Characterisation of the PQQ cofactor radical in quinoprotein ethanol dehydrogenase of <i>Pseudomonas aeruginosa</i> by electron paramagnetic resonance spectroscopy

Kay, Christopher W. M.; Mennenga, Bina; Görisch, Helmut; Bittl, Robert

doi:10.1016/s0014-5793(04)00317-5

Cited by 17 publications

(22 citation statements)

References 37 publications

(43 reference statements)

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“…Its g-value and lines width were typical for an organic radical, and it was assigned to the PQQ-semiquinone. Similar spectra were described for the PQQ moiety in purified ADH complexes from Pseudomonas aeruginosa (16) and Comamonas testosteroni (6).…”

Section: Bad905751)supporting

confidence: 56%

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Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Gómez-Manzo¹,

Chávez‐Pacheco²,

Contreras-Zentella³

et al. 2010

J Bacteriol

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Several aldehyde dehydrogenase (ALDH) complexes have been purified from the membranes of acetic acid bacteria. The enzyme structures and the chemical nature of the prosthetic groups associated with these enzymes remain a matter of debate. We report here on the molecular and catalytic properties of the membrane-bound ALDH complex of the diazotrophic bacterium Gluconacetobacter diazotrophicus. The purified ALDH complex is a heterodimer comprising two subunits of 79.7 and 50 kDa, respectively. Reversed-phase high-pressure liquid chromatography (HPLC) and electron paramagnetic resonance spectroscopy led us to demonstrate, for the first time, the unequivocal presence of a pyrroloquinoline quinone prosthetic group associated with an ALDH complex from acetic acid bacteria. In addition, heme b was detected by UV-visible light (UV-Vis) spectroscopy and confirmed by reversed-phase HPLC. The smaller subunit bears three cytochromes c. Aliphatic aldehydes, but not formaldehyde, were suitable substrates. Using ferricyanide as an electron acceptor, the enzyme showed an optimum pH of 3.5 that shifted to pH 7.0 when phenazine methosulfate plus 2,6-dichlorophenolindophenol were the electron acceptors. Acetaldehyde did not reduce measurable levels of the cytochrome b and c centers; however, the dithionite-reduced hemes were conveniently oxidized by ubiquinone-1; this finding suggests that cytochrome b and the cytochromes c constitute an intramolecular redox sequence that delivers electrons to the membrane ubiquinone.

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Section: Bad905751)supporting

confidence: 56%

“…Earlier, this technique had been successfully applied to investigate the PQQ prosthetic group in bacterial ADHs complexes (16,6). To our knowledge, ALDH complexes of acetic acid bacteria have yet to be analyzed by EPR spectroscopy.…”

Section: Bad905751)mentioning

confidence: 99%

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Gómez-Manzo¹,

Chávez‐Pacheco²,

Contreras-Zentella³

et al. 2010

J Bacteriol

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“…PQQ is bonded to the Ca 2? ion and exhibits an EPR spectrum with the Dg xx = 3,531, Dg yy = 2,861, and Dg zz = -199 components [10,11]. The magnitude of the Dg-shifts predicted by us for the model o-semiquinone complexes is similar, despite the chemical differences.…”

Section: G Tensormentioning

confidence: 57%

“…Electron paramagnetic resonance (EPR) spectroscopy has established its important position in investigation of semiquinone radicals in laboratory conditions and in their natural surroundings [8][9][10][11][12][13][14]. Also, the formation of a complex between diamagnetic metal ions and semiquinone radicals can be efficiently investigated using the EPR techniques since the g and A tensors are sensitive to the radical-metal ions interaction [5,6,[15][16][17][18].…”

Section: Introductionmentioning

confidence: 99%

DFT insight into o-semiquinone radicals and Ca2+ ion interaction: structure, g tensor, and stability

Witwicki

Jezierska

2013

Theor Chem Acc

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Density functional theory methods were employed to elucidate the interactions between calcium ions and various o-semiquinone radicals mimicking the interactions occurring in biochemical systems. Predicted changes in the molecular and electronic structures of the radicals on Ca 2? coordination were correlated with the changes of g tensor and compared with those exerted by Mg 2? ions (reported by us previously). In order to broaden the insight into the differences between the Mg 2? and Ca 2?complexes, their relative stability was estimated on the basis of theoretically predicted Gibbs energies for the process of the complex formation.

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“…38 The wildtype as well as the mutant protein were active in a functional assay using an artificial electron acceptor, implying that the disulfide bridge does not play a role in the general mechanism of the enzyme. The X-band continuous-wave EPR spectrum of the PQQ semiquinone radical in the wild-type QEDH enzyme was found to be slightly asymmetric with a line centered at g iso ¼ 2:0043, and a peak-to-peak line width of 0.5 mT, which is similar to that observed in GDH, 39 in quinohaemoprotein ethanol dehydrogenase, 40 methylamine dehydrogenase (MADH), 41 and in MDH.…”

Section: -12mentioning

confidence: 97%

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

Weber¹,

Bittl²

2007

Bulletin of the Chemical Society of Japan

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The elucidation of structural and electronic aspects of paramagnetic organic cofactors in their protein surroundings and related model compounds is a topic of widespread interest. In this field, electron paramagnetic resonance (EPR) spectroscopy, especially in conjunction with high-resolution electron-nuclear double resonance (ENDOR) is an important tool, allowing us to analyze details of the distribution of the unpaired electron spin, which is influenced by the interaction between the cofactor and its immediate environment, as well as to characterize paramagnetic intermediate states in the course of the protein action. In this account, investigations are reported in which EPR/ENDOR methods have contributed significantly to answering questions on mechanistic and structural aspects of protein function. Emphasis is given to in-depth characterizations of electronic and spatial structures of radical cofactors and their positioning relative to substrate molecules. After a brief introduction, covering some of the trends of modern EPR/ENDOR method development, we will focus, as examples, on characterizations of quinone-type and flavin cofactors in two proteins, (i) quinoprotein alcohol dehydrogenase, and (ii) DNA photolyase.

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Characterisation of the PQQ cofactor radical in quinoprotein ethanol dehydrogenase of Pseudomonas aeruginosa by electron paramagnetic resonance spectroscopy

Cited by 17 publications

References 37 publications

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

Molecular and Catalytic Properties of the Aldehyde Dehydrogenase of Gluconacetobacter diazotrophicus , a Quinoheme Protein Containing Pyrroloquinoline Quinone, Cytochrome b , and Cytochrome c

DFT insight into o-semiquinone radicals and Ca2+ ion interaction: structure, g tensor, and stability

Studies of Organic Protein Cofactors Using Electron Paramagnetic Resonance

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