EPR, ENDOR and TRIPLE Resonance and MO Studies on Ubiquinones (Q-n): Comparison of Radical Anions and Cations of Coenzymes Q-10 and Q-6 with the Model Compounds Q-2 and Q-0.

Joela, Heikki; Kasa, Seppo; Lehtovuori, Pekka; Bech, Martin

doi:10.3891/acta.chem.scand.51-0233

Cited by 26 publications

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“…This is the largest isotropic constant reported for these protons for ubiquinones bound to proteins or in solution. Hyperfine coupling of 5.5-6.5 MHz has been reported for ubiquinone anion-radicals in different solvents (25)(26)(27). The value changes only slightly in the Q A and Q B sites of the photosynthetic reaction center (28).…”

Section: Discussionmentioning

confidence: 94%

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Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Yap

Samoilova

Gennis

et al. 2006

Journal of Biological Chemistry

149

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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. Two-dimensional electron spin echo envelope modulation has been applied to explore the exchangeable protons involved in hydrogen bonding to the semiquinone by substitution of 1 H 2 O by 2 H 2 O. Three exchangeable protons possessing different isotropic and anisotropic hyperfine couplings were identified. The strength of the hyperfine interaction with one proton suggests a significant covalent O-H binding of carbonyl oxygen O1 that is a characteristic of a neutral radical, an assignment that is also supported by the unusually large hyperfine coupling to the methyl protons. The second proton with a large anisotropic coupling also forms a strong hydrogen bond with a carbonyl oxygen. This second hydrogen bond, which has a significant out-of-plane character, is from an NH 2 or NH nitrogen, probably from an arginine (Arg-71) known to be in the quinone binding site. Assignment of the third exchangeable proton with smaller anisotropic coupling is more ambiguous, but it is clearly not involved in a direct hydrogen bond with either of the carbonyl oxygens. The results support a model that the semiquinone is bound to the protein in a very asymmetric manner by two strong hydrogen bonds from Asp-75 and Arg-71 to the O1 carbonyl, while the O4 carbonyl is not hydrogen-bonded to the protein.Cytochrome bo 3 (cyt bo 3 ) 3 is a terminal oxidase in the aerobic respiratory chain of Escherichia coli. It catalyzes the two-electron oxidation of ubiquinol-8 with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to solution. The available evidence suggests that the cyt bo 3 ubiquinol oxidase has two Q binding sites (1-3): a low affinity site (Q L ) where the substrate quinol is oxidized and the product is released, and a high affinity site where the bound quinone species acts as a conduit for electrons, similar to the role of the Q A site in the bacterial reaction center (3-7, 9). The substrate (QH 2 ) site, referred to as the low affinity site (Q L ), is equilibrated with the quinone pool in the membrane. The high affinity quinone-binding site (Q H ), from which Q is not readily removed, stabilizes the SQ. The quinone bound at the Q H site functions as a tightly bound cofactor. Pulse radiolysis studies (10) have shown that the tightly bound quinone can be rapidly reduced to the semiquinone species and that the tightly bound quinone is essential for rapid electron transfer to heme b. The first order rate constant for the reduction of heme b is 1.5 ϫ 10 3 s Ϫ1 , which is approximately the turnover rate of the enzyme. It is reasonably assumed that there is a rapid (Ͼ10 4 s Ϫ1 ) two-electron reduction of Q H by the bound substrate Q L H 2 , followed by two one-electr...

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Section: Discussionmentioning

confidence: 94%

“…Thus, we assign cross-peaks 1 to the protons H1 of the methyl group with the couplings a ϭ 10 MHz, and T ϭ 1.8 MHz. The choice of absolute signs follows from the triple ENDOR experiments (25,26).…”

Section: Appendixmentioning

confidence: 99%

Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Yap

Samoilova

Gennis

et al. 2006

Journal of Biological Chemistry

149

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“…All previous ENDOR and ESEEM studies of cytochrome bo 3 (14 -16) have found a ϭ 10 -11 MHz for the methyl protons, which is the largest isotropic hyperfine constant reported for the methyl protons for ubisemiquinones bound to proteins or in solution. Hyperfine coupling of 5.5-6.5 MHz has been reported for ubiquinone anion radicals in different solvents (31)(32)(33). The proton isotropic constant of the rotating methyl group is directly proportional to the -spin density on the attached carbon atom, as described by the McConnel relation a ϭ 81 (34).…”

Section: The Nature Of H-bonded Nitrogensmentioning

confidence: 99%

Characterization of Mutants That Change the Hydrogen Bonding of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Yap

Samoilova

Gennis

et al. 2007

Journal of Biological Chemistry

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The cytochrome bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O 2 to water. This enzyme has a high affinity quinone binding site (Q H ), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (Q L ), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the Q H site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the Q H site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function. Escherichia coli cytochrome (cyt)3 bo 3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol and the fourelectron reduction of O 2 to water. The enzyme contains three redox-active metal centers: a low spin heme b, which is involved in quinol oxidation, and the heme o 3 /Cu B bimetallic center, which is the site where O 2 binds and is reduced to water. The ubiquinol oxidation occurs with a semiquinone (SQ) intermediate in an overall reaction that releases two protons to the periplasm. The enzyme contains two Q sites (1-6): a low affinity site (Q L ), which is equilibrated with the quinone pool in the membrane and functions as the substrate (QH 2 ) binding site, and a high affinity (Q H ) site, from which Q is not readily removed, and that stabilizes a SQ (7-10). The quinone bound at the high-affinity site appears to function as a tightly bound cofactor, similar to the Q A site of the reaction centers. Rapid kinetic studies show that the quinone bound at the Q H site is important for rapid reduction of heme b but not for rapid electron transfer from heme b to the heme o 3 /Cu B binuclear center (11,12). The heme o 3 /Cu B site is where O 2 is reduced to H 2 O using the electrons provided by the oxidation of quinol. Hence, the suggested electron transfer sequence is as follows in Reaction 1.The x-ray structure of cytochrome bo 3 (13) does not contain any bound quinone, but site-directed mutagenesis studies (2-4, 13) have identified residues that modulate the properties of the Q H site. The model of the Q H quinone binding site (Fig. 1), including Arg-71, Asp-75, His-98, and Gln-101 residues (13), has...

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“…However, when decylubiquinone, whose side chain is saturated, was used as a model compound for Q-2, a similar effect was not noticed (6).…”

Section: Introductionmentioning

confidence: 97%

ENDOR Spectroscopic and Molecular Orbital Study of the Dynamical Properties of the Side Chain in Radical Anions of Ubiquinones Q-1, Q-2, Q-6, and Q-10

Lehtovuori

Joela

2000

Journal of Magnetic Resonance

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EPR, ENDOR and TRIPLE Resonance and MO Studies on Ubiquinones (Q-n): Comparison of Radical Anions and Cations of Coenzymes Q-10 and Q-6 with the Model Compounds Q-2 and Q-0.

Cited by 26 publications

References 0 publications

Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Characterization of the Exchangeable Protons in the Immediate Vicinity of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

Characterization of Mutants That Change the Hydrogen Bonding of the Semiquinone Radical at the QH Site of the Cytochrome bo3 from Escherichia coli

ENDOR Spectroscopic and Molecular Orbital Study of the Dynamical Properties of the Side Chain in Radical Anions of Ubiquinones Q-1, Q-2, Q-6, and Q-10

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