RESUMEN
We have explored a strategy to identify potential proton transfer channels using computational analysis of a protein structure based on Voronoi partitioning and applied it for the analysis of proton transfer pathways in redox-driven proton-pumping respiratory complex I. The analysis results in a network of connected voids/channels, which represent the dual structure of the protein; we then hydrated the identified channels using our water placement program Dowser++. Many theoretical water molecules found in the channels perfectly match the observed experimental water molecules in the structure; some other predicted water molecules have not been resolved in the experiments. The channels are of varying cross sections. Some channels are big enough to accommodate water molecules that are suitable to conduct protons; others are too narrow to hold water but require only minor conformational changes to accommodate proton transfer. We provide a preliminary analysis of the proton conductivity of the network channels, classifying the proton transfer channels as open, closed, and partially open, and discuss possible conformational changes that can modulate, i.e., open and close, the channels.
RESUMEN
Proton circuits within biological membranes, the foundation of natural bioenergetic systems, are significantly influenced by the lipid compositions of different biological membranes. In this study, we investigate the influence of mixed lipid membrane composition on the proton transfer (PT) properties on the surface of the membrane. We track the excited-state PT (ESPT) process from a tethered probe to the membrane with timescales and length scales of PT relevant to bioenergetic systems. Two processes can happen during ESPT: the initial PT from the probe to the membrane at short timescales, followed by diffusion of dissociated protons around the probe on the membrane, and the possible geminate recombination with the probe at longer timescales. Here, we use membranes composed of mixtures of phosphatidylcholine (PC) and phosphatidic acid (PA). We show that the changes in the ESPT properties are not monotonous with the concentration of the lipid mixture; at a low concentration of PA in PC, we find that the membrane is a poor proton acceptor. Molecular dynamics simulations indicate that the membrane is more structured at this specific lipid mixture, with the least number of defects. Accordingly, we suggest that the structure of the membrane is an important factor in facilitating PT. We further show that the composition of the membrane affects the geminate proton diffusion around the probe, whereas, on a timescale of tens of nanoseconds, the dissociated proton is mostly lateral restricted to the membrane plane in PA membranes, while in PC, the diffusion is less restricted by the membrane.
RESUMEN
In all resolved structures of complex I, there exists a tunnel-like Q-chamber for ubiquinone binding and reduction. The entrance to the Q-chamber in ND1 subunit forms a narrow bottleneck, which is rather tight and requires thermal conformational changes for ubiquinone to get in and out of the binding chamber. The substitution of alanine with threonine at the bottleneck (AlaThr MUT), associated with 3460/ND1 mtDNA mutation in human complex I, is implicated in Leber's Hereditary Optic Neuropathy (LHON). Here, we show the AlaThr MUT further narrows the Q-chamber entrance cross-section area by almost 30%, increasing the activation free energy barrier of quinone passage by approximately 5 kJ mol-1. This severely disrupts quinone binding and reduction as quinone passage through the bottleneck is slowed down almost tenfold. Our estimate of the increase in free energy barrier is entirely due to the bottleneck narrowing, leading to a reduction of the transition state entropy between WT and MUT, and thus more difficult quinone passage. Additionally, we investigate details of possible water exchange between the Q-chamber and membrane. We find water exchange is dynamic in WT but may be severely slowed in MUT. We propose that LHON symptoms caused by 3460/ND1 mtDNA mutation are due to slowed quinone binding. This leads to an increased production of reactive oxidative species due to upstream electron backup at the FMN site of complex I, thus resulting in a mt bioenergetic defect.
Asunto(s)
Complejo I de Transporte de Electrón , Atrofia Óptica Hereditaria de Leber , Humanos , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , ADN Mitocondrial/genética , Ubiquinona/metabolismo , Mitocondrias/metabolismo , Mutación , Atrofia Óptica Hereditaria de Leber/genética , Agua/metabolismoRESUMEN
Autoxidation of tartaric acid in air-saturated aqueous solutions in the presence of Fe(II) at low pH, 2.5, shows autocatalytic behavior with distinct initiation, propagation, and termination phases. With increasing pH, the initiation phase speeds up, while the propagation phase shortens and reduces to none. We show that the propagation phase is a chain reaction that occurs via activation of oxygen in the initiation stage with the production of hydrogen peroxide. The subsequent Fenton oxidation that regenerates hydrogen peroxide with a positive feedback is typical of a self-sustained chain reaction. The conditions for such a chain reaction are shown to be similar to those of a dynamical system with critical behavior; namely, the system becomes unstable when the kinetic matrix of pseudo-first-order reaction becomes negatively defined with a negative eigenvalue giving the rate of exponential (chain) growth of the reactive species.
RESUMEN
Complex I is a key proton-pumping enzyme in bacterial and mitochondrial respiratory electron transport chains. Using quantum chemistry and electrostatic calculations, we have examined the pKa of the reduced quinone QH-/QH2 in the catalytic cavity of complex I. We find that pKa (QH-/QH2) is very high, above 20. This means that the energy of a single protonation reaction of the doubly reduced quinone (i.e. the reduced semiquinone QH-) is sufficient to drive four protons across the membrane with a potential of 180 mV. Based on these calculations, we propose a possible scheme of redox-linked proton pumping by complex I. The model explains how the energy of the protonation reaction can be divided equally among four pumping units of the pump, and how a single proton can drive translocation of four additional protons in multiple pumping blocks.
Asunto(s)
Complejo I de Transporte de Electrón , Protones , Complejo I de Transporte de Electrón/metabolismo , Oxidación-Reducción , Quinonas , Transporte de ElectrónRESUMEN
Proton migration on biological membranes plays a major role in cellular respiration and photosynthesis, but it is not yet fully understood. Here we show that proton dissociation kinetics and related geminate recombination can be used as a probe of such proton migration mechanisms. We develop a simple model for the process and apply it to analyze the results obtained using a photo-induced proton release probe (chemically modified photoacid) tethered to phosphatidylcholine membranes. In our theoretical model, we apply approximate treatment for the diffusional cloud of the geminate proton around the dissociated photoacid and consider arbitrary dimension of the system, 1 < d < 3. We observe that in d > 2, there is a kinetic phase transition between an exponential and a power-law kinetic phases. The existence of an exponential decay phase at the beginning of the proton dissociation is a signature of d > 2 systems. In most other cases, the exponential decay phase is not present, and the kinetics follows a diffusional power-law P(t) â¼ t-d/2 that develops after a short initiation time. Specifically, in a 1D case, which corresponds to the desorption of a proton from the surface, the dissociation occurs by the slow power-law â¼1/t and explains the abnormally slow desorption rate reported recently in experiments.
Asunto(s)
Protones , Recombinación Genética , Difusión , CinéticaRESUMEN
We apply linear response theory to calculate mechanical allosteric couplings in respiratory complex I between the iron sulfur cluster N2, located in the catalytic cavity, and the membrane part of the enzyme, separated from it by more than 50 Å. According to our hypothesis, the redox reaction of ubiquinone in the catalytic cavity of the enzyme generates an unbalanced charge that via repulsion of the charged redox center N2 produces local mechanical stress that transmits into the membrane part of the enzyme where it induces proton pumping. Using coarse-grained simulations of the enzyme, we calculated mechanistic allosteric couplings that reveal the pathways of the mechanical transmission of the stress along the enzyme. The results shed light on the recent experimental studies where a stabilization of the enzyme with an introduced disulfide bridge resulted in the abolishing of proton pumping. Simulation of the disulfide bond action indicates a dramatic change of the mechanistic coupling pathways in line with experimental findings.
Asunto(s)
Complejo I de Transporte de Electrón , Proteínas Hierro-Azufre , Disulfuros/metabolismo , Complejo I de Transporte de Electrón/química , Proteínas Hierro-Azufre/química , Oxidación-Reducción , ProtonesRESUMEN
At the joint between the membrane and hydrophilic arms of the enzyme, the structure of the respiratory complex I reveals a tunnel-like Q-chamber for ubiquinone binding and reduction. The narrow entrance of the quinone chamber located in ND1 subunit forms a bottleneck (eye of a needle) which in all resolved structures was shown to be too small for a bulky quinone to pass through, and it was suggested that a conformational change is required to open the channel. The closed bottleneck appears to be a well-established feature of all structures reported so-far, both for the so-called open and closed states of the enzyme, with no indication of a stable open state of the bottleneck. We propose a squeeze-in mechanism of the bottleneck passage, where dynamic thermal conformational fluctuations allow quinone to get in and out. Here, using molecular dynamics simulations of the bacterial enzyme, we have identified collective conformational changes that open the quinone chamber bottleneck. The model predicts a significant reduction-due to a need for a rare opening of the bottleneck-of the effective bi-molecular rate constant, in line with the available kinetic data. We discuss possible reasons for such a tight control of the quinone passage into the binding chamber and mechanistic consequences for the quinone two-electron reduction.
Asunto(s)
Complejo I de Transporte de Electrón , Ubiquinona , Sitios de Unión , Transporte de Electrón , Complejo I de Transporte de Electrón/metabolismo , Conformación Molecular , Oxidación-Reducción , Quinonas , Ubiquinona/metabolismoRESUMEN
The structure of the entire respiratory complex I is now known at reasonably high resolution for many species - bacteria, yeast, and several mammals, including human. The structure reveals an almost 30 angstrom tunnel-like chamber for ubiquinone binding in the core part of the enzyme, at the joint between the membrane and hydrophilic arms of the enzyme. Here we characterize the geometric bottleneck forming the entrance of the quinone reaction chamber. Computer simulations of quinone/quinol passage through the bottleneck suggest that in all structures available, from bacterial to human, this bottleneck is too narrow for the quinone or quinol to pass and that a conformational change is required to open the channel. Moreover, the bottleneck is too narrow even for isoprenoid tail free passage. The closed structure can be an artifact of the crystallization packing forces, low temperature, or other unnatural conditions occurring in the structural data acquisition procedure that affect this flexible part of the enzyme. Two of the helices forming the bottleneck are in direct contact with the subunit (ND3) that was recently demonstrated to be involved in conformational changes during the redox proton pumping cycle, which indicates flexibility of that part of the enzyme. We conclude that the published structures are all locked in the unfunctional states and do not represent correctly the functional enzyme; we discuss possible ways to open the structure in the context of possible mechanisms of the enzyme.
Asunto(s)
Proteínas Bacterianas/química , Benzoquinonas/química , Complejo I de Transporte de Electrón/química , Proteínas Fúngicas/química , Thermus thermophilus/enzimología , Yarrowia/enzimología , Animales , Proteínas Bacterianas/metabolismo , Benzoquinonas/metabolismo , Complejo I de Transporte de Electrón/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Ratones , Modelos Moleculares , Dominios Proteicos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , OvinosRESUMEN
The kinetics of the autoxidation reaction of tartaric acid in an air-saturated solution in the presence of Fe(II) show autocatalytic behavior with distinct initiation, propagation, and termination phases. The initiation phase, which involves activation of dissolved oxygen, decreases with increasing pH, over the test range of pH of 2.5-4.5, indicating that activation of oxygen is catalyzed by an Fe(II)-tartrate complex. The autocatalytic nature of this reaction indicates the presence of a catalytic intermediate that is produced during the initiation phase and regenerated during the propagation phase. The addition of catalase, as well as direct measurements, provided evidence of the presence and kinetic action of hydrogen peroxide as one of the intermediates. Direct addition of hydrogen peroxide resulted in shortening of the initiation stage and the propagation phase with similar rates as in the autoxidation reaction at low pH. The propagation is approximately a zero order reaction with respect to oxygen and iron. The kinetic analysis suggests that an intermediate catalytic complex(s) involving a ferryl ion (FeO2+) controls the rate of the propagation reaction. The Fe(III) formation shows autocatalytic behavior that mirrors the dissolved oxygen consumption patterns under all pH conditions studied. At pH values of 2.5 and 3.0, Fe(III) accumulated to a maximum, before it was partially consumed. This maximum coincided with the depletion of dissolved oxygen. The consumption of Fe(III), or the reduction of Fe(III) back to Fe(II), reflects the catalytic nature of Fe(II) and the essential role of tartaric acid in the initiation phase of Fenton's original reaction.
RESUMEN
Complexes I to IV, with the exception of Complex II, are redox-driven proton pumps that convert redox energy of oxygen reduction to proton gradient across the mitochondrial or bacterial membrane; in turn, the created electrochemical gradient drives the adenosine triphosphate synthesis in the cells by utilizing complex V of the chain. Here we address a general question of the efficiency of such enzymes, considering them as molecular machines that couple endergonic and exergonic reactions and converting one form of free energy into another. One well-known example of the efficiency is given by Carnot's theorem for heat engines. Here we extend the concept to respiratory enzymes and specifically focus on the proton pumping by Complex I of the respiratory chain, nicotinamide adenine dinucleotide dehydrogenase. To discuss the efficiency issues, we develop a model of enzyme kinetics, which generalizes the Michaelis-Menten model. Our model includes several substrates and products and, in general, can be considered as Generalized Michaelis-Menten Kinetic model. The model might be useful for describing complex enzyme kinetics, regardless of the efficiency issues that are addressed in this paper.
Asunto(s)
Complejo I de Transporte de Electrón/química , Complejo I de Transporte de Electrón/metabolismo , Modelos Químicos , Enzimas/química , Enzimas/metabolismo , Cinética , Modelos Moleculares , Oxidación-Reducción , ProtonesRESUMEN
Complex I (CI) is the first enzyme of the mitochondrial respiratory chain and couples the electron transfer with proton pumping. Mutations in genes encoding CI subunits can frequently cause inborn metabolic errors. We applied proteome and metabolome profiling of patient-derived cells harboring pathogenic mutations in two distinct CI genes to elucidate underlying pathomechanisms on the molecular level. Our results indicated that the electron transfer within CI was interrupted in both patients by different mechanisms. We showed that the biallelic mutations in NDUFS1 led to a decreased stability of the entire N-module of CI and disrupted the electron transfer between two iron-sulfur clusters. Strikingly interesting and in contrast to the proteome, metabolome profiling illustrated that the pattern of dysregulated metabolites was almost identical in both patients, such as the inhibitory feedback on the TCA cycle and altered glutathione levels, indicative for reactive oxygen species (ROS) stress. Our findings deciphered pathological mechanisms of CI deficiency to better understand inborn metabolic errors.
Asunto(s)
Transporte de Electrón/genética , Metabolismo Energético/genética , Errores Innatos del Metabolismo/genética , Metaboloma/genética , NADH Deshidrogenasa/genética , Estudios de Casos y Controles , Células Cultivadas , Reprogramación Celular/genética , Análisis Mutacional de ADN , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Errores Innatos del Metabolismo/metabolismo , Errores Innatos del Metabolismo/patología , Modelos Moleculares , NADH Deshidrogenasa/química , NADH Deshidrogenasa/metabolismo , Embarazo , Cultivo Primario de Células , Conformación Proteica , Proteoma/genéticaRESUMEN
Respiratory complex I catalyzes two-electron/two-proton reduction of a ubiquinone (Q) substrate bound at its Q-binding pocket; upon reduction, ubiquinole carries electrons further down the electron transport chain. The mechanism of this two-electron transfer reaction is poorly understood. Here we consider a hypothetical scheme in which two electrons transfer together with two protons in a concerted fashion. On one side, a coupled electron/proton transfer occurs from the reduced N2 FeS cluster and protonated His38 residue, respectively, while on the other side a hydrogen atom transfer occurs from the neutral Tyr87 residue, generating a tyrosyl radical. A method to evaluate the coupling matrix element that corresponds to a concerted tunneling of two electrons was developed. Overall, our calculations indicate that the concerted reaction is feasible, in which case a transient tyrosyl radical is formed during the catalytic cycle of the enzyme.
Asunto(s)
Complejo I de Transporte de Electrón/química , Ubiquinona/química , Sitios de Unión , Biocatálisis , Teoría Funcional de la Densidad , Transporte de Electrón , Complejo I de Transporte de Electrón/metabolismo , Electrones , Hidrógeno/química , Hidrógeno/metabolismo , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Oxidación-Reducción , Protones , Ubiquinona/metabolismoRESUMEN
In aerobic cells, the proton gradient that drives ATP synthesis is created by three different proton pumps-membrane enzymes of the respiratory electron transport chain known as complex I, III, and IV. Despite the striking dissimilarity of structures and apparent differences in molecular mechanisms of proton pumping, all three enzymes have much in common and employ the same universal physical principles of converting redox energy to proton pumping. In this study, we describe a simple mathematical model that illustrates the general principles of redox-driven proton pumps and discuss their implementation in complex I, III, and IV of the respiratory chain.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Modelos Biológicos , Bombas de Protones/metabolismo , Transporte de Electrón , TermodinámicaRESUMEN
We developed a unique integrated software package (called Electron Tunneling in Proteins Program or ETP) which provides an environment with different capabilities such as tunneling current calculation, semi-empirical quantum mechanical calculation, and molecular modeling simulation for calculation and analysis of electron transfer reactions in proteins. ETP program is developed as a cross-platform client-server program in which all the different calculations are conducted at the server side while only the client terminal displays the resulting calculation outputs in the different supported representations. ETP program is integrated with a set of well-known computational software packages including Gaussian, BALLVIEW, Dowser, pKip, and APBS. In addition, ETP program supports various visualization methods for the tunneling calculation results that assist in a more comprehensive understanding of the tunneling process. © 2016 Wiley Periodicals, Inc.
Asunto(s)
Electrones , Proteínas/química , Programas Informáticos , Transporte de Electrón , Modelos Moleculares , Teoría CuánticaRESUMEN
A new binding site and potential novel inhibitors of the respiratory complex III are described. The site is located at the opposite side of the enzyme with respect to ubiquinol binding site (Qo site), and distinctly different from both Qo and Qi sites (hence designated as Non-Q binding site, NQ). NQ site binding pocket extends up close to Phe90 residue, an internal switch (LH switch) that regulates electron transfer between heme bL and heme bH of the low potential redox chain. Docking studies and molecular dynamics simulations of different molecules to the NQ site revealed potential ligands which exhibit a novel inhibitory effect for bc1 complex by switching the LH switch to "off" conformation, thereby shutting down electron transfer in the low potential redox chain. Moreover, the novel inhibitors have lower binding affinity for both Qo and Qi sites, and hence do not interfere with binding of the natural ligands to those sites. The inhibitory activity of those novel ligands in bc1 complex is suggested to promote the production of reactive oxygen species (ROS) at the Qo site. Hence those ligands are potential candidates for designing new "mitocan" drugs.
Asunto(s)
Complejo III de Transporte de Electrones/antagonistas & inhibidores , Complejo III de Transporte de Electrones/química , Inhibidores Enzimáticos/farmacología , Sitios de Unión/efectos de los fármacos , Transporte de Electrón , Complejo III de Transporte de Electrones/metabolismo , Inhibidores Enzimáticos/química , Ligandos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica MolecularRESUMEN
Monte Carlo (MC) simulations of conformational changes and protonation of Glu-242, a key residue that shuttles protons in cytochrome c oxidase (CcO), are reported. Previous studies suggest that this residue may play a role of the valve of the enzyme proton pump. Here we examine how sensitive the results of simulations are to the computational method used. We applied both molecular mechanic (MM) and hybrid quantum mechanic:molecular mechanic (QM:MM) methods and find that the results are qualitatively different. The results indicate that the mechanism for proton gating in CcO is still an open issue.
Asunto(s)
Complejo IV de Transporte de Electrones/química , Ácido Glutámico/química , Enlace de Hidrógeno , Modelos Moleculares , Método de Montecarlo , Teoría CuánticaRESUMEN
In different X-ray crystal structures of bc1 complex, some of the key residues of electron tunneling pathways are observed in different conformations; here we examine their relative importance in modulating electron transfer and propose their possible gating function in the Q-cycle. The study includes inter-monomeric electron transfer; here we provide atomistic details of the reaction, and discuss the possible roles of inter-monomeric electronic communication in bc(1) complex. Binding of natural ligands or inhibitors leads to local conformational changes which propagate through protein and control the conformation of key residues involved in the electron tunneling pathways. Aromatic-aromatic interactions are highly utilized in the communication network since the key residues are aromatic in nature. The calculations show that there is a substantial change of the electron transfer rates between different redox pairs depending on the different conformations acquired by the key residues of the complex.
Asunto(s)
Citocromos c/química , Complejo III de Transporte de Electrones/química , Hemo/química , Conformación Proteica , Cristalografía por Rayos X , Citocromos c/metabolismo , Transporte de Electrón , Complejo III de Transporte de Electrones/genética , Complejo III de Transporte de Electrones/metabolismo , Electrones , Hemo/metabolismo , Modelos Químicos , Modelos Moleculares , Estructura Molecular , Mutación , Oxidación-Reducción , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Unión ProteicaRESUMEN
The most detailed and comprehensive to date study of electron transfer reactions in the respiratory complex III of aerobic cells, also known as bc1 complex, is reported. In the framework of the tunneling current theory, electron tunneling rates and atomistic tunneling pathways between different redox centers were investigated for all electron transfer reactions comprising different stages of the proton-motive Q-cycle. The calculations reveal that complex III is a smart nanomachine, which under certain conditions undergoes conformational changes gating electron transfer, or channeling electrons to specific pathways. One-electron tunneling approximation was adopted in the tunneling calculations, which were performed using hybrid Broken-Symmetry (BS) unrestricted DFT/ZINDO levels of theory. The tunneling orbitals were determined using an exact biorthogonalization scheme that uniquely separates pairs of tunneling orbitals with small overlaps out of the remaining Franck-Condon orbitals with significant overlap. Electron transfer rates in different redox pairs show exponential distance dependence, in agreement with the reported experimental data; some reactions involve coupled proton transfer. Proper treatment of a concerted two-electron bifurcated tunneling reaction at the Q(o) site is given.
Asunto(s)
Transporte de Electrón , Teoría Cuántica , Modelos MolecularesRESUMEN
The transition flux formula for the coupling matrix element of long-distance electron transfer reactions is discussed. Here we present a new derivation which is based on the Golden Rule approach. The electronic Franck-Condon factor that appears in the multielectronic formulation of the coupling element is discussed using the concept of tunneling time. An application of the tunneling flux theory to electron transfer reactions in a model system based on the low-potential heme and high-potential heme (heme bL)/(heme bH) redox pair of ubiquinol:cytochrome c oxidoreductase complex is described; the results are compared to those obtained by measuring energy splitting of the donor/acceptor multielectronic states and the direct calculation method.