Elucidating mechanisms in haemcopperoxidases: The high-affinity Q<sub>H</sub>binding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

MacMillan, Fraser; Kacprzak, Sylwia; Hellwig, Petra; Grimaldi, Stéphane; Michel, Hartmut; Kaupp, Martin

doi:10.1039/c005149g

Cited by 14 publications

(39 citation statements)

References 93 publications

(218 reference statements)

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“…Remarkably, the two other protein-bound semiquinones with redox properties most strongly affected by the protein environment as compared with the corresponding species in alcoholic solvents are the very low potential (E m ϳϪ750 mV) phyllosemiquinone anion A 1 ⅐ Ϫ in photosystem I and the high affinity ubisemiquinone at the Q H site of cytochrome bo 3 , which has a high stability, albeit 10 times smaller than that measured for MSQ D . In both cases, the semiquinones interact with the protein environment in a very asymmetric manner (46,(51)(52)(53). This contrasts to the much lower stability of the more symmetrically bound semiquinones at the Q B site of photosynthetic bacterial RC (48,54) or at the Q i site in R. sphaeroides bc 1 complex (47,55).…”

Section: Single Exchangeable Proton Coupling With Peculiar Hyperfinementioning

confidence: 50%

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Grimaldi¹,

Arias-Cartin²,

Lanciano³

et al. 2012

Journal of Biological Chemistry

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Background: Escherichia coli nitrate reductase A highly stabilizes a semiquinone catalytic intermediate. Results:Three proton hyperfine couplings to this radical with atypical characteristics are characterized. Conclusion: Semiquinone binding is strongly asymmetric and occurs via a single short in-plane H-bond. Significance: Learning how the protein environment tunes the semiquinone properties is crucial for understanding the quinol utilization mechanism by energy-transducing enzymes.

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Section: Single Exchangeable Proton Coupling With Peculiar Hyperfinementioning

confidence: 50%

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Grimaldi¹,

Arias-Cartin²,

Lanciano³

et al. 2012

Journal of Biological Chemistry

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“…In contrast, an essentially anisotropic coupling to Lys 15 Nz consistent with at hrough-space magnetic interaction was resolved. This suggestst hat MSK does not form ah ydrogen bond with the side chain of the nearbyL ys86 residue.…”

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confidence: 97%

“…N selectivel abeling of His 15 Nd or Lys 15 Nz in combination with hyperfine sublevelc orrelation (HYSCORE) spectroscopyu nambiguously identified His 15 Nd as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys 15 Nz consistent with at hrough-space magnetic interaction was resolved.…”

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confidence: 99%

“…Assigning the detected 1 Ha nd 14 Nn ucleit oaparticular chemical group is not straightforward. For this purpose, various strategies have been used, including (i)uniform enrichment of the protein in 2 H [12][13][14] or 15 N [15][16][17][18][19][20][21][22] or of the solvent thankst oH 2 O/ 2 H 2 Oe xchange experiments; [12,[23][24][25][26][27][28] (ii)theoretical calculations of hyperfine and quadrupolar parameters, [13,[29][30][31][32][33] (iii)reconstitution experimentsu sing exogenous quinones with various substituents or chemically labeled with 2 H, [29,34] and (iv) comparison with spectroscopic data obtained on model SQs measured in organic solvents. [35][36][37][38][39] Ad irect and more physiological strategy relies on in vivo isotope labeling of individual amino acids or sets of amino acids,w hich,h owever,r equires engineered auxotrophics trains supplemented with isotopically enriched amino acids.…”

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confidence: 99%

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Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective ²H and ¹⁵N Labeling, HYSCORE Spectroscopy, and DFT Modeling

et al. 2017

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In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15N selective labeling of His15Nδ or Lys15Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15Nδ as the direct hydrogen‐bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15Nζ consistent with a through‐space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2H—and hence of the 1H—methyl isotropic hyperfine coupling by 2H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen‐bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one‐sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.

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“…These data indicate the substantial shift of the unpaired spin density toward the O1 side of the Q H SQ. Unfortunately, very recent calculations devoted to the comparison of the unpaired spin density distributions in neutral and anionic SQ species of UQ (54,55) do not report the values of the 13 C methyl couplings, precluding any comparison of the values shown in Table 2 with theoretical values.…”

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confidence: 99%

Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents

Lin

Shubin

Samoilova

et al. 2011

Journal of Biological Chemistry

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The cytochrome bo 3 ubiquinol oxidase from Escherichia coli resides in the bacterial cytoplasmic membrane and catalyzes the two-electron oxidation of ubiquinol-8 and four-electron reduction of O 2 to water. The one-electron reduced semiquinone forms transiently during the reaction, and the enzyme has been demonstrated to stabilize the semiquinone. The semiquinone is also formed in the D75E mutant, where the mutation has little influence on the catalytic activity, and in the D75H mutant, which is virtually inactive. In this work, wild-type cytochrome bo 3 as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 13 C-labeled selectively at the methyl and two methoxy groups. This was accomplished by expressing the proteins in a methionine auxotroph in the presence of L-methionine with the side chain methyl group 13 C-labeled. The 13 Clabeled quinone isolated from cytochrome bo 3 was also used for the generation of model anion radicals in alcohol. Two-dimensional pulsed EPR and ENDOR were used for the study of the 13 C methyl and methoxy hyperfine couplings in the semiquinone generated in the three proteins indicated above and in the model system. The data were used to characterize the transferred unpaired spin densities on the methyl and methoxy substituents and the conformations of the methoxy groups. In the wild type and D75E mutant, the constraints on the configurations of the methoxy side chains are similar, but the D75H mutant appears to have altered methoxy configurations, which could be related to the perturbed electron distribution in the semiquinone and the loss of enzymatic activity. Ubiquinone (UQ)3 functions as a membrane-soluble twoelectron carrier. It is often found as a cofactor in many respiratory and photosynthetic complexes and plays an important role in biological oxidation-reduction reactions. Many of these reactions consist of one-electron transfer steps and involve the one-electron reduced ubisemiquinone species as the intermediates. Knowledge of how the semiquinone (SQ) radicals are stabilized in these complexes is critical to understand their reaction mechanisms. The quinone binding sites located in the respiratory and photosynthetic enzymes are significantly different (1), and each protein fine tunes the properties of the bound quinone cofactor by providing a unique environment in order to accomplish its own electron transfer process (2). The well characterized quinone binding sites include those from bacterial photosynthetic reaction centers (3), cytochrome bo 3 ubiquinol oxidase (cyt bo 3 ) from Escherichia coli (4), cytochrome bc 1 complexes (5), photosystems I and II, and some others (6 -9).A SQ can bind within a protein site in a manner that favors either the neutral (QH ⅐ ) or anionic (Q . ) form or intermediate states with partial charge remaining on the SQ. The proton locations along the hydrogen bonds to the quinone carbonyls determine the net charge on the SQ. The formation of hydrogen bonds of different strengths to the quinone oxygens usually leads to an asym...

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Elucidating mechanisms in haemcopperoxidases: The high-affinity Q_Hbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

Cited by 14 publications

References 93 publications

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective ²H and ¹⁵N Labeling, HYSCORE Spectroscopy, and DFT Modeling

Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents

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Elucidating mechanisms in haemcopperoxidases: The high-affinity QHbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

Cited by 14 publications

References 93 publications

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective 2H and 15N Labeling, HYSCORE Spectroscopy, and DFT Modeling

Exploring by Pulsed EPR the Electronic Structure of Ubisemiquinone Bound at the QH Site of Cytochrome bo3 from Escherichia coli with in Vivo 13C-Labeled Methyl and Methoxy Substituents

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Elucidating mechanisms in haemcopperoxidases: The high-affinity Q_Hbinding site in quinol oxidase as studied by DONUT-HYSCOREspectroscopy and density functional theory

Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective ²H and ¹⁵N Labeling, HYSCORE Spectroscopy, and DFT Modeling