Quinol:fumarate Reductase Research Articles

Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na+-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

Mathematical Modeling of ROS Production and Diode-like Behavior in the SDHA/SDHB Subcomplex of Succinate Dehydrogenases in Reverse Quinol-Fumarate Reductase Direction

Succinate dehydrogenase (SDH) plays an important role in reverse electron transfer during hypoxia/anoxia, in particular, in ischemia, when blood supply to an organ is disrupted, and oxygen is not available. It was detected in the voltammetry studies about three decades ago that the SDHA/SDHB subcomplex of SDH can have such a strong nonlinear property as a "tunnel-diode" behavior in reverse quinol-fumarate reductase direction. The molecular and kinetic mechanisms of this phenomenon, that is, a strong drop in the rate of fumarate reduction as the driving force is increased, are still unclear. In order to account for this property of SDH, we developed and analyzed a mechanistic computational model of reverse electron transfer in the SDHA/SDHB subcomplex of SDH. It was shown that a decrease in the rate of succinate release from the active center during fumarate reduction quantitatively explains the experimentally observed tunnel-diode behavior in SDH and threshold values of the electrode potential of about -80 mV. Computational analysis of ROS production in the SDHA/SDHB subcomplex of SDH during reverse electron transfer predicts that the rate of ROS production decreases when the tunnel-diode behavior appears. These results predict a low rate of ROS production by the SDHA/SDHB subcomplex of SDH during ischemia.

Production and characterization of a broad-spectrum antimicrobial 5-butyl-2-pyridine carboxylic acid from Aspergillus fumigatus nHF-01

The present study aims at the production optimization, purification, and characterization of a potent broad-spectrum antimicrobial compound (AMC) produced by Aspergillus fumigatus nHF-01 (GenBank Ac. No. MN190286). The culture conditions were optimized for a higher amount of AMC. The AMC was solvent extracted and characterized by UV–Vis, FT–IR, ESI–MS, and 1H-NMR spectroscopy. The MIC, MBC and mode of action were determined against a set of Gram-positive and Gram-negative human pathogenic bacteria. Its antibiofilm, synergistic and cytotoxic effects were also tested. The putative target site of action was evaluated through in silico molecular docking study. The stain A. fumigatus nHF-01 produced the maximum AMC (5-butyl-2-pyridine carboxylic acid) in 2% MEB (w/v) and 4% YE (w/v) at pH 6.0 and 20 °C temperature with 100 rpm agitation for ten days. It caused complete lethality of the Gram-positive and Gram-negative human pathogenic bacteria at a 129 µg/mL dose by rupture and entire dissolution of cell integrity. It showed moderate antibiofilm activity and had a synergistic activity with streptomycin and additive effects with ciprofloxacin and vancomycin. It targets a respiratory enzyme, Quinol-Fumarate Reductase (1l0v), with the highest binding affinities. It had cytotoxicity against human lung carcinoma A549 cell line and was stable up to 100 °C. Thus, the study revealed that the strain A. fumigatus nHF-01 produces a potent broad-spectrum AMC 5-butyl-2-pyridine carboxylic acid that could be used against human food and topical pathogenic bacteria. This is the first report of such a compound produced from the A. fumigatus.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia.

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

Structural insights into the electron/proton transfer pathways in the quinol:fumarate reductase from Desulfovibrio gigas

The membrane-embedded quinol:fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/proton-transfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6 Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structural insights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.

Three-dimensional models of Mycobacterium tuberculosis proteins Rv1555, Rv1554 and their docking analyses with sildenafil, tadalafil, vardenafil drugs, suggest interference with quinol binding likely to affect protein\u2019s function

BackgroundEarlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the ‘TB-drugome’ the Rv1555 protein is ‘druggable’ with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein’s function.ResultsThe three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs.ConclusionsThe predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

New crystal forms of the integral membrane Escherichia coli quinol:fumarate reductase suggest that ligands control domain movement.

Quinol:fumarate reductase (QFR) is an integral membrane protein and a member of the respiratory Complex II superfamily. Although the structure of Escherichia coli QFR was first reported almost twenty years ago, many open questions of catalysis remain. Here we report two new crystal forms of QFR, one grown from the lipidic cubic phase and one grown from dodecyl maltoside micelles. QFR crystals grown from the lipid cubic phase processed as P1, merged to 7.5 Å resolution, and exhibited crystal packing similar to previous crystal forms. Crystals grown from dodecyl maltoside micelles processed as P21, merged to 3.35 Å resolution, and displayed a unique crystal packing. This latter crystal form provides the first view of the E. coli QFR active site without a dicarboxylate ligand. Instead, an unidentified anion binds at a shifted position. In one of the molecules in the asymmetric unit, this is accompanied by rotation of the capping domain of the catalytic subunit. In the other molecule, this is associated with loss of interpretable electron density for this same capping domain. Analysis of the structure suggests that the ligand adjusts the position of the capping domain.

Design, Synthesis, and Biological Testing of Novel Naphthoquinones as Substrate-Based Inhibitors of the Quinol/Fumarate Reductase from Wolinella succinogenes

Novel naphthoquinones were designed, synthesized, and tested as substrate-based inhibitors against the membrane-embedded protein quinol/fumarate reductase (QFR) from Wolinella succinogenes, a target closely related to QFRs from the human pathogens Helicobacter pylori and Campylobacter jejuni. For a better understanding of the hitherto structurally unexplored substrate binding pocket, a structure-activity relationship (SAR) study was carried out. Analogues of lawsone (2-hydroxy-1,4-naphthoquinone 3a) were synthesized that vary in length and size of the alkyl side chains (3b-k). A combined study on the prototropic tautomerism of 2-hydroxy-1,4-naphthoquinones series indicated that the 1,4-tautomer is the more stable and biologically relevant isomer and that the presence of the hydroxyl group is crucial for inhibition. Furthermore, 2-bromine-1,4-naphthoquinone (4a-c) and 2-methoxy-1,4-naphthoquinone (5a-b) series were also discovered as novel and potent inhibitors. Compounds 4a and 4b showed IC50 values in low micromolar range in the primary assay and no activity in the counter DT-diaphorase assay.

Hydrogen-Bonded Networks Along and Bifurcation of the E-Pathway in Quinol:Fumarate Reductase

The E-pathway of transmembrane proton transfer has been demonstrated previously to be essential for catalysis by the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes. Two constituents of this pathway, Glu-C180 and heme bD ring C (bD-C-) propionate, have been validated experimentally. Here, we identify further constituents of the E-pathway by analysis of molecular dynamics simulations. The redox state of heme groups has a crucial effect on the connectivity patterns of mobile internal water molecules that can transiently support proton transfer from the bD-C-propionate to Glu-C180. The short H-bonding paths formed in the reduced states can lead to high proton conduction rates and thus provide a plausible explanation for the required opening of the E-pathway in reduced QFR. We found evidence that the bD-C-propionate group is the previously postulated branching point connecting proton transfer to the E-pathway from the quinol-oxidation site via interactions with the heme bD ligand His-C44. An essential functional role of His-C44 is supported experimentally by site-directed mutagenesis resulting in its replacement with Glu. Although the H44E variant enzyme retains both heme groups, it is unable to catalyze quinol oxidation. All results obtained are relevant to the QFR enzymes from the human pathogens Campylobacter jejuni and Helicobacter pylori.

Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: Gene targeting of the Fp subunit

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite.

Limited reversibility of transmembrane proton transfer assisting transmembrane electron transfer in a dihaem-containing succinate:quinone oxidoreductase

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential (Δ p), 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 Δ p-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 Δ p 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 ɛ-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 Δ p. 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.

Electroneutral And Electrogenic Catalysis By Diheme-Containing Succinate:Quinone Oxidoreductases

Membrane proteins 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. In the case of the diheme-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes [1], both theoretical and experimental results, reviewed in [2], supported, but did not prove, a previous hypothesis [3] that both of these mechanisms are combined in a single membrane protein complex, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. Results of measurements on proteoliposomes will be presented, providing evidence for the presence of this unprecedented transmembrane proton transfer pathway (“E-pathway”) in the wild-type enzyme and its non-functionality in a variant QFR where a key glutamate residue has been replaced [4]. The “E-pathway”, discussed on the basis of the 1.78-A-resolution crystal structure of QFR, is essential for life under the conditions of fumarate respiration. Results will be compared to those obtained with the diheme-containing succinate:menaquinone reductase from the Gram-positive bacterium Bacillus licheniformis [5,6].

Electroneutral and electrogenic catalysis by dihaem-containing succinate:quinone oxidoreductases

Membrane protein complexes can support both the generation and utilization of a transmembrane electrochemical proton potential (Deltap), either by supporting transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by supporting transmembrane proton transfer. Regarding the first mechanism, this has been unequivocally demonstrated to be operational for Deltap-dependent catalysis of succinate oxidation by quinone in the case of the dihaem-containing SQR (succinate:menaquinone reductase) from the Gram-positive bacterium Bacillus licheniformis. This is physiologically relevant in that it allows the transmembrane Deltap 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 QFR (quinol:fumarate reductase) of the epsilon-proteobacterium Wolinella succinogenes, evidence has been obtained indicating 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'). This is necessary because, although the reduction of fumarate by menaquinol is exergonic, it is obviously not exergonic enough to support the generation of a Deltap. This compensatory E-pathway appears to be required by all dihaem-containing QFR enzymes and the conservation of the essential acidic residue on transmembrane helix V (Glu-C180 in W. succinogenes QFR) is a useful key for the sequence-based discrimination of these QFR enzymes from the dihaem-containing SQR enzymes.

A Threonine on the Active Site Loop Controls Transition State Formation in Escherichia coli Respiratory Complex II

In Escherichia coli, the complex II superfamily members succinate:ubiquinone oxidoreductase (SQR) and quinol:fumarate reductase (QFR) participate in aerobic and anaerobic respiration, respectively. Complex II enzymes catalyze succinate and fumarate interconversion at the interface of two domains of the soluble flavoprotein subunit, the FAD binding domain and the capping domain. An 11-amino acid loop in the capping domain (Thr-A234 to Thr-A244 in quinol:fumarate reductase) begins at the interdomain hinge and covers the active site. Amino acids of this loop interact with both the substrate and a proton shuttle, potentially coordinating substrate binding and the proton shuttle protonation state. To assess the loop's role in catalysis, two threonine residues were mutated to alanine: QFR Thr-A244 (act-T; Thr-A254 in SQR), which hydrogen-bonds to the substrate at the active site, and QFR Thr-A234 (hinge-T; Thr-A244 in SQR), which is located at the hinge and hydrogen-bonds the proton shuttle. Both mutations impair catalysis and decrease substrate binding. The crystal structure of the hinge-T mutation reveals a reorientation between the FAD-binding and capping domains that accompanies proton shuttle alteration. Taken together, hydrogen bonding from act-T to substrate may coordinate with interdomain motions to twist the double bond of fumarate and introduce the strain important for attaining the transition state.

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

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 epsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions.

Differences in Protonation of Ubiquinone and Menaquinone in Fumarate Reductase from Escherichia coli

Escherichia coli quinol-fumarate reductase operates with both natural quinones, ubiquinone (UQ) and menaquinone (MQ), at a single quinone binding site. We have utilized a combination of mutagenesis, kinetic, EPR, and Fourier transform infrared methods to study the role of two residues, Lys-B228 and Glu-C29, at the quinol-fumarate reductase quinone binding site in reactions with MQ and UQ. The data demonstrate that Lys-B228 provides a strong hydrogen bond to MQ and is essential for reactions with both quinone types. Substitution of Glu-C29 with Leu and Phe caused a dramatic decrease in enzymatic reactions with MQ in agreement with previous studies, however, the succinate-UQ reductase reaction remains unaffected. Elimination of a negative charge in Glu-C29 mutant enzymes resulted in significantly increased stabilization of both UQ-* and MQ-* semiquinones. The data presented here suggest similar hydrogen bonding of the C1 carbonyl of both MQ and UQ, whereas there is different hydrogen bonding for their C4 carbonyls. The differences are shown by a single point mutation of Glu-C29, which transforms the enzyme from one that is predominantly a menaquinol-fumarate reductase to one that is essentially only functional as a succinate-ubiquinone reductase. These findings represent an example of how enzymes that are designed to accommodate either UQ or MQ at a single Q binding site may nevertheless develop sufficient plasticity at the binding pocket to react differently with MQ and UQ.

A tale of two ferredoxins: sequence similarity and structural differences

BackgroundSequence similarity between proteins is usually considered a reliable indicator of homology. Pyruvate-ferredoxin oxidoreductase and quinol-fumarate reductase contain ferredoxin domains that bind [Fe-S] clusters and are involved in electron transport. Profile-based methods for sequence comparison, such as PSI-BLAST and HMMer, suggest statistically significant similarity between these domains.ResultsThe sequence similarity between these ferredoxin domains resides in the area of the [Fe-S] cluster-binding sites. Although overall folds of these ferredoxins bear no obvious similarity, the regions of sequence similarity display a remarkable local structural similarity. These short regions with pronounced sequence motifs are incorporated in completely different structural environments. In pyruvate-ferredoxin oxidoreductase (bacterial ferredoxin), the hydrophobic core of the domain is completed by two β-hairpins, whereas in quinol-fumarate reductase (α-helical ferredoxin), the cluster-binding motifs are part of a larger all-α-helical globin-like fold core.ConclusionFunctionally meaningful sequence similarity may sometimes be reflected only in local structural similarity, but not in global fold similarity. If detected and used naively, such similarities may lead to incorrect fold predictions.

Heterologous production in Wolinella succinogenes and characterization of the quinol:fumarate reductase enzymes from Helicobacter pylori and Campylobacter jejuni.

The epsilon-proteobacteria Helicobacter pylori and Campylobacter jejuni are both human pathogens. They colonize mucosal surfaces causing severe diseases. The membrane protein complex QFR (quinol:fumarate reductase) from H. pylori has previously been established as a potential drug target, and the same is likely for the QFR from C. jejuni. In the present paper, we describe the cloning of the QFR operons from the two pathogenic bacteria H. pylori and C. jejuni and their expression in Wolinella succinogenes, a non-pathogenic -proteobacterium. To our knowledge, this is the first documentation of heterologous membrane protein production in W. succinogenes. We demonstrate that the replacement of the homologous enzyme from W. succinogenes with the heterologous enzymes yields mutants where fumarate respiration is fully functional. We have isolated and characterized the heterologous QFR enzymes. The high quality of the enzyme preparation enabled us to determine unequivocally by analytical ultracentrifugation the homodimeric state of the three detergent-solubilized heterotrimeric QFR enzymes, to accurately determine the different oxidation-reduction ('redox') midpoint potentials of the six prosthetic groups, the Michaelis constants for the quinol substrate, maximal enzymatic activities and the characterization of three different anti-helminths previously suggested to be inhibitors of the QFR enzymes from H. pylori and C. jejuni. This characterization allows, for the first time, a detailed comparison of the QFR enzymes from C. jejuni and H. pylori with that of W. succinogenes.

Probing Heme Propionate Involvement in Transmembrane Proton Transfer Coupled to Electron Transfer in Dihemic Quinol:Fumarate Reductase by 13C-Labeling and FTIR Difference Spectroscopy

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.

FTIR Difference Spectra ofWolinella succinogenesQuinol:Fumarate Reductase Support a Key Role of Glu C180 within the “E-Pathway Hypothesis” of Coupled Transmembrane Electron and Proton Transfer

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.