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Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the 15N HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wild-type and mutant D75H cytochrome bo3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Δa, and parameter, δ = |2a + T| - 4v15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems. [ABSTRACT FROM AUTHOR]

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (∼9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nϵ of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Nδ in other quinone-processing sites. The experiments with 15N showed that the Nϵ of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nϵ of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38 ± 0.13 Å. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N2211, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover. [ABSTRACT FROM AUTHOR]

Exchangeable protons in the immediate neighborhood of the semiquinone (SQ) at the Qi-site of the bc1 complex (ubihydroquinone:cytochrome c oxidoreductase (EC 1.10.2.2)) from Rhodobacter sphaeroides have been characterized using electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) and visualized by substitution of H2O by ²H2O. Three exchangeable protons interact with the electron spin of the SQ. They possess different isotropic and anisotropic hyperfine couplings that allow a clear distinction between them. The strength of interactions indicates that the protons are involved in hydrogen bonds with SQ. The hyperfine couplings differ from values typical for in-plane hydrogen bonds previously observed in model experiments. It is suggested that the two stronger couplings involve formation of hydrogen bonds with carbonyl oxygens, which have a significant out-of-plane character due to the combined influence of bulky substituents and the protein environment. These two hydrogen bonds are most probably to side chains suggested from crystallographic structures (His-217 and Asp-252 in R. sphaeroides). Assignment of the third hydrogen bond is more ambiguous but may involve either a bond between Asn-221 and a methoxy O-atom or a bond to water. The structural and catalytic roles of the exchangeable protons are discussed in the context of three high resolution crystallographic structures for mitochondrial bc1 complexes. Potential H-bonds, including those to water molecules, form a network connecting the quinone (ubiquinone) occupant and its ligands to the propionates of heme bH and the external aqueous phase. They provide pathways for exchange of protons within the site and with the exteriors, needed to accommodate the different hydrogen bonding requirements of different quinone species during catalysis. [ABSTRACT FROM AUTHOR]

Ubiquinone forms an integral part of the electron transport chain in cellular respiration and photosynthesis across a vast number of organisms. Prior experimental results have shown that the photosynthetic reaction center (RC) from Rhodobacter sphaeroides is only fully functional with a limited set of methoxy-bearing quinones, suggesting that specific interactions with this substituent are required to drive electron transport and the formation of quinol. The nature of these interactions has yet to be determined. Through parameterization of a CHARMM-compatible quinone force field and subsequent molecular dynamics simulations of the quinone-bound RC, we have investigated and characterized the interactions of the protein with the quinones in the QA and QB sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the QB site and the amide nitrogen of GlyL225 that we implicate in locking the orientation of the 2-methoxy group, thereby tuning the redox potential difference between the quinones occupying the QA and QB sites. Disruption of this interaction leads to weaker binding in a ubiquinone analogue that lacks a 2-methoxy group, a finding supported by reverse electron transfer electron paramagnetic resonance experiments of the QA-QB- biradical and competitive binding assays. [ABSTRACT FROM AUTHOR]

When the superoxide radical Due to image rights restrictions, multiple line equation(s) is generated on reaction of KO2 with water in dimethyl sulfoxide, the decay of the radical is dramatically accelerated by inclusion of quinones in the reaction mix. For quinones with no or short hydrophobic tails, the radical product is a semiquinone at much lower yield, likely indicating reduction of quinone by superoxide and loss of most of the semiquinone product by disproportionation. In the presence of ubiquinone-10, a different species (I) is generated, which has the EPR spectrum of superoxide radical. However, pulsed EPR shows spin interaction with protons in fully deuterated solvent, indicating close proximity to the ubinquinone-10. We discuss the nature of species I, and possible roles in the physiological reactions through which ubisemiquinone generates superoxide by reduction of O2 through bypass reactions in electron transfer chains. [ABSTRACT FROM AUTHOR]

Since available structures of native bc1 complexes show a vacant Qo-site, occupancy by substrate and product must be investigated by kinetic and spectroscopic approaches. In this brief review, we discuss recent advances using these approaches that throw new light on the mechanism. The rate-limiting reaction is the first electron transfer after formation of the enzyme–substrate complex at the Qo-site. This is formed by binding of both ubiquinol (QH2) and the dissociated oxidized iron–sulfur protein (ISPox). A binding constant of ∼14 can be estimated from the displacement of Em or pK for quinone or ISPox, respectively. The binding likely involves a hydrogen bond, through which a proton-coupled electron transfer occurs. An enzyme–product complex is also formed at the Qo-site, in which ubiquinone (Q) hydrogen bonds with the reduced ISP (ISPH). The complex has been characterized in ESEEM experiments, which detect a histidine ligand, likely His-161 of ISP (in mitochondrial numbering), with a configuration similar to that in the complex of ISPH with stigmatellin. This special configuration is lost on binding of myxothiazol. Formation of the H-bond has been explored through the redox dependence of cytochrome c oxidation. We confirm previous reports of a decrease in Em of ISP on addition of myxothiazol, and show that this change can be detected kinetically. We suggest that the myxothiazol-induced change reflects loss of the interaction of ISPH with Q, and that the change in Em reflects a binding constant of ∼4. We discuss previous data in the light of this new hypothesis, and suggest that the native structure might involve a less than optimal configuration that lowers the binding energy of complexes formed at the Qo-site so as to favor dissociation. We also discuss recent results from studies of the bypass reactions at the site, which lead to superoxide (SO) production under aerobic conditions, and provide additional information about intermediate states. [Copyright &y& Elsevier]

Abstract: Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with 13C- and 15N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins. [Copyright &y& Elsevier]

In the photosynthetic reaction center from Rhodobacter sphaeroides, the primary (QA) and secondary (QB) electron acceptors are both ubiquinone-10, but with very different properties and functions. To investigate the protein environment that imparts these functional differences, we have applied X-band HYSCORE, a 2D pulsed EPR technique, to characterize the exchangeable protons around the semiquinone (SQ) in the QA and QB sites, using samples of 15N-labeled reaction centers, with the native high spin Fe2+ exchanged for diamagnetic Zn2+, prepared in 1H20 and 2H20 solvent. The powder HYSCORE method is first validated against the orientation-selected Q-band ENDOR study of the QB. SQBy Flores et a!. (Biophys. J. 2007, 92, 671-682), with good agreement for two exchangeable protons with anisotropic hyperfine tensor components, T, both in the range 4.6-5.4 MHz. HYSCORE was then applied to the QB SQ where we found proton lines corresponding to T ≈ 5.2, 3.7 MHz and T ≈ 1.9 MHz. Density functional-based quantum mechanics/molecular mechanics (QM/ MM) calculations, employing a model of the QB site, were used to assign the observed couplings to specific hydrogen bonding interactions with the QB SQ These calculations allow us to assign the T = 5.2 MHz proton to the His-L190 NδH · · · O4 (carbonyl) hydrogen bonding interaction. The T = 3.7 MHz spectral feature most likely results from hydrogen bonding interactions of 01 (carbonyl) with both Gly-L225 peptide NH and Ser-L223 hydroxyl OH, which possess calculated couplings very close to this value. The smaller 1.9 MHz coupling is assigned to a weakly bound peptide NH proton of Ile-L224. The calculations performed with this structural model of the QB site show less asymmetric distribution of unpaired spin density over the SQ than seen for the QA site, consistent with available experimental data for 13C and 17O carbonyl hyperfine couplings. The implications of these interactions for QB function and comparisons with the QA site are discussed. [ABSTRACT FROM AUTHOR]

The cytochrome bo3 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 O2 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 bo3 as well as the D75E and D75H mutant proteins were prepared with ubiquinone-8 13C-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 13C-labeled. The 13C- labeled quinone isolated from cytochrome bo3 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 13C 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. [ABSTRACT FROM AUTHOR]

Photosynthetic reaction centers from Rhodobacter sphaeroides have identical ubiquinone-l0 molecules functioning as primary (QA) and secondary (QB) electron acceptors. X-band 2D pulsed EPR spectroscopy, called HYSCORE, was applied to study the interaction of the QB site semiquinone with nitrogens from the local protein environment in natural and 15N uniformly labeled reactions centers. 14N and 15N HYSCORE spectra of the QB semiquinone show the interaction with two nitrogens carrying transferred unpaired spin density. Quadrupole coupling constants estimated from 14N HYSCORE spectra indicate them to be a protonated nitrogen of an imidazole residue and amide nitrogen of a peptide group. 15N HYSCORE spectra allowed estimation of the isotropic and anisotropic couplings with these nitrogens. From these data, we calculated the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and analyzed the contribution of different factors to the anisotropic hyperfine tensors. The hyperfine coupling of other protein nitrogens with the semiquinone is weak (<0.1 MHz). These results clearly indicate that the QB semiquinone forms hydrogen bonds with two nitrogens and provide quantitative characteristics of the hyperfine couplings with these nitrogens, which can be used in theoretical modeling of the QB site. On the basis of the quadrupole coupling constant, one nitrogen can only be assigned to Nδ of His-L190, consistent with all existing structures. However, we cannot specify between two candidates the residue corresponding to the second nitrogen. Further work employing multifrequency spectroscopic approaches or selective isotope labeling would be desirable for unambiguous assignment of this nitrogen. [ABSTRACT FROM AUTHOR]

Cytochrome aa3-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O2 to 2H2O. Cytochrome aa3-600 is of interest because it is a very close homologue of the cytochrome bo3 ubiquinol oxidase from Escherichia coli, except that it uses menaqninol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiqninone. The current work initiates studies to characterize the equivalent site in cytochrome aa3-600. Cytochrome aa3-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaqninone-7 and that the enzyme stabilizes a menasemiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa3-600 in comparison with the ubi-semiquinone in cytochrome bo3. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa3-600 in comparison with ubiquinone in cytochrome bo3. In addition, the electronic structure of the semiquinone cyt aa3-600 is more shifted toward the anionic form from the neutral state in cyt bo3. [ABSTRACT FROM AUTHOR]

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), 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 (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gin-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH 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. [ABSTRACT FROM AUTHOR]

The cytochrome bo3 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 O2 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 ¹H2O by ²H2O. 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 NH2 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. [ABSTRACT FROM AUTHOR]

Pulsed EPR spectroscopy was used to explore the structural neighborhood of the semiquinone (SQ) stabilized at the Q[sub i] site of the bc[sub 1] complex of Rhodobacter sphaeroides (EC 1.10.2.2) and to demonstrate that the nitrogen atom of a histidine imidazole group donates an H-bond to the SQ. Crystallographic structures show two different configurations for the binding of ubiquinone at the Q[sub i] site of mitochondrial bc[sub 1] complexes in which histidine (His-201 in bovine sequence) is either a direct H-bond donor or separated by a bridging water. The paramagnetic properties of the SQ formed at the site provide an independent method for studying the liganding of this intermediate species. The antimycin-sensitive SQ formed at the Q[sub i] site by either equilibrium redox titration, reduction of the oxidized complex by ascorbate, or addition of decylubihydroquinone to the oxidized complex in the presence of myxothiazol all showed similar properties. The electron spin echo envelope modulation spectra in the [sup 14]N region were dominated by lines with frequencies at 1.7 and 3.1 MHz. Hyperfine sublevel correlation spectroscopy spectra showed that these were contributed by a single nitrogen. Further analysis showed that the [sup 14]N nucleus was characterized by an isotropic hyperfine coupling of ∼0.8 MHz and a quadrupole coupling constant of ∼0.35 MHz. The nitrogen was identified as the N-∈ or N-δ imidazole nitrogen of a histidine (it is likely to be His-217, or His-201 in bovine sequence). A distance of 2.5-3.1 Å for the O-N distance between the carbonyl of SQ and the nitrogen was estimated. The mechanistic significance is discussed in the context of a dynamic role for the movement of His-217 in proton transfer to the site. [ABSTRACT FROM AUTHOR]

Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. 13C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with quantum mechanics calculations of the 13C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160–195 mV. [ABSTRACT FROM AUTHOR]