Quinone Binding Research Articles

Binding of the respiratory chain inhibitor ametoctradin to the mitochondrial bc1 complex.

Ametoctradin is an agricultural fungicide that inhibits the mitochondrial bc1 complex of oomycetes. The bc1 complex has two quinone binding sites that can be addressed by inhibitors. Depending on their binding sites and binding modes, the inhibitors show different degrees of cross-resistance that need to be considered when designing spray programmes for agricultural fungicides. The binding site of ametoctradin was unknown. Cross-resistance analyses, the reduction of isolated Pythium sp. bc1 complex in the presence of different inhibitors and molecular modelling studies were used to analyse the binding site and binding mode of ametoctradin. All three approaches provide data supporting the argument that ametoctradin binds to the Pythium bc1 complex similarly to stigmatellin. The binding mode of ametoctradin differs from other agricultural fungicides such as cyazofamid and the strobilurins. This explains the lack of cross-resistance with strobilurins and related inhibitors, where resistance is mainly caused by G143A amino acid exchange. Accordingly, mixtures or alternating applications of these fungicides and ametoctradin can help to minimise the risk of the emergence of new resistant isolates.

Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I.

Amilorides, well-known inhibitors of Na(+)/H(+) antiporters, were previously shown to inhibit bacterial and mitochondrial NADH-quinone oxidoreductase (complex I) but were markedly less active for complex I. Because membrane subunits ND2, ND4, and ND5 of bovine complex I are homologous to Na(+)/H(+) antiporters, amilorides have been thought to bind to any or all of the antiporter-like subunits; however, there is currently no direct experimental evidence that supports this notion. To identify the binding site of amilorides in bovine complex I, we synthesized two photoreactive amilorides (PRA1 and PRA2), which have a photoreactive azido (-N3) group and terminal alkyne (-C≡CH) group at the opposite ends of the molecules, respectively, and conducted photoaffinity labeling with bovine heart submitochondrial particles. The terminal alkyne group allows various molecular tags to covalently attach to it via Cu(+)-catalyzed click chemistry, thereby allowing purification and/or detection of the labeled peptides. Proteomic analyses revealed that PRA1 and PRA2 label none of the antiporter-like subunits; they specifically label the accessory subunit B14.5a and core subunit 49 kDa (N-terminal region of Thr25-Glu115), respectively. Suppressive effects of ordinary inhibitors (bullatacin, fenpyroximate, and quinazoline), which bind to the putative quinone binding pocket, on labeling were fairly different between the B14.5a and 49 kDa subunits probably because the binding positions of the three inhibitors differ within the pocket. The results of this study clearly demonstrate that amilorides inhibit complex I activity by occupying the quinone binding pocket rather than directly blocking translocation of protons through the antiporter-like subunits (ND2, ND4, and ND5). The accessory subunit B14.5a may be located adjacent to the N-terminal region of the 49 kDa subunits. The structural features of the quinone binding pocket in bovine complex I were discussed on the basis of these results.

Computational modeling analysis of mitochondrial superoxide production under varying substrate conditions and upon inhibition of different segments of the electron transport chain

A computational mechanistic model of superoxide (O2•−) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•− production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•− formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•− production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron–sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues.

Replacing Arg70 by Histidine in The Cytochrome Aa3‐600 Menaquinol Oxidase from Bacillus Subtilis Changes The Nitrogen Interacting with The Semiquinone Formed at The Qh Site But Does Not Eliminate Catalytic Function

Cytochrome aa3‐600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses as its electron donor menaquinol instead of cytochrome c, when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made R70H mutant in the high affinity quinone binding site (Qh) and have characterized the menasemiquinone radical by advanced EPR in X‐ and Q‐band. The bound SQ state at Qh site has a strong isotropic hyperfine coupling (14A ~ 2.1 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on determined quadrupolar parameters (κ = 0.36 MHz, η = 0.25) typical for protonated imidazole nitrogens. In contrast, the SQ stabilized in the wild type is hydrogen bonded with Nε from Arg70 side chain. The mutation also changes the number and strength of the hydrogen bonds in the SQ environment, based on the analysis of the 1H 2D ESEEM spectra. Despite the alterations in the immediate environment of the SQ, the R70H remains catalytically active. This contrasts to the equivalent mutation in the close homologue, the cytochrome bo3 ubiquinol oxidase from E. coli, where the R75H mutation eliminates function.

Redox potential tuning through differential quinone binding in the photosynthetic reaction center of Rhodobacter sphaeroides.

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 Q(A) and Q(B) sites using both equilibrium simulation and thermodynamic integration. In particular, we identify a specific interaction between the 2-methoxy group of ubiquinone in the Q(B) 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 Q(A) and Q(B) 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 Q(A)⁻Q(B)⁻ biradical and competitive binding assays.

Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I).

The NuoD segment (homologue of mitochondrial 49 kDa subunit) of the proton-translocating NADH:quinone oxidoreductase (complex I/NDH-1) from Escherichia coli is in the hydrophilic domain and bears many highly conserved amino acid residues. The three-dimensional structural model of NDH-1 suggests that the NuoD segment, together with the neighboring subunits, constitutes a putative quinone binding cavity. We used the homologous DNA recombination technique to clarify the role of selected key amino acid residues of the NuoD segment. Among them, residues Tyr273 and His224 were considered candidates for having important interactions with the quinone headgroup. Mutant Y273F retained partial activity but lost sensitivity to capsaicin-40. Mutant H224R scarcely affected the activity, suggesting that this residue may not be essential. His224 is located in a loop near the N-terminus of the NuoD segment (Gly217–Phe227) which is considered to form part of the quinone binding cavity. In contrast to the His224 mutation, mutants G217V, P218A, and G225V almost completely lost the activity. One region of this loop is positioned close to a cytosolic loop of the NuoA subunit in the membrane domain, and together they seem to be important in keeping the quinone binding cavity intact. The structural role of the longest helix in the NuoD segment located behind the quinone binding cavity was also investigated. Possible roles of other highly conserved residues of the NuoD segment are discussed.

Quinone Binding in Bacterial Photosynthetic Reaction Center

Quinones play a crucial role in transmembrane electron and proton transfer in proteins, thereby creating a gradient that is utilized for energy conversion. The medicinal properties of these molecules render them important for drug design. The quinone binding site varies considerably across different proteins and any strict binding motif is not defined yet. The bacterial photosynthetic reaction center (BRC) is one such protein with two quinone binding sites - QA and QB. Kinetic studies have shown differently substituted foreign quinones to be good binders at the QA site, however there are no protein structures available with the foreign quinones. The goal of this computational study is to derive a BRC-quinone interaction signature, with different quinones docked at the QA site. The derived signature could pave the way for future computer aided drug design, targeted at quinone binding proteins.Sets of substituted foreign quinones were built and optimized in Gaussian09 and docked into the BRC QA site with AutoDock4.2. The protein-ligand vanderwaals overlap was calculated in Chimera. Analyses of the van der waals interaction of the complexes brought out a pattern of favorable interactions of the ligand with the protein, that directly correlate with high binding affinity. The benzoquinone strong binders showed favorable interaction with TrpM252, MetM256 and MetM262 of the BRC. The methyl and methoxy flanking a carbonyl formed a benzoquinone ligand-motif for strong binding. LeuM215, TrpM252, Met256, PheM258, MetM262, and IleM265 of the receptor favorably interacted with the strong naptho binders. Future work includes Molecular dynamics studies that will involve the FEP method on the docked complexes in NAMD2.9.

Stigmatellin Probes the Electrostatic Potential in the QB Site of the Photosynthetic Reaction Center

The electrostatic potential in the secondary quinone (QB) binding site of the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides determines the rate and free energy change (driving force) of electron transfer to QB. It is controlled by the ionization states of residues in a strongly interacting cluster around the QB site. Reduction of the QB induces change of the ionization states of residues and binding of protons from the bulk. Stigmatellin, an inhibitor of the mitochondrial and photosynthetic respiratory chain, has been proven to be a unique voltage probe of the QB binding pocket. It binds to the QB site with high affinity, and the pK value of its phenolic group monitors the local electrostatic potential with high sensitivity. Investigations with different types of detergent as a model system of isolated RC revealed that the pK of stigmatellin was controlled overwhelmingly by electrostatic and slightly by hydrophobic interactions. Measurements showed a high pK value (>11) of stigmatellin in the QB pocket of the dark-state wild-type RC, indicating substantial negative potential. When the local electrostatics of the QB site was modulated by a single mutation, L213Asp→Ala, or double mutations, L213Asp-L212Glu→Ala-Ala (AA), the pK of stigmatellin dropped to 7.5 and 7.4, respectively, which corresponds to a >210 mV increase in the electrostatic potential relative to the wild-type RC. This significant pK drop (ΔpK > 3.5) decreased dramatically to (ΔpK > 0.75) in the RC of the compensatory mutant (AA+M44Asn→AA+M44Asp). Our results indicate that the L213Asp is the most important actor in the control of the electrostatic potential in the QB site of the dark-state wild-type RC, in good accordance with conclusions of former studies using theoretical calculations or light-induced charge recombination assay.

Covalent binding of quinones activates the Ah receptor in Hepa1c1c7 cells.

Highly reactive quinone species produced by photooxidation and/or metabolic activation of mono- or bi-aromatic hydrocarbons modulate cellular homeostasis and electrophilic signal transduction pathways through the covalent modification of proteins. Polycyclic aromatic hydrocarbons, but not mono- or bi-aromatic hydrocarbons, are well recognized as ligands for the aryl hydrocarbon receptor (AhR). However, quinone species produced from mono- and bi-aromatic hydrocarbons could potentially cause AhR activation. To clarify the AhR response to mono- and bi-aromatic hydrocarbon quinones, we studied Cyp1a1 (cytochrome P450 1A1) induction and AhR activation by these quinones. We detected Cyp1a1 induction during treatment with quinones in Hepa1c1c7 cells, but not their parent compounds. Nine of the twelve quinones with covalent binding capability for proteins induced Cyp1a1. Cyp1a1 induction mediated by 1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone (1,4-BQ) and tert-butyl-1,4-BQ was suppressed by a specific AhR inhibitor and was not observed in c35 cells, which do not have a functional AhR. These quinones stimulated AhR nuclear translocation and interaction with the AhR nuclear translocator. Interestingly, 1,2-NQ covalently modified AhR, which was detected by an immunoprecipitation assay using a specific antibody against 1,2-NQ, resulting in enhancement of xenobiotic responsive element (XRE)-derived luciferase activity and binding of AhR to the Cyp1a1 promoter region. While mono- and bi-aromatic hydrocarbons are generally believed to be poor ligands for AhR and hence unable to induce Cyp1a1, our study suggests that the quinones of these molecules are able to modify AhR and activate the AhR/XRE pathway, thereby inducing Cyp1a1. Since we previously reported that 1,2-NQ and tert-butyl-1,4-BQ also activate NF-E2-related factor 2, it seems likely that some of quinones are bi-functional inducers for phase-I and phase-II reaction of xenobiotics.

Electronic connection between the quinone and cytochrome C redox pools and its role in regulation of mitochondrial electron transport and redox signaling.

Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria.

Structure and binding efficiency relations of QB site inhibitors of photosynthetic reaction centres.

Many herbicides employed in agriculture and also some antibiotics bind to a specific site of the reaction centre protein (RC) blocking the photosynthetic electron transport. Crystal structures showed that all these compounds bind at the secondary ubiquinone (QB) site albeit to slightly different places. Different herbicide molecules have different binding affinities (evaluated as inhibition constants, KI, and binding enthalpy values, ΔHbind). The action of inhibitors depends on the following parameters: (i) herbicide molecular structure; (ii) interactions between herbicide and quinone binding site; (iii) protein environment. In our investigations KI and ΔHbind were determined for several inhibitors. Bound herbicide structures were optimized and their intramolecular charge distributions were calculated. Experimental and calculated data were compared to those available from databank crystal structures. We can state that the herbicide inhibition efficiency depends on steric and electronic, i.e. geometry of binding with the protein and molecular charge distribution, respectively. Apolar bulky groups on N-7 atom of the inhibitor molecule (like t-buthyl in terbutryn) are preferable for establishing stronger interactions with QB site, while such substituents are not recommended on N-8. The N-4,7,8 nitrogen atoms maintain a larger electron density so that more effective H-bonds are formed between the inhibitor and the surrounding amino acids of the protein.

Direct evidence for the atovaquone action on the Plasmodium cytochrome bc1 complex

Atovaquone, a coenzyme Q analogue has been indicated to specifically target the cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the quinone binding site of the cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to atovaquone. There is no direct evidence of relation between the mutations and resistance to atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance.The establishment of a mitochondrial isolation method for the malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to atovaquone directly. We have tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant clones in the presence of a wide concentration range of atovaquone. Our results show the IC50 of P. berghei atovaquone resistant clones is much higher (1.5 up to 40nM) in comparison to the atovaquone sensitive clones (0.132–0.465nM). The highest IC50 was revealed in clones carrying Y268C and Y268N mutations (which play an important role in atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the quinone binding site of the cytochrome b gene and that provide a direct evidence for the atovaquone inhibitory mechanism in the cytochrome bc1 complex of the parasite.

Overcoming drug-resistant malaria through rational drug design in cytochrome bc1

Over three billion people live in regions affected by malaria and there are over one million deaths each year [1]. Malaria is caused by the Plasmodium parasite and various drugs are currently used in both treatment and prophylaxis but resistant strains are rapidly emerging. One of the most commonly used anti-malarial drugs is Atovaquone, a hydroxynapthoquinone that is currently used in combination with Proguanil and sold as Malarone™. Atovaquone targets cytochrome bc1 (Complex III, ubiquinol-cytochrome c oxidoreductase), a multi subunit electron transfer protein complex embedded in the inner mitochondrial membrane [2]. Drug resistance rises through a single point mutation in cytochrome b at the Qo site, one of two quinone binding sites. By visualising compounds bound to cytochrome bc1 through x-ray crystallography, it may be possible to modify the compounds to both bind stronger and more specifically. We have worked on compounds that recently failed phase I clinical trials due to cross-reactivity with human cytochrome bc1 [3]. Our structural studies have shown that these compounds appear to bind at the Qi site, which would overcome current drug-resistant strains. Further work here could produce a novel class of anti-malarial drug.

The binding of quinone to the photosynthetic reaction centers: kinetics and thermodynamics of reactions occurring at the QB-site in zwitterionic and anionic liposomes.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

Q-Site Occupancy Defines Heme Heterogeneity in Escherichia coli Nitrate Reductase A (NarGHI)

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.

ND3, ND1 and 39 kDa subunits are more exposed in the de-active form of bovine mitochondrial complex I

An intriguing feature of mitochondrial complex I from several species is the so-called A/D transition, whereby the idle enzyme spontaneously converts from the active (A) form to the de-active (D) form. The A/D transition plays an important role in tissue response to the lack of oxygen and hypoxic deactivation of the enzyme is one of the key regulatory events that occur in mitochondria during ischaemia. We demonstrate for the first time that the A/D conformational change of complex I does not affect the macromolecular organisation of supercomplexes in vitro as revealed by two types of native electrophoresis. Cysteine 39 of the mitochondrially-encoded ND3 subunit is known to become exposed upon de-activation. Here we show that even if complex I is a constituent of the I+III2+IV (S1) supercomplex, cysteine 39 is accessible for chemical modification in only the D-form. Using lysine-specific fluorescent labelling and a DIGE-like approach we further identified two new subunits involved in structural rearrangements during the A/D transition: ND1 (MT-ND1) and 39kDa (NDUFA9). These results clearly show that structural rearrangements during de-activation of complex I include several subunits located at the junction between hydrophilic and hydrophobic domains, in the region of the quinone binding site. De-activation of mitochondrial complex I results in concerted structural rearrangement of membrane subunits which leads to the disruption of the sealed quinone chamber required for catalytic turnover.

Characterization of 2- and 3-Methoxy in Ubiquinone Binding and Redox Tuning within the Photosynthetic Reaction Center of Rhodobacter Sphaeroides

Ubiquinone is a universal, membrane-associated redox mediator. The redox properties of ubiquinone in vivo are closely controlled both by its electrostatic environment within the binding sites of proteins and by steric interactions determining its conformation. Recent experimental work suggests that differences in the angle of the two methoxy side chains with respect to the quinone ring plane dictate the redox potential difference between the two quinone binding sites, however the specific quinone-protein interactions that alter the redox potential could not be identified.In order to ascertain which specific interactions between the quinones and their environment tune the redox potential, molecular dynamics simulations of ubiquinone bound to the QA and QB sites of the photosynthetic reaction center (RC) of Rhodobacter Sphaeroides were compared with complementary studies done on ubiquinone analogs lacking either the 2- or 3-methoxy group. Since prior ubiquinone parameters were biased towards non-native methoxy angles, systematically parameterized ubiquinone and its analogues to accurately describe the structure and dynamics of the methoxy groups.The removal any methoxy group showed little impact on the QA binding. However, both the binding conformation and the methoxy dihedral angle distribution in the QB site were significantly altered upon removal of the 2-methoxy group. We observe that the 2-methoxy group in the QB site is oriented approximately normal to the ring plane. This orientation is favored by a weak hydrogen bond between the 2-methoxy oxygen and the amine of GLY225. A more out of plane methoxy (as imposed by the protein environment) would correspond to a higher electron affinity of QB over QA. We propose this hydrogen bond as the mechanism whereby the redox potential is tuned to promote inter-quinone electron transfer in the RC.

Enhancing Electron Transfer Form Photosynthetic Reaction Centers to Electrodes by Exposing Quinone Binding Pocket

Due to their potential for bio-energy generation and as bio-sensors, photosynthetic reaction centers (RCs) have been characterized quite extensively for their light induced electroactivity on electrode surfaces. Most such studies have depended on the presence of redox mediators to achieve higher current densities. We are aiming to effect direct electron transfer from both the acceptor and donor sides of the RC to electrodes and to determine the distance and orientation dependence of this process. The RC from Rhodobacter sphaeroides is a model system for photosynthesis research and is generally more robust than its counterparts from plants, in vitro. It consists of three subunits, L, M and H. The H subunit is not involved in the scaffold for electron transfer cofactors and can be removed without compromising the light dependent reduction of the primary quinone. We have developed an improved protocol to remove the H subunit, rendering a LM dimer that behaves as previously report (Debus et al. (1985) Biochemistry 24, 2488). Preliminary data suggest that unlike the complete RC, reduced semiquinone in LM dimers can donate electrons to a gold electrode without any redox mediators present. We are now working to obtain direct linkage between electrode and RC, in various orientations, through covalent gold-thiol bonds to site-directed cysteines on the LM surface and by randomly generated and selected peptides that bind RCs in a very different manner. By comparing and modeling the light induced current, we hope to arrive at a quantitative description of the electron transfer events between the RC and electrode.

Synthesis and Evaluation of Heterocyclic Analogues of Bromoxynil

One attractive strategy to discover more active and/or crop-selective herbicides is to make structural changes to currently registered compounds. This strategy is especially appealing for those compounds with limited herbicide resistance and whose chemistry is accompanied with transgenic tools to enable herbicide tolerance in crop plants. Bromoxynil is a photosystem II (PSII) inhibitor registered for control of broadleaf weeds in several agronomic and specialty crops. Recently at the University of Tennessee-Knoxville several analogues of bromoxynil were synthesized including a previously synthesized pyridine (2,6-dibromo-5-hydroxypyridine-2-carbonitrile sodium salt), a novel pyrimidine (4,6-dibromo-5-hydroxypyrimidine-2-carbonitrile sodium salt), and a novel pyridine N-oxide (2,6-dibromo-1-oxidopyridin-1-ium-4-carbonitrile). These new analogues of bromoxynil were also evaluated for their herbicidal activity on soybean (Glycine max), cotton (Gossypium hirsutum), redroot pigweed (Amaranthus retroflexus), velvetleaf (Abutilon theophrasti), large crabgrass (Digitaria sanguinalis), and pitted morningglory ( Ipomoea lacunose ) when applied at 0.28 kg ha(-1). A second study was conducted on a glyphosate-resistant weed (Amaranthus palmeri) with the compounds being applied at 0.56 kg ha(-1). Although all compounds were believed to inhibit PSII by binding in the quinone binding pocket of D1, the pyridine and pyridine-N-oxide analogues were clearly more potent than bromoxynil on Amaranthus retroflexus. However, application of the pyrimidine herbicide resulted in the least injury to all species tested. These variations in efficacy were investigated using molecular docking simulations, which indicate that the pyridine analogue may form a stronger hydrogen bond in the pocket of the D1 protein than the original bromoxynil. A pyridine analogue was able to control the glyphosate-resistant Amaranthus palmeri with >80% efficacy. The pyridine analogues of bromoxynil showed potential to have a different weed control spectrum compared to bromoxynil. A pyridine analogue of bromoxynil synthesized in this research controlled several weed species greater than bromoxynil itself, potentially due to enhanced binding within the PSII binding pocket. Future research should compare this analogue to bromoxynil using optimized formulations at higher application rates.

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na+-translocating NADH:Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na(+)-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na(+)-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na(+)-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na(+)-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na(+)-NQR.