RESUMEN
The reaction of benzylsuccinate synthase, the radical-based addition of toluene to a fumarate cosubstrate, is initiated by hydrogen transfer from a conserved cysteine to the nearby glycyl radical in the active center of the enzyme. In this study, we analyze this step by comprehensive computer modeling, predicting (i) the influence of bound substrates or products, (ii) the energy profiles of forward- and backward hydrogen-transfer reactions, (iii) their kinetic constants and potential mechanisms, (iv) enantiospecificity differences, and (v) kinetic isotope effects. Moreover, we support several of the computational predictions experimentally, providing evidence for the predicted H/D-exchange reactions into the product and at the glycyl radical site. Our data indicate that the hydrogen transfer reactions between the active site glycyl and cysteine are principally reversible, but their rates differ strongly depending on their stereochemical orientation, transfer of protium or deuterium, and the presence or absence of substrates or products in the active site. This is particularly evident for the isotope exchange of the remaining protium atom of the glycyl radical to deuterium, which appears dependent on substrate or product binding, explaining why the exchange is observed in some, but not all, glycyl-radical enzymes.
Asunto(s)
Biocatálisis , Cinética , Liasas de Carbono-Azufre/química , Liasas de Carbono-Azufre/metabolismo , Dominio Catalítico , Modelos Moleculares , Cisteína/química , Cisteína/metabolismo , Hidrógeno/química , Radicales Libres/química , Radicales Libres/metabolismo , Liasas de Carbono-CarbonoRESUMEN
Here, we address the problem of the antioxidant activity of carotenoids in biomembranes. The activity of lutein and zeaxanthin in the quenching of singlet oxygen generated by photosensitization was monitored in lipid vesicles using a singlet oxygen-sensitive fluorescent probe and with the application of fluorescence lifetime imaging microscopy. The antioxidant activity of xanthophylls was interpreted on the basis of electron paramagnetic resonance oximetry results showing that xanthophylls constitute a barrier to the penetration of molecular oxygen into lipid membranes: to a greater extent in the 13-cis configuration than in all-trans. These results are discussed in relation to the trans-cis photoisomerization of xanthophylls observed in the human retina. It can be concluded that photoisomerization of xanthophylls is a regulatory mechanism that is important for both the modulation of light filtration through the macula and photoprotection by quenching singlet oxygen and creating a barrier to oxygen permeation to membranes.
Asunto(s)
Antioxidantes , Xantófilas , Humanos , Xantófilas/metabolismo , Oxígeno Singlete , Estrés Oxidativo , Oxígeno , LípidosRESUMEN
Experiments based on fast reagent mixing and observation of reaction progress are considered a powerful tool for investigating the kinetics of chemical and enzymatic reactions. Various spectroscopic methods are used in monitoring the reaction progress, which require different sample preparation methods. Stopped-flow is the most widespread method, where the reaction in the liquid phase is observed by optical absorption spectroscopy. Albeit less popular, the freeze-quench method is also used, in which the reaction is rapidly stopped by freezing the sample at a given time point after the reaction onset. The frozen droplets of the sample are collected and measured at low temperatures in the solid state. Currently, many commercial solutions are available for freeze-quench or stopped-flow experiments, but despite the high price of the devices, most of these do not allow combining both these methods in a single experiment. This study presents a relatively simple solution that combines both these methods, thus making a complete study of chemical or enzymatic reactions possible. Besides, the presented solution enables sequential double mixing of reagents, which is generally problematic and cannot be done using commercial instruments.
RESUMEN
Plants use solar energy to power cellular metabolism. The oxidation of plastoquinol and reduction of plastocyanin by cytochrome b6f (Cyt b6f) is known as one of the key steps of photosynthesis, but the catalytic mechanism in the plastoquinone oxidation site (Qp) remains elusive. Here, we describe two high-resolution cryo-EM structures of the spinach Cyt b6f homodimer with endogenous plastoquinones and in complex with plastocyanin. Three plastoquinones are visible and line up one after another head to tail near Qp in both monomers, indicating the existence of a channel in each monomer. Therefore, quinones appear to flow through Cyt b6f in one direction, transiently exposing the redox-active ring of quinone during catalysis. Our work proposes an unprecedented one-way traffic model that explains efficient quinol oxidation during photosynthesis and respiration.
Asunto(s)
Citocromos b , Plastocianina , Citocromos b/metabolismo , Plastocianina/metabolismo , Microscopía por Crioelectrón , Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/metabolismo , Oxidación-Reducción , Fotosíntesis , Plantas/metabolismo , Quinonas , Transporte de ElectrónRESUMEN
Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Qn and Qp). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b6f, the redox midpoint potentials (Ems) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b6f and revealed that Em of heme bn is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Qp and Qn sites and modulate the efficiency of cyclic and linear ET in photosynthesis.
Asunto(s)
Citocromos b , Hemo , Electrones , Transporte Biológico , CatálisisRESUMEN
Magnetic linear actuators have a wide range of applications. Their main advantage is the ease with which they can be controlled by regulating the current. However, high electrical power is required for obtaining a large continuous force, acceleration, and stroke from a device with small dimensions. In this study, we developed a comprehensive open-source system consisting of simple movable iron magnetic actuators, a four-channel controller, and dedicated software. The graphical user interface facilitates the designing of the sequence of piston strokes, including the start time, duration of movement, and force for each stroke separately. The controller generates a high current of pulses, which allows achieving a high force, acceleration, and stroke from small-sized coils while maintaining a relatively safe voltage. The system was originally designed as a reagent syringe driver to control the rapid mixing process used for studying the kinetics of enzymatic reactions. However, this driver may also be applied in various other scientific as well as nonscientific applications.
RESUMEN
Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.
Asunto(s)
Grupo Citocromo b/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Rhodobacter capsulatus/metabolismo , Antimicina A/análogos & derivados , Grupo Citocromo b/genética , Espectroscopía de Resonancia por Spin del Electrón , Complejo III de Transporte de Electrones/genética , Enlace de Hidrógeno , Modelos Moleculares , Mutación , Oxidación-Reducción , Conformación Proteica , Análisis Espectral/métodosRESUMEN
While metal-organic frameworks (MOFs) are at the forefront of cutting-edge porous materials, extraordinary sorption properties can also be observed in Prussian Blue Analogs (PBAs) and related materials comprising extremely short bridging ligands. Herein, we present a bimetallic nonporous cyanide-bridged coordination polymer (CP) {[Mn(imH)]2[Mo(CN)8]} n (1Mn; imH = imidazole) that can efficiently and reversibly capture and release water molecules over tens of cycles without any fatigue despite being based on one of the shortest bridging ligands known - the cyanide. The sorption performance of {[Mn(imH)]2[Mo(CN)8]} n matches or even outperforms MOFs that are typically selected for water harvesting applications with perfect sorption reversibility and very low desorption temperatures. Water sorption in 1Mn is possible due to the breathing effect (accompanied by a dramatic cyanide-framework transformation) occurring in three well-defined steps between four different crystal phases studied structurally by X-ray diffraction structural analysis. Moreover, the capture of H2O by 1Mn switches the EPR signal intensity of the MnII centres, which has been demonstrated by in situ EPR measurements and enables monitoring of the hydration level of 1Mn by EPR. The sorption of water in 1Mn controls also its photomagnetic behavior at the cryogenic regime, thanks to the presence of the [MoIV(CN)8]4- photomagnetic chromophore in the structure. These observations demonstrate the extraordinary sorption potential of cyanide-bridged CPs and the possibility to merge it with the unique physical properties of this class of compounds arising from their bimetallic character (e.g. photomagnetism and long-range magnetic ordering).
RESUMEN
Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.
Asunto(s)
Proteínas Bacterianas/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Flavobacterium/metabolismo , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , Citocromos b6/genética , Citocromos b6/metabolismo , Transporte de Electrón , Complejo III de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/genética , Flavobacterium/genética , Flavobacterium/ultraestructura , Oxidación-Reducción , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
Cytochrome bc 1 (mitochondrial complex III) catalyzes electron transfer from quinols to cytochrome c and couples this reaction with proton translocation across lipid membrane; thus, it contributes to the generation of protonmotive force used for the synthesis of ATP. The energetic efficiency of the enzyme relies on a bifurcation reaction taking place at the Qo site which upon oxidation of ubiquinol directs one electron to the Rieske 2Fe2S cluster and the other to heme b L. The molecular mechanism of this reaction remains unclear. A semiquinone spin-coupled to the reduced 2Fe2S cluster (SQo-2Fe2S) was identified as a state associated with the operation of the Qo site. To get insights into the mechanism of the formation of this state, we first constructed a mutant in which one of the histidine ligands of the iron ion of heme b L Rhodobacter capsulatus cytochrome bc 1 was replaced by asparagine (H198N). This converted the low-spin, low-potential heme into the high-spin, high-potential species which is unable to support enzymatic turnover. We performed a comparative analysis of redox titrations of antimycin-supplemented bacterial photosynthetic membranes containing native enzyme and the mutant. The titrations revealed that H198N failed to generate detectable amounts of SQo-2Fe2S under neither equilibrium (in dark) nor nonequilibrium (in light), whereas the native enzyme generated clearly detectable SQo-2Fe2S in light. This provided further support for the mechanism in which the back electron transfer from heme b L to a ubiquinone bound at the Qo site is mainly responsible for the formation of semiquinone trapped in the SQo-2Fe2S state in R. capusulatus cytochrome bc 1.
RESUMEN
This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc1/b6f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.
Asunto(s)
Complejo de Citocromo b6f/química , Complejo de Citocromo b6f/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Animales , Catálisis , Humanos , Membranas/química , Membranas/enzimología , Simulación de Dinámica Molecular , Fotosíntesis , Conformación Proteica , Respiración , Rhodobacter capsulatus , TermodinámicaRESUMEN
Cytochromes bc1 and c b6f are part of respiratory or photosynthetic machinery. The main role of these enzymes is to build proton motive force across the bioenergetic membranes by coupling the proton translocations with electron transfer from the pool of membrane-soluble quinones to water-soluble redox proteins. Despite many years of research, the mechanism of quinol oxidation is not fully understood. It is assumed that unstable form of a partially oxidized quinol semiquinone is an intermediate state of this process and that it is also a potential electron donor in the side reaction of superoxide generation. This semiquinone has remained experimentally elusive over years but recently a semiquinone interacting with the reduced iron-sulfur cluster was identified as a new state of the enzyme. The results indicate that semiquinone coupled to the iron-sulfur cluster is most probably an additional state that can prevent side reactions, including superoxide generation.
Asunto(s)
Benzoquinonas/química , Benzoquinonas/metabolismo , Electrones , Citocromos/metabolismo , Transporte de Electrón , Oxidación-Reducción , Superóxidos/metabolismoRESUMEN
The rational design of coordination frameworks combining more than two different metal ions using a self-assembly approach is challenging because it rarely offers sufficient control over the building blocks at the actual self-assembly stage. In this work, we present a successful two-step strategy toward heterotrimetallic coordination frameworks by employing a new bimetallic [(NC)7MoIV-CN-PtIV(NH3)4-NC-MoIV(CN)7]4- secondary building unit (SBU). This anionic moiety has been isolated and characterized as a simple salt with an organic dppipH22+ cation (dppipH2)2[(NC)7MoIV-CN-PtIV(NH3)4-NC-MoIV(CN)7]·15H2O (1) (dppip = 1,4-di(4-pyridinyl)piperazine). The salt presents a second-order phase transition related to cation conformational change around 250 K and a photomagnetic effect after irradiation with 450 nm light at 10 K. When combined with aqueous solutions of MnII or CuII complexes, it forms either a one-dimensional chain [MnII(dpop)][MnII(dpop)(H2O)][(NC)7MoIV-CN-PtIV(NH3)4-NC-MoIV(CN)7]·36H2O (2) (dpop = 2,13-dimethyl-3,6,9,12,18-pentaazabicyclo-[12.3.1]octadeca-1(18),2,12,14,16-pentaene) or a photomagnetic two-dimensional honeycomb network [CuII(cyclam)]2[(NC)7MoIV-CN-PtIV(NH3)4-NC-MoIV(CN)7]·40.89H2O (3) (cyclam = 1,4,8,11-tetraazacyclotetradecane), both characterized by very large cavities in their structure filled with solvent molecules. Both 2 and 3 incorporate three different transition-metal ions and constitute a new family of 3d-4d-5d coordination frameworks. Moreover, compound 3 inherits the photomagnetic properties of the MoPtMo SBU.
RESUMEN
Quinone reduction site (Qi) of cytochrome bc1 represents one of the canonical sites used to explore the enzymatic redox reactions involving semiquinone (SQ) states. However, the mechanism by which Qi allows the completion of quinone reduction during the sequential transfers of two electrons from the adjacent heme bH and two protons to C1- and C4-carbonyl remains unclear. Here we established that the SQ coupled to an oxidized heme bH is a dominant intermediate of catalytic forward reaction and, contrary to the long-standing assumption, represents a significant population of SQ detected across pH 5-9. The pH dependence of its redox midpoint potential implicated proton exchange with histidine. Complementary quantum mechanical calculations revealed that the SQ anion formed after the first electron transfer undergoes charge and spin polarization imposed by the electrostatic field generated by histidine and the aspartate/lysine pair interacting with the C4- and C1-carbonyl, respectively. This favors a barrierless proton exchange between histidine and the C4-carbonyl, which continues until the second electron reaches the SQi. Inversion of charge polarization facilitates the uptake of the second proton by the C1-carbonyl. Based on these findings we developed a comprehensive scheme for electron and proton transfers at Qi featuring the equilibration between the anionic and neutral states of SQi as means for a leak-proof stabilization of the radical intermediate. The key catalytic role of the initial charge/spin polarization of the SQ anion at the active site, inherent to the proposed mechanism, may also be applicable to the other quinone oxidoreductases.
Asunto(s)
Complejo III de Transporte de Electrones/metabolismo , Quinonas/metabolismo , Sitios de Unión , Complejo III de Transporte de Electrones/química , Electrones , Modelos Moleculares , Oxidación-Reducción , Conformación ProteicaRESUMEN
A homoleptic gadolinium(III) complex with the smallest helicene-type ligand, 1,10-phenanthroline-N,N'-dioxide (phendo) [Gd(phendo)4](NO3)3·xMeOH (phendo = 1,10-phenanthroline-N,N'-dioxide, MeOH = methanol), shows slow relaxation of the magnetization characteristic for Single Ion Magnets (SIM), despite negligible magnetic anisotropy, confirmed by ab initio calculations. Solid state dilution magnetic and EPR studies reveal that the magnetization dynamics of the [Gd(phendo)4]3+ cation is controlled mainly by a Raman process. Pulsed EPR experiments demonstrate long phase memory times (up to 2.7 µs at 5 K), enabling the detection of Rabi oscillations at 20 K, which confirms coherent control of its spin state.
RESUMEN
Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc1 , the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin-spin-coupled state between SQ and the Rieske cluster (SQ-FeS) associated with catalysis of the quinol oxidation site of cytochrome bc1 . We reveal that mutants that preclude direct interaction between SQ and the Rieske cluster do not form SQ-FeS and release enhanced superoxide. In the enzymes generating SQ-FeS, little or no superoxide is detected. We propose that SQ-FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically relevant conditions slowing electron flow through the enzyme.
Asunto(s)
Benzoquinonas/metabolismo , Complejo III de Transporte de Electrones/química , Complejo III de Transporte de Electrones/metabolismo , Rhodobacter capsulatus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Complejo III de Transporte de Electrones/genética , Mutación , Unión Proteica , Rhodobacter capsulatus/química , Rhodobacter capsulatus/genética , Superóxidos/metabolismoRESUMEN
Dimeric cytochromes bc are central components of photosynthetic and respiratory electron transport chains. In their catalytic core, four hemes b connect four quinone (Q) binding sites. Two of these sites, Qi sites, reduce quinone to quinol (QH2) in a step-wise reaction, involving a stable semiquinone intermediate (SQi). However, the interaction of the SQi with the adjacent hemes remains largely unexplored. Here, by revealing the existence of two populations of SQi differing in paramagnetic relaxation, we present a new mechanistic insight into this interaction. Benefiting from a clear separation of these SQi species in mutants with a changed redox midpoint potential of hemes b, we identified that the fast-relaxing SQi (SQiF) corresponds to the form magnetically coupled with the oxidized heme bH (the heme b adjacent to the Qi site), while the slow-relaxing SQi (SQiS) reflects the form present alongside the reduced (and diamagnetic) heme bH. This so far unreported SQiF calls for a reinvestigation of the thermodynamic properties of SQi and the Qi site. The existence of SQiF in the native enzyme reveals a possibility of an extended electron equilibration within the dimer, involving all four hemes b and both Qi sites. This substantiates the predicted earlier electron transfer acting to sweep the b-chain of reduced hemes b to diminish generation of reactive oxygen species by cytochrome bc1. In analogy to the Qi site, we anticipate that the quinone binding sites in other enzymes may contain yet undetected semiquinones which interact magnetically with oxidized hemes upon progress of catalytic reactions.
Asunto(s)
Proteínas Bacterianas/química , Complejo III de Transporte de Electrones/química , Electrones , Hemo/química , Quinonas/química , Especies Reactivas de Oxígeno/química , Antimicina A/análogos & derivados , Antimicina A/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/aislamiento & purificación , Complejo III de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/química , Expresión Génica , Hemo/metabolismo , Cinética , Metacrilatos/química , Mutación , Oxidación-Reducción , Potenciometría , Unión Proteica , Multimerización de Proteína , Quinonas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rhodobacter capsulatus/química , Rhodobacter capsulatus/enzimología , Termodinámica , Tiazoles/químicaRESUMEN
One of the less understood parts of the catalytic cycle of cytochrome bc1/b6f complexes is the mechanism of electronic bifurcation occurring within the hydroquinone oxidation site (Qo site). Several models describing this mechanism invoke a phenomenon of formation of an unstable semiquinone. Recent studies with isolated cytochrome bc1 or b6f revealed that a relatively stable semiquinone spin-coupled to the reduced Rieske cluster (SQ-FeS) is generated at the Qo site during the oxidation of ubi- or plastohydroquinone analogs under conditions of continuous turnover. Here, we identified the EPR transition of SQ-FeS formed upon oxidation of ubihydroquinone in native photosynthetic membranes from purple bacterium Rhodobacter capsulatus. We observed a significant amount of SQ-FeS generated when the antimycin-inhibited enzyme experiences conditions of non-equilibrium caused by the continuous light activation of the reaction center. We also noted that SQ-FeS cannot be detected under equilibrium redox titrations in dark. The non-equilibrium redox titrations of SQ-FeS indicate that this center has a higher apparent redox midpoint potential when compared to the redox midpoint potential of the quinone pool. This suggests that SQ-FeS is stabilized, which corroborates a recently proposed mechanism in which the SQ-FeS state is metastable and functions to safely hold electrons at the local energy minimum during the oxidation of ubihydroquinone and limits superoxide formation. Our results open new possibilities to study the formation and properties of this state in cytochromes bc under close to physiological conditions in which non-equilibrium is attained by the light activation of bacterial reaction centers or photosystems.
Asunto(s)
Benzoquinonas/química , Membrana Celular/enzimología , Complejo III de Transporte de Electrones/química , Luz , Rhodobacter capsulatus/enzimología , Benzoquinonas/metabolismo , Dominio Catalítico , Complejo III de Transporte de Electrones/metabolismo , Oxidación-Reducción/efectos de la radiaciónRESUMEN
Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b6f This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.
Asunto(s)
Complejo de Citocromo b6f/química , Complejo III de Transporte de Electrones/química , Catálisis , Espectroscopía de Resonancia por Spin del Electrón , Oxígeno/química , Fotosíntesis , Rhodobacter capsulatus , Spinacia oleraceaRESUMEN
The cytochrome (cyt) bc1 complex is an integral component of the respiratory electron transfer chain sustaining the energy needs of organisms ranging from humans to bacteria. Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme's substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone - a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.