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1.
Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the “E-pathway hypothesis” is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180 → Gln variant. A possible haem propionate involvement has recently been supported by combining 13C-haem propionate labelling with redox-induced FTIR difference spectroscopy. 相似文献
2.
Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the "E-pathway hypothesis" is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180-->Gln variant. A possible haem propionate involvement has recently been supported by combining (13)C-haem propionate labelling with redox-induced FTIR difference spectroscopy. 相似文献
3.
Quinol:fumarate reductase (QFR) is the terminal enzyme of anaerobic fumarate respiration. This membrane protein complex couples the oxidation of menaquinol to menaquinone to the reduction of fumarate to succinate. Although the diheme-containing QFR from Wolinella succinogenes is known to catalyze an electroneutral process, its three-dimensional structure at 2.2 A resolution and the structural and functional characterization of variant enzymes revealed locations of the active sites that indicated electrogenic catalysis. A solution to this apparent controversy was proposed with the so-called "E-pathway hypothesis". According to this, transmembrane electron transfer via the heme groups is strictly coupled to a parallel, compensatory transfer of protons via a transiently established pathway, which is inactive in the oxidized state of the enzyme. Proposed constituents of the E-pathway are the side chain of Glu C180 and the ring C propionate of the distal heme. Previous experimental evidence strongly supports such a role of the former constituent. Here, we investigate a possible heme-propionate involvement in redox-coupled proton transfer by a combination of specific (13)C-heme propionate labeling and Fourier transform infrared (FTIR) difference spectroscopy. The labeling was achieved by creating a W. succinogenes mutant that was auxotrophic for the heme-precursor 5-aminolevulinate and by providing [1-(13)C]-5-aminolevulinate to the medium. FTIR difference spectroscopy revealed a variation on characteristic heme propionate vibrations in the mid-infrared range upon redox changes of the distal heme. These results support a functional role of the distal heme ring C propionate in the context of the proposed E-pathway hypothesis of coupled transmembrane electron and proton transfer. 相似文献
4.
Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Δp), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. The first mechanism has been unequivocally demonstrated to be operational for Δp-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing succinate:menaquinone reductase (SQR) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane potential Δp to drive the endergonic oxidation of succinate by menaquinone by the dihaem-containing SQR of Gram-positive bacteria. In the case of a related but different respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of the ?-proteobacterium Wolinella succinogenes, evidence has been obtained that both mechanisms are combined, so as to facilitate transmembrane electron transfer by proton transfer via a both novel and essential compensatory transmembrane proton transfer pathway (“E-pathway”). Although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Δp. This compensatory “E-pathway” appears to be required by all dihaem-containing QFR enzymes and results in the overall reaction being electroneutral. However, here we show that the reverse reaction, the oxidation of succinate by quinone, as catalysed by W. succinogenes QFR, is not electroneutral. The implications for transmembrane proton transfer via the E-pathway are discussed. 相似文献
5.
C.Roy D. Lancaster 《生物化学与生物物理学报:生物膜》2002,1565(2):215-231
The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-Å resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The “four-helix” dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the “two-helix motif” of the cytochrome bc1 complex and the “three-helix motif” of the formate dehydrogenase/hydrogenase group. The γ-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the “E-pathway”) in concert with the transfer of two electrons via the membrane-bound haem groups. According to this “E-pathway hypothesis”, the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential. 相似文献
6.
Calculated coupling of transmembrane electron and proton transfer in dihemic quinol:fumarate reductase
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The quinol:fumarate reductase of Wolinella succinogenes binds a low- and a high-potential heme b group in its transmembrane subunit C. Both hemes are part of the electron transport chain between the two catalytic sites of this redox enzyme. The oxidation-reduction midpoint potentials of the hemes are well established but their assignment in the structure has not yet been determined. By simulating redox titrations, using continuum electrostatics calculations, it was possible to achieve an unequivocal assignment of the low- and high-potential hemes to the distal and proximal positions in the structure, respectively. Prominent features governing the differences in midpoint potential between the two hemes are the higher loss of reaction field energy for the proximal heme and the stronger destabilization of the oxidized form of the proximal heme due to several buried Arg and Lys residues. According to the so-called "E-pathway hypothesis", quinol:fumarate reductase has previously been postulated to exhibit a novel coupling of transmembrane electron and proton transfer. Simulation of heme b reduction indicates that the protonation state of the conserved residue Glu C180, predicted to play a key role in this process, indeed depends on the redox state of the hemes. This result clearly supports the E-pathway hypothesis. 相似文献
7.
Electrochemically induced static FTIR difference spectroscopy has been employed to investigate redox-driven protonation changes of individual amino acid residues in the quinol:fumarate reductase (QFR) from Wolinella succinogenes. The difference spectra presented were taken in the mid-infrared region from 1800 to 1000 cm(-1), and the signals obtained represent transitions between the reduced and oxidized states of the enzyme. Analysis of the difference spectra shows evidence for structural reorganizations of the polypeptide backbone upon the induced redox reaction. Furthermore, spectral contributions were found above 1710 cm(-1) where stretching vibrations of protonated carboxyl groups from aspartic or glutamic acid side chains absorb. With the help of site-directed mutagenesis and hydrogen/deuterium isotope exchange, it was possible to identify amino acid residue Glu C180, which is located in the membrane-spanning, diheme-containing subunit C of QFR, as being partially responsible for the derivative-shaped spectral feature with a peak/trough at 1741/1733 cm(-1) in the reduced-minus-oxidized difference spectrum. This signal pattern is interpreted as a superposition of a protonation/deprotonation and a change of the hydrogen-bonding environment of Glu C180. This residue is the principal constituent of the recently proposed "E-pathway hypothesis" of coupled transmembrane proton and electron transfer in QFR [Lancaster, C. R. D. (2002) Biochim. Biophys. Acta 1565, 215-231]. Thus, the study presented yields experimental evidence which supports a key role of Glu C180 within the framework of the E-pathway hypothesis. 相似文献
8.
Cytochrome bd is a tri-heme (b
558, b
595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in one-electron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (τ<1.5 µs) electron transfer from heme d to heme b
595 in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b
558 (τ∼16 µs). It returns from the b-hemes to heme d with τ∼180 µs. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (τ∼16 µs) yielding a transient hexacoordinate state (CO-Fe2+-L). Then the ligand slowly (τ∼30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (τ∼20 µs), some heme b
558 (τ∼0.2–3 ms), and finally migrates from heme d to heme b
595 (τ∼24 ms) in ∼5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions. 相似文献
9.
Rodrigues ML Scott KA Sansom MS Pereira IA Archer M 《Journal of molecular biology》2008,381(2):341-350
Membrane-bound cytochrome c quinol dehydrogenases play a crucial role in bacterial respiration by oxidizing menaquinol and transferring electrons to various periplasmic oxidoreductases. In this work, the menaquinol oxidation site of NrfH was characterized by the determination of the X-ray structure of Desulfovibrio vulgaris NrfHA nitrite reductase complex bound to 2-heptyl-4-hydroxyquinoline-N-oxide, which is shown to act as a competitive inhibitor of NrfH quinol oxidation activity. The structure, at 2.8-Å resolution, reveals that the inhibitor binds close to NrfH heme 1, where it establishes polar contacts with two essential residues: Asp89, the residue occupying the heme distal ligand position, and Lys82, a strictly conserved residue. The menaquinol binding cavity is largely polar and has a wide opening to the protein surface. Coarse-grained molecular dynamics simulations suggest that the quinol binding site of NrfH and several other respiratory enzymes lie in the head group region of the membrane, which probably facilitates proton transfer to the periplasm. Although NrfH is not a multi-span membrane protein, its quinol binding site has several characteristics similar to those of quinone binding sites previously described. The data presented here provide the first characterization of the quinol binding site of the cytochrome c quinol dehydrogenase family. 相似文献
10.
Lancaster CR 《Biochimica et biophysica acta》2002,1565(2):215-231
The structure of the respiratory membrane protein complex quinol:fumarate reductase (QFR) from Wolinella succinogenes has been determined by X-ray crystallography at 2.2-A resolution [Nature 402 (1999) 377]. Based on the structure of the three protein subunits A, B, and C and the arrangement of the six prosthetic groups (a covalently bound FAD, three iron-sulfur clusters, and two haem b groups), a pathway of electron transfer from the quinol-oxidising dihaem cytochrome b in the membrane to the site of fumarate reduction in the hydrophilic subunit A has been proposed. The structure of the membrane-integral dihaem cytochrome b reveals that all transmembrane helical segments are tilted with respect to the membrane normal. The "four-helix" dihaem binding motif is very different from other dihaem-binding transmembrane four-helix bundles, such as the "two-helix motif" of the cytochrome bc(1) complex and the "three-helix motif" of the formate dehydrogenase/hydrogenase group. The gamma-hydroxyl group of Ser C141 has an important role in stabilising a kink in transmembrane helix IV. By combining the results from site-directed mutagenesis, functional and electrochemical characterisation, and X-ray crystallography, a residue was identified which was found to be essential for menaquinol oxidation [Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 13051]. The distal location of this residue in the structure indicates that the coupling of the oxidation of menaquinol to the reduction of fumarate in dihaem-containing succinate:quinone oxidoreductases could in principle be associated with the generation of a transmembrane electrochemical potential. However, it is suggested here that in W. succinogenes QFR, this electrogenic effect is counterbalanced by the transfer of two protons via a proton transfer pathway (the "E-pathway") in concert with the transfer of two electrons via the membrane-bound haem groups. According to this "E-pathway hypothesis", the net reaction catalysed by W. succinogenes QFR does not contribute directly to the generation of a transmembrane electrochemical potential. 相似文献
11.
Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic varepsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions. 相似文献
12.
Frederik A.J. Rotsaert 《BBA》2008,1777(3):239-249
We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (bH heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the bH heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the bH heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function. 相似文献
13.
The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b595, have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b595 and heme b558. It is concluded that 1) O2 binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O2 binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b595, respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm]?→?E107?→?E99 (heme b595)?→?E445 (heme d)?→?oxygenated heme d. 相似文献
14.
Energy transduction in the cytochrome bc1 complex is achieved by catalyzing opposite oxido-reduction reactions at two different quinone binding sites. We have determined the pre-steady state kinetics of cytochrome b and c1 reduction at varying quinol/quinone ratios in the isolated yeast bc1 complex to investigate the mechanisms that minimize inhibition of quinol oxidation at center P by reduction of the bH heme through center N. The faster rate of initial cytochrome b reduction as well as its lower sensitivity to quinone concentrations with respect to cytochrome c1 reduction indicated that the bH hemes equilibrated with the quinone pool through center N before significant catalysis at center P occurred. The extent of this initial cytochrome b reduction corresponded to a level of bH heme reduction of 33%–55% depending on the quinol/quinone ratio. The extent of initial cytochrome c1 reduction remained constant as long as the fast electron equilibration through center N reduced no more than 50% of the bH hemes. Using kinetic modeling, the resilience of center P catalysis to inhibition caused by partial pre-reduction of the bH hemes was explained using kinetics in terms of the dimeric structure of the bc1 complex which allows electrons to equilibrate between monomers. 相似文献
15.
Grimaldi S Arias-Cartin R Lanciano P Lyubenova S Szenes R Endeward B Prisner TF Guigliarelli B Magalon A 《The Journal of biological chemistry》2012,287(7):4662-4670
Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (QD) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the QD site (MSQD) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with 1H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQD binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme bD. Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the QD site are discussed, in light of the unusually high thermodynamic stability of MSQD. 相似文献
16.
Heme-copper oxidases (HCOs) are terminal electron acceptors in aerobic respiration. They catalyze the reduction of molecular oxygen to water with concurrent pumping of protons across the mitochondrial and bacterial membranes. Protons required for oxygen reduction chemistry and pumping are transferred through proton uptake channels. Recently, the crystal structure of the first C-type member of the HCO superfamily was resolved [Buschmann et al. Science 329 (2010) 327–330], but crystallographic water molecules could not be identified. Here we have used molecular dynamics (MD) simulations, continuum electrostatic approaches, and quantum chemical cluster calculations to identify proton transfer pathways in cytochrome cbb3. In MD simulations we observe formation of stable water chains that connect the highly conserved Glu323 residue on the proximal side of heme b3 both with the N- and the P-sides of the membrane. We propose that such pathways could be utilized for redox-coupled proton pumping in the C-type oxidases. Electrostatics and quantum chemical calculations suggest an increased proton affinity of Glu323 upon reduction of high-spin heme b3. Protonation of Glu323 provides a mechanism to tune the redox potential of heme b3 with possible implications for proton pumping. 相似文献
17.
The Q-cycle mechanism of the bc1 complex explains how the electron transfer from ubihydroquinone (quinol, QH2) to cytochrome (cyt) c (or c2 in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Qo-site of the complex. This directs the two electrons from QH2 down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme bL and bH to the Qi-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc1 complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Qo-site reaction. However, previous research has strongly suggested that SQ is the reductant for O2 in generation of superoxide at the Qo-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Qo-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme bL when the latter is reduced. This allows rapid and reversible QH2 oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation. 相似文献
18.
Chang-An Yu Xiaowei Cen He-Wen Ma Ying Yin Linda Yu Lothar Esser Di Xia 《BBA》2008,1777(7-8):1038-1043
Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc1 complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the QP site with both electrons transferred simultaneously to ISP and cyt bL when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc1 demonstrates that the reduced ISP-ED moves to the c1-position to reduce cyt c1 only after the reduced cyt bL is oxidized by cyt bH. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of QP site inhibitors, Pm and Pf, under various redox states of the bc1 complex, suggest that the electron transfer from heme bL to bH is the driving force for the releasing of the reduced ISP-ED from the b-position to c1-position to reduce cyt c1. 相似文献
19.
Matthias Schultze Björn Forberich Sascha Rexroth Nina Gwendolyn Dyczmons Matthias Roegner Jens Appel 《BBA》2009,1787(12):1479-1485
The cytochrome b6f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b6f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase. 相似文献
20.
Quinol oxidation at center P of the cytochrome bc1
complex involves bifurcated electron transfer to the Rieske iron-sulfur
protein and cytochrome b. It is unknown whether both electrons are
transferred from the same domain close to the Rieske protein, or if an
unstable semiquinone anion intermediate diffuses rapidly to the vicinity of
the bL heme. We have determined the pre-steady state rate
and activation energy (Ea) for quinol oxidation in
purified yeast bc1 complexes harboring either a Y185F
mutation in the Rieske protein, which decreases the redox potential of the FeS
cluster, or a E272Q cytochrome b mutation, which eliminates the
proton acceptor in cytochrome b. The rate of the bifurcated reaction
in the E272Q mutant (<10% of the wild type) was even lower than that of the
Y185F enzyme (∼20% of the wild type). However, the E272Q enzyme showed the
same Ea (61 kJ mol-1) with respect to the wild
type (62 kJ mol-1), in contrast with the Y185F mutation, which
increased Ea to 73 kJ mol-1. The rate and
Ea of the slow reaction of quinol with oxygen that are
observed after cytochrome b is reduced were unaffected by the E272Q
substitution, whereas the Y185F mutation modified only its rate. The
Y185F/E272Q double mutation resulted in a synergistic decrease in the rate of
quinol oxidation (0.7% of the wild type). These results are inconsistent with
a sequential “movable semiquinone” mechanism but are consistent
with a model in which both electrons are transferred simultaneously from the
same domain in center P.The cytochrome bc1 complex couples the oxidation of a
two-electron carrier molecule of quinol to the movement of protons across the
inner mitochondrial or bacterial membrane. The key reaction in this
energy-conserving mechanism, known as the Q-cycle
(1,
2), is the bifurcation of
electrons at the active site located closer to the positive side of the
membrane, termed center P or Qo site. One of the electrons from
quinol is transferred to a chain of one-electron carriers with relatively high
redox potentials that include the FeS cluster of the Rieske protein and the
hemes of cytochromes c1 and c. The other electron
is donated to the low potential (bL) heme of cytochrome
b, from which it crosses most of the membrane width to the high
potential bH heme, located close to another active site
(center N or Qi site), where quinone is reduced to quinol after two
center P turnovers. Proton release and uptake at each active site are achieved
by taking advantage of the chemistry of quinol and quinone, which can only
stably exist at physiological pH in the protonated and deprotonated forms,
respectively.Critical to the electron bifurcation reaction at center P is the
arrangement of protonatable groups (His181 of the Rieske protein
and Glu272 of cytochrome b) close to the electron
acceptors at opposite sides of the substrate (see
Fig. 1). However, the exact
mechanism of electron bifurcation at center P is still an unresolved issue.
Proposed models have ranged from strictly concerted mechanisms in which both
electrons from quinol are extracted simultaneously
(3,
4) to those that postulate a
highly stabilized semiquinone intermediate
(5). Between these two extremes
are mechanisms that propose the formation of an unstable semiquinone
intermediate after a first electron transfer from quinol to the Rieske protein
(6–8),
which seem to be supported by recent reports that claim to have detected low
concentrations of semiquinone at center P when reoxidation of cytochrome
b is impeded under special conditions
(9,
10). One version of the
unstable semiquinone mechanism proposes that this intermediate diffuses from
the vicinity of the Rieske protein to a location within center P located
closer to the bL heme, which would allow non-rate-limiting
rates of bL reduction to occur even at very low
semiquinone occupancy (11). In
this proposal, the movement of the unstable semiquinone would be allowed by
protonation and rotation of Glu272 in cytochrome b, which
occupies different conformations in crystallographic structures
(Fig. 1)
(11–14).Open in a separate windowFIGURE 1.Electron and proton acceptors involved in quinol oxidation at center
P. Crystallographic structures 1EZV
(12) and 1P84
(13) show stigmatellin
(A) or 5-n-heptyl-6-hydroxy-4,7-dioxobenzothiazole
(B) bound at center P forming a hydrogen bond to the
His181 residue of the Rieske protein, which is a ligand to the FeS
cluster. The Tyr185 residue in the Rieske protein influences the
Em value of the FeS cluster
(22). On the side pointing to
the bL heme, a bound water molecule is also
hydrogen-bonded to the inhibitor, either to the Glu272 carboxylate
in cytochrome b (A), or to its backbone amino group
(B), when the side chain is rotated toward a water network that
connects to the propionate of the bL heme and to
Arg79 of cytochrome b.An important prediction of the movable semiquinone model
(11) is that mutation of
Glu272 should impede diffusion of the anionic semiquinone, forcing
electron transfer to the bL heme to occur through a longer
distance from the position closer to the Rieske FeS cluster
(15), thereby shifting the
rate-limiting step from the first to the second electron transfer. Although it
has already been reported that different mutations at Glu272
partially slow down quinol oxidation at center P
(15–17),
no effort has been made so far to evaluate whether the rate-limiting step
changes upon inhibition of the deprotonation of quinol (or of a putative
semiquinone intermediate) by mutation of the cytochrome b
Glu272. In the present work, we analyze the energy of activation of
quinol oxidation at center P and show that the rate-limiting step when
Glu272 is mutated to glutamine, although slower, is still
determined by the driving force for electron transfer to the Rieske protein.
We also show that decreasing this driving force enhances the relative
inhibition caused by mutating Glu272, suggesting a tight coupling
of reactions involved in quinol oxidation and deprotonation. In contrast,
reactions with oxygen that bypass the electron bifurcation at center P, which
are likely to involve a semiquinone intermediate, are independent of
Glu272 and go through an energetic barrier different from that of
the bifurcated reaction. We discuss how these results support a mechanism in
which both electron transfer events from quinol to the Rieske protein and the
bL heme occur from the same position and at the same
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