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The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

ABSTRACT: Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.

SUBMITTER: Hong S

PROVIDER: S-EPMC4179594 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex.

Project description: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 (approximately 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 Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon 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 Nepsilon 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+/-0.13A. 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, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

| S-EPMC3060708 | biostudies-literature

The road to the crystal structure of the cytochrome bc1 complex from the anoxigenic, photosynthetic bacterium Rhodobacter sphaeroides.

Project description:The advantages of using bacterial systems to study the mechanism and function of cytochrome bc (1) complexes do not extend readily to their structural investigations. High quality crystals of bacterial complexes have been difficult to obtain despite the enzymes' smaller sizes and simpler subunit compositions compared to their mitochondrial counterparts. In the course of the structure determination of the bc (1) complex from R. sphaeroides, we observed that the growth of only low quality crystals correlated with low activity and stability of the purified complex, which was mitigated in part by introducing a double mutations to the enzyme. The S287R(cyt b)/V135S(ISP) mutant shows 40% increase in electron transfer activity and displays a 4.3 degrees C increase in thermal stability over wild-type enzyme. The amino acid histidine was found important in maintaining structural integrity of the bacterial complex, while the respiratory inhibitors such as stigmatellin are required for immobilization of the iron-sulfur protein extrinsic domain. Crystal quality of the R. sphaeroides bc (1) complex can be improved further by the presence of strontium ions yielding crystals that diffracted X-rays to better than 2.3 A resolution. The improved crystal quality can be understood in terms of participation of strontium ions in molecular packing arrangement in crystal.

| S-EPMC4788495 | biostudies-literature

Trapped conformational states of semiquinone (D+*QB-*) formed by B-branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers.

Project description:The reaction center (RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones QA and QB, involving a conformational gating step. In this work, conformational states of D+*QB-* were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D+*QB-* formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D+*QB-* in mutant RCs illuminated while freezing formed in approximately 95% of the sample did not decay (tau >107 s) at 80 K (also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active QB-*'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (approximately 70%) of QB, structurally located in the distal site, was not stably reducible, whereas the minority (approximately 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D+*QB-* states is attributed to the relaxation of protein residues and internal water molecules that stabilize D+*QB-*. These results demonstrate energetically significant conformational changes involved in stabilizing the D+*QB-* state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.

| S-EPMC2259235 | biostudies-literature

Features of Rhodobacter sphaeroides CcmFH.

Project description:In this study, the in vivo function and properties of two cytochrome c maturation proteins, CcmF and CcmH from Rhodobacter sphaeroides, were analyzed. Strains lacking CcmH or both CcmF and CcmH are unable to grow under anaerobic conditions where c-type cytochromes are required, demonstrating their critical role in the assembly of these electron carriers. Consistent with this observation, strains lacking both CcmF and CcmH are deficient in c-type cytochromes when assayed under permissive growth conditions. In contrast, under permissive growth conditions, strains lacking only CcmH contain several soluble and membrane-bound c-type cytochromes, albeit at reduced levels, suggesting that this bacterium has a CcmH-independent route for their maturation. In addition, the function of CcmH that is needed to support anaerobic growth can be replaced by adding cysteine or cystine to growth media. The ability of exogenous thiol compounds to replace CcmH provides the first physiological evidence for a role of this protein in thiol chemistry during c-type cytochrome maturation. The properties of R. sphaeroides cells containing translational fusions between CcmF and CcmH and either Escherichia coli alkaline phosphatase or beta-galactosidase suggest that they are each integral cytoplasmic membrane proteins with their presumed catalytic domains facing the periplasm. Analysis of CcmH shows that it is synthesized as a higher-molecular-weight precursor protein with an N-terminal signal sequence.

| S-EPMC145331 | biostudies-literature

rne Rhodobacter sphaeroides

Project description:Comparison of wild type and mutant strain with temperature-sensitive RNase E. Goal: to study the effect of RNase E on the transcriptome

2018-07-27 | GSE104278 | GEO

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid.

Project description:Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

| S-EPMC5805856 | biostudies-literature

Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: I. Equilibrium and modeling studies.

Project description:We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c(1) in detergent-solubilized bc(1) complex from Rhodobacter sphaeroides. Imidazole binding to cyt c(1) substantially lowers the midpoint potential of the heme and fully inhibits bc(1) complex activity. Temperature dependences showed that binding of Im (K(d) approximately 330 microM, 25 degrees C, pH 8) is enthalpically driven (DeltaH(0) = -56 kJ/mol, DeltaS(0) = -121 J/mol/K), whereas binding of MeIm is 30 times weaker (K(d) approximately 9.3 mM) and is entropically driven (DeltaH(0) = 47 kJ/mol, DeltaS(0)(o) = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c(1) triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c(2) suggested that Im binding to cyt c(1) is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c(2), and c(1), which showed hydrogen bonds formed between the N(delta)H of Im and the cyt c(1) protein, or with a water molecule sequestered with the ligand in the heme cleft.

| S-EPMC2903416 | biostudies-literature

Complete genome sequence of Rhodobacter sphaeroides KD131.

Project description:Rhodobacter sphaeroides is a purple nonsulfur photosynthetic bacterium that is considered a possible source of H(2) production. R. sphaeroides KD131, which was isolated from sea mud in South Korea, was found to produce high levels of H(2). Here we report the complete and annotated genome sequence of R. sphaeroides KD131.

| S-EPMC2632106 | biostudies-literature

ENDOR spectroscopy reveals light induced movement of the H-bond from Ser-L223 upon forming the semiquinone (Q(B)(-)(*)) in reaction centers from Rhodobacter sphaeroides.

Project description:Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.

| S-EPMC2597558 | biostudies-literature

Binding of imidazole to the heme of cytochrome c1 and inhibition of the bc1 complex from Rhodobacter sphaeroides: II. Kinetics and mechanism of binding.

Project description:The kinetics of imidazole (Im) and N-methylimidazole (MeIm) binding to oxidized cytochrome (cyt) c(1) of detergent-solubilized bc(1) complex from Rhodobacter sphaeroides are described. The rate of formation of the cyt c(1)-Im complex exhibited three separated regions of dependence on the concentration of imidazole: (i) below 8 mM Im, the rate increased with concentration in a parabolic manner; (ii) above 20 mM, the rate leveled off, indicating a rate-limiting conformational step with lifetime approximately 1 s; and (iii) at Im concentrations above 100 mM, the rate substantially increased again, also parabolically. In contrast, binding of MeIm followed a simple hyperbolic concentration dependence. The temperature dependences of the binding and release kinetics of Im and MeIm were also measured and revealed very large activation parameters for all reactions. The complex concentration dependence of the Im binding rate is not consistent with the popular model for soluble c-type cytochromes in which exogenous ligand binding is preceded by spontaneous opening of the heme cleft, which becomes rate-limiting at high ligand concentrations. Instead, binding of ligand to the heme is explained by a model in which an initial and superficial binding facilitates access to the heme by disruption of hydrogen-bonded structures in the heme domain. For imidazole, two separate pathways of heme access are indicated by the distinct kinetics at low and high concentration. The structural basis for ligand entry to the heme cleft is discussed.

| S-EPMC2903381 | biostudies-literature

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