively]. The reaction was sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), a menaquinol analogue (Rothery and Weiner 1998). The presence of 0.5 mM HOQNO in the assay caused a 98.8% decrease of the fumarate reduction rate.…”

Section: Acetate Oxidation In Pure Culturementioning

confidence: 99%

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

Galushko

Schink

2000

Archives of Microbiology

136

163

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Geobacter sulfurreducens strain PCA oxidized acetate to CO 2 via citric acid cycle reactions during growth with acetate plus fumarate in pure culture, and with acetate plus nitrate in coculture with Wolinella succinogenes. Acetate was activated by succinyl-CoA:acetate CoA-transferase and also via acetate kinase plus phosphotransacetylase. Citrate was formed by citrate synthase. Soluble isocitrate and malate dehydrogenases reduced NADP + and NAD + , respectively. Oxidation of 2-oxoglutarate was measured as benzyl viologen reduction and was strictly CoA-dependent; a low activity was also observed with NADP + . Succinate dehydrogenase and fumarate reductase both were membrane-bound. Succinate oxidation was coupled to NADP + reduction whereas fumarate reduction was coupled to NADPH and NADH oxidation. Coupling of succinate oxidation to NADP + or cytochrome(s) reduction required an ATP-dependent reversed electron transport. Net ATP synthesis proceeded exclusively through electron transport phosphorylation. During fumarate reduction, both NADPH and NADH delivered reducing equivalents into the electron transport chain, which contained a menaquinone. Overall, acetate oxidation with fumarate proceeded through an open loop of citric acid cycle reactions, excluding succinate dehydrogenase, with fumarate reductase as the key reaction for electron delivery, whereas acetate oxidation in the syntrophic coculture required the complete citric acid cycle.

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Interaction of a menaquinol binding site with the [3Fe‐4S] cluster of Escherichia coli fumarate reductase

Cited by 24 publications

References 12 publications

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

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

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

Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in syntrophic coculture

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