Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Pennoyer, Jeffrey D.; Ohnishi, Tomo̧ko; Trumpower, Bernard L.

doi:10.1016/0005-2728(88)90216-2

Cited by 47 publications

(33 citation statements)

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“…2 Enzyme Purification-SQR was purified by thawing the stored cell packs using 150 -200 g of material each time. The purification procedure was as described previously (6), with modifications similar to those described previously (14,15). The enzyme was concentrated, and the salt and Triton X-100 concentrations of the final samples were reduced, the latter to ϳ0.05% (w/v), by repeated exchange in Centricon 100-kDa cutoff concentrators against 100 mM Hepes, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

“…13543 strain. The enzyme was considered sufficiently pure (Ͼ90%) for use in our experiments by criteria of optical spectra (negligible absorption due to hemes other than b 557 ) and SDS-polyacrylamide gel electrophoresis (6).…”

Section: Methodsmentioning

confidence: 99%

“…Cytochrome b concentrations were determined from dithionite reducedminus-oxidized difference spectra in a pyridine hemochrome assay mixture, using ⌬⑀ 557-540 ϭ 24.0 mM Ϫ1 cm Ϫ1 (17). The SQR activity was measured with a large excess of ubiquinone-2 (Q 2 ; 20 M) and dichlorophenol-indophenol as the primary and terminal electron acceptors, respectively (6,18); activation of the enzyme was achieved by incubating the enzyme (in Triton X-100) for 20 min at 298 K in 50 mM Tris buffer, 50 mM sodium succinate, 0.2 mM dodecyl-␤-D-maltoside, pH 7.5. Typical turnover numbers (moles of succinate/mol of SQR) of the purified enzyme at 310 K were 300 -350 s Ϫ1 based on the FAD concentrations of the samples (see also Ref.…”

Section: Methodsmentioning

confidence: 99%

“…P. denitrificans appears to be the closest bacterial homologue to this organelle (11,12), and its SQR is amenable to molecular genetic techniques. The purification and basic biochemical properties of SQR from this bacterium have also been reported (6).…”

mentioning

confidence: 96%

“…These anchoring subunits confer reactivity with the bound Qs; for SQR from bovine heart (3, 4) and a variety of higher plants (5), the existence of two Q sites has been established. SQR from Paracoccus denitrificans contains covalently bound FAD; the membrane anchor consists of two polypeptides with a mono-heme cytochrome b (6). Despite extensive efforts (for reviews see Refs.…”

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confidence: 99%

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Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Waldeck¹,

Stowell²,

Lee³

et al. 1997

Journal of Biological Chemistry

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Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic "signatures" accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochrome b, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4 -10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate-and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity ( 2 nm) of the high potential 3Fe-4S cluster, situated in the succinate dehydrogenase part of the enzyme, and the low potential, low spin b-heme in the membrane anchor of the enzyme.Succinate:ubiquinone oxidoreductase, SQR, 1 is the only membrane-bound enzyme in the tricarboxylic acid cycle. As "complex II," it performs the two-electron oxidation of succinate to produce fumarate, while transferring the electrons to quinone (Q) to yield quinol (QH 2 ). The reverse process is mediated by quinol:fumarate oxidoreductases (QFR), which occur in anaerobic and some facultative organisms. The two enzymes are related and are capable of catalyzing their respective reverse reactions under suitable conditions (1, 2).SQR contains three or four polypeptides depending on the organism. The largest subunit, a flavoprotein, contains the dicarboxylate binding site and one flavin moiety (FAD); the latter is covalently bound in most cases. The iron-sulfur protein is intermediate in size and contains three iron-sulfur clusters of type 2Fe-2S, 4Fe-4S, and 3Fe-4S, often referred to as S-1, S-2, and S-3, respectively, in the case of SQR. These two hydrophilic subunits protrude into the cytosol (prokaryotic enzyme) or the mitochondrial matrix (eukaryotic enzyme), and together catalyze the succinate dehydrogenase activity of the enzyme. They are anchored to the membrane by one or two hydrophobic quinone polypeptides (QP), which may contain zero, one, or two b-heme(s). These anchoring subunits confer reactivity with the bound Qs; for SQR from bovine heart (3, 4) and a variety of higher plants (5), the existence of two Q sites has been established. S...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 96%

mentioning

confidence: 99%

See 3 more Smart Citations

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Waldeck¹,

Stowell²,

Lee³

et al. 1997

Journal of Biological Chemistry

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Succinate:Quinone Oxidoreductases

Lancaster

2004

Handbook of Metalloproteins

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Succinate:quinone oxidoreductases encompass the succinate:quinone reductase (SQR) enzymes of aerobic respiration (respiratory complex II) and the quinol:fumarate reductase (QFR) enzymes of anaerobic respiration. These enzymes couple the two‐electron oxidation of succinate to fumarate to the two‐electron reduction of quinone to hydroquinone (quinol), and also catalyse the reverse reaction, the reduction of fumarate by quinol. Members of the superfamily are composed of two hydrophilic subunits, a large flavoprotein subunit A and a smaller subunit B containing three iron–sulfur centers, and one large subunit C or two small subunits C and D hydrophobic polypeptides that are integral membrane proteins and bind two, one, or no heme b groups. On the basis of the available crystal structures of members of the superfamily, structural and functional aspects of succinate:quinone oxidoreductases are comprehensively discussed.

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Succinate:Quinone Oxidoreductases

Lancaster

2004

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Purification and properties of succinate-ubiquinone oxidoreductase complex from Paracoccus denitrificans

Cited by 47 publications

References 46 publications

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Electron Paramagnetic Resonance Studies of Succinate:Ubiquinone Oxidoreductase from Paracoccus denitrificans

Succinate:Quinone Oxidoreductases

Succinate:Quinone Oxidoreductases

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