RESUMO
Cytochrome bd-I from Escherichia coli belongs to the superfamily of prokaryotic bd-type oxygen reductases. It contains three hemes, b558, b595 and d, and couples oxidation of quinol by dioxygen with the generation of a proton-motive force. The enzyme exhibits resistance to various stressors and is considered as a target protein for next-generation antimicrobials. By using electronic absorption and MCD spectroscopy, this work shows that cyanide binds to heme d2+ in the isolated fully reduced cytochrome bd-I. Cyanide-induced difference absorption spectra display changes near the heme d2+ α-band, a minimum at 633 nm and a maximum around 600 nm, and a W-shaped response in the Soret region. Apparent dissociation constant (Kd) of the cyanide complex of heme d2+ is â¼0.052 M. Kinetics of cyanide binding is monophasic, indicating the presence of a single ligand binding site in the enzyme. Consistently, MCD data show that cyanide binds to heme d2+ but not to b5582+ or b5952+. This agrees with the published structural data that the enzyme's active site is not a di-heme site. The observed rate of binding (kobs) increases as the concentration of cyanide is increased, giving a second-order rate constant (kon) of â¼0.1 M-1 s-1.
Assuntos
Cianetos , Proteínas de Escherichia coli , Escherichia coli , Heme , Oxirredutases , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Escherichia coli/enzimologia , Cianetos/metabolismo , Cianetos/química , Heme/metabolismo , Heme/química , Oxirredutases/metabolismo , Oxirredutases/química , Oxirredução , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos b/química , Cinética , Citocromos/metabolismo , Citocromos/química , Sítios de Ligação , Ligação ProteicaRESUMO
The cytochrome bd oxygen reductase catalyzes the four-electron reduction of dioxygen to two water molecules. The structure of this enzyme reveals three heme molecules in the active site, which differs from that of heme-copper cytochrome c oxidase. The quantum chemical cluster approach was used to uncover the reaction mechanism of this intriguing metalloenzyme. The calculations suggested that a proton-coupled electron transfer reduction occurs first to generate a ferrous heme b595. This is followed by the dioxygen binding at the heme d center coupled with an outer-sphere electron transfer from the ferrous heme b595 to the dioxygen moiety, affording a ferric ion superoxide intermediate. A second proton-coupled electron transfer produces a heme d ferric hydroperoxide, which undergoes efficient O-O bond cleavage facilitated by an outer-sphere electron transfer from the ferrous heme b595 to the O-O σ* orbital and an inner-sphere proton transfer from the heme d hydroxyl group to the leaving hydroxide. The synergistic benefits of the two types of hemes rationalize the highly efficient oxygen reduction repertoire for the multi-heme-dependent cytochrome bd oxygen reductase family.
Assuntos
Proteínas de Escherichia coli , Oxirredutases , Oxirredutases/química , Oxigênio/química , Prótons , Elétrons , Grupo dos Citocromos b/metabolismo , Proteínas de Escherichia coli/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Citocromos/química , Oxirredução , Heme/química , FerroRESUMO
Cytochrome bd-I from Escherichia coli is a terminal oxidase in the respiratory chain that plays an important role under stress conditions. Cytochrome bd-I was thought to consist of the major subunits CydA and CydB plus the small CydX subunit. Recent high-resolution structures of cytochrome bd-I demonstrated the presence of an additional subunit, CydH/CydY (called CydH here), the function of which is unclear. In this report, we show that in the absence of CydH, cytochrome bd-I is catalytically active, can sustain bacterial growth and displays haem spectra and susceptibility for haem-binding inhibitors comparable to the wild-type enzyme. Removal of CydH did not elicit catalase activity of cytochrome bd-I in our experimental system. Taken together, in the absence of the CydH subunit cytochrome bd-I retained key enzymatic properties.
Assuntos
Proteínas de Escherichia coli , Escherichia coli , Grupo dos Citocromos b/genética , Grupo dos Citocromos b/química , Citocromos/genética , Citocromos/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , HemeRESUMO
Respiration is a core biological energy-converting process whose last steps are carried out by a chain of multisubunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate Tetrahymena thermophila (Tt). Using cryo-electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt complex I (Tt-CI) and Tt-CIII2 (Tt-SC I+III2) and a structure of Tt-CIV2. Tt-SC I+III2 (~2.3 megadaltons) is a curved assembly with structural and functional symmetry breaking. Tt-CIV2 is a ~2.7-megadalton dimer with more than 50 subunits per protomer, including mitochondrial carriers and a TIM83-TIM133-like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, thereby expanding our understanding of core metabolism.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons , Transporte de Elétrons , Membranas Mitocondriais , Tetrahymena thermophila , Microscopia Crioeletrônica , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Tetrahymena thermophila/metabolismoRESUMO
The last steps of respiration, a core energy-harvesting process, are carried out by a chain of multi-subunit complexes in the inner mitochondrial membrane. Several essential subunits of the respiratory complexes are RNA-edited in plants, frequently leading to changes in the encoded amino acids. While the impact of RNA editing is clear at the sequence and phenotypic levels, the underlying biochemical explanations for these effects have remained obscure. Here, we used the structures of plant respiratory complex I, complex III2 and complex IV to analyze the impact of the amino acid changes of RNA editing in terms of their location and biochemical features. Through specific examples, we demonstrate how the structural information can explain the phenotypes of RNA-editing mutants. This work shows how the structural perspective can bridge the gap between sequence and phenotype and provides a framework for the continued analysis of RNA-editing mutants in plant mitochondria and, by extension, in chloroplasts.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Edição de RNA , RNA de Plantas/química , RNA de Plantas/metabolismo , Modelos Moleculares , Mutação/genética , Proteínas de Plantas/química , Proteínas de Plantas/genéticaRESUMO
The mitochondrial oxidative phosphorylation system is central to cellular metabolism. It comprises five enzymatic complexes and two mobile electron carriers that work in a mitochondrial respiratory chain. By coupling the oxidation of reducing equivalents coming into mitochondria to the generation and subsequent dissipation of a proton gradient across the inner mitochondrial membrane, this electron transport chain drives the production of ATP, which is then used as a primary energy carrier in virtually all cellular processes. Minimal perturbations of the respiratory chain activity are linked to diseases; therefore, it is necessary to understand how these complexes are assembled and regulated and how they function. In this Review, we outline the latest assembly models for each individual complex, and we also highlight the recent discoveries indicating that the formation of larger assemblies, known as respiratory supercomplexes, originates from the association of the intermediates of individual complexes. We then discuss how recent cryo-electron microscopy structures have been key to answering open questions on the function of the electron transport chain in mitochondrial respiration and how supercomplexes and other factors, including metabolites, can regulate the activity of the single complexes. When relevant, we discuss how these mechanisms contribute to physiology and outline their deregulation in human diseases.
Assuntos
Mitocôndrias/metabolismo , Animais , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Humanos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Fosforilação OxidativaRESUMO
New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.
Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos d/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Oxirredutases/química , Conformação Proteica , Subunidades Proteicas , Vitamina K 2/análogos & derivados , Vitamina K 2/químicaRESUMO
Cytochromes bd are ubiquitous amongst prokaryotes including many human-pathogenic bacteria. Such complexes are targets for the development of antimicrobial drugs. However, an understanding of the relationship between the structure and functional mechanisms of these oxidases is incomplete. Here, we have determined the 2.8 Å structure of Mycobacterium smegmatis cytochrome bd by single-particle cryo-electron microscopy. This bd oxidase consists of two subunits CydA and CydB, that adopt a pseudo two-fold symmetrical arrangement. The structural topology of its Q-loop domain, whose function is to bind the substrate, quinol, is significantly different compared to the C-terminal region reported for cytochromes bd from Geobacillus thermodenitrificans (G. th) and Escherichia coli (E. coli). In addition, we have identified two potential oxygen access channels in the structure and shown that similar tunnels also exist in G. th and E. coli cytochromes bd. This study provides insights to develop a framework for the rational design of antituberculosis compounds that block the oxygen access channels of this oxidase.
Assuntos
Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica/métodos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Mycobacterium smegmatis/enzimologia , Oxirredutases/ultraestrutura , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Heme/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/genética , Oxirredutases/química , Oxirredutases/metabolismo , Oxigênio/metabolismo , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Especificidade por SubstratoRESUMO
Native electrophoresis is a powerful tool to analyze the mitochondrial electron transport chain complexes (Cx) I-V and their assembly into supercomplexes. Valuable information regarding the composition and bioenergetic regulation in physiological and pathological conditions can be obtained. This chapter compares different types of native electrophoresis to analyze mitochondrial supercomplexes.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel Bidimensional/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Immunoblotting/métodos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Animais , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Humanos , Proteínas Mitocondriais/químicaRESUMO
Numerous lines of evidence have shown that the interaction between the nuclear and mitochondrial genomes ensures the efficient functioning of the OXPHOS complexes, with substantial implications in bioenergetics, adaptation, and disease. Their interaction is a fascinating and complex trait of the eukaryotic cell that MitImpact explores with its third major release. MitImpact expands its collection of genomic, clinical, and functional annotations of all non-synonymous substitutions of the human mitochondrial genome with new information on putative Compensated Pathogenic Deviations and co-varying amino acid sites of the Respiratory Chain subunits. It further provides evidence of energetic and structural residue compensation by techniques of molecular dynamics simulation. MitImpact is freely accessible at http://mitimpact.css-mendel.it.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/química , Subunidades Proteicas/química , Software , Substituição de Aminoácidos , Animais , Cetáceos , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Ontologia Genética , Humanos , Internet , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Anotação de Sequência Molecular , Mutação , Fosforilação Oxidativa , Primatas , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RoedoresRESUMO
Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV2+) and anthraquinone sulfonate (AQS-) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Proteínas Fúngicas/química , Neurospora crassa/enzimologia , Nitrato Redutase/química , Benzil Viologênio/química , OxirreduçãoRESUMO
BACKGROUND: Mitochondrial succinate accumulation has been suggested as key event for ischemia reperfusion injury in mice. No specific data are however available on behavior of liver mitochondria during ex situ machine perfusion in clinical transplant models. METHODS: We investigated mitochondrial metabolism of isolated perfused rat livers before transplantation. Livers were exposed to warm and cold ischemia to simulate donation after circulatory death (DCD) and organ transport. Subsequently, livers were perfused with oxygenated Belzer-MPS for 1h, at hypothermic or normothermic conditions. Various experiments were performed with supplemented succinate and/or mitochondrial inhibitors. The perfusate, liver tissues, and isolated mitochondria were analyzed by mass-spectroscopy and fluorimetry. Additionally, rat DCD livers were transplanted after 1h hypothermic or normothermic oxygenated perfusion. In parallel, perfusate samples were analysed during HOPE-treatment of human DCD livers before transplantation. FINDINGS: Succinate exposure during rat liver perfusion triggered a dose-dependent release of mitochondrial Flavin-Mononucleotide (FMN) and NADH in perfusates under normothermic conditions. In contrast, perfusate FMN was 3-8 fold lower under hypothermic conditions, suggesting less mitochondrial injury during cold re-oxygenation compared to normothermic conditions. HOPE-treatment induced a mitochondrial reprogramming with uploading of the nucleotide pool and effective succinate metabolism. This resulted in a clear superiority after liver transplantation compared to normothermic perfusion. Finally, the degree of mitochondrial injury during HOPE of human DCD livers, quantified by perfusate FMN and NADH, was predictive for liver function. INTERPRETATION: Mitochondrial injury determines outcome of transplanted rodent and human livers. Hypothermic oxygenated perfusion improves mitochondrial function, and allows viability assessment of liver grafts before implantation. FUNDING: detailed information can be found in Acknowledgments.
Assuntos
Hipotermia Induzida , Transplante de Fígado , Fígado/metabolismo , Oxigênio/metabolismo , Perfusão , Traumatismo por Reperfusão/prevenção & controle , Condicionamento Pré-Transplante , Animais , Biomarcadores , Reprogramação Celular , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético , Humanos , Hipotermia Induzida/métodos , Fígado/cirurgia , Testes de Função Hepática , Transplante de Fígado/métodos , Mitocôndrias/metabolismo , Modelos Animais , NAD , NADP , Perfusão/métodos , Ratos , Relação Estrutura-Atividade , Temperatura , Condicionamento Pré-Transplante/métodosRESUMO
This review focuses on the electrochemical and spectroelectrochemical studies that gave insight into redox potentials of the four mitochondrial complexes and their homologues from bacterial respiratory chains using O2 as a terminal acceptor, thus providing crucial information about their reaction mechanism. Advantages and limitations of the use of the different techniques for the study of membrane proteins are presented. Electrocatalytic experiments are described that revealed specific features of the reaction with the substrates and inhibitors. An overview is given on the great variability of the redox and catalytic properties of the enzymes in different organisms that may be due to adaptation to the specific environments in which these enzymes function. The adaptation of the redox chain to the different types of quinone and substrates is analyzed, and future studies are discussed.
Assuntos
Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Cristalografia por Raios X , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , OxirreduçãoRESUMO
Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.
Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Alanina/genética , Sequência de Aminoácidos , Membrana Celular/metabolismo , Grupo dos Citocromos b/isolamento & purificação , Complexo de Proteínas da Cadeia de Transporte de Elétrons/isolamento & purificação , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/isolamento & purificação , Heme/metabolismo , Mutagênese/genética , Proteínas Mutantes/química , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Oxirredutases/isolamento & purificação , Consumo de Oxigênio , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Cytochrome bd-I oxidase is a terminal reductase of bacterial respiratory chains produced under low oxygen concentrations, oxidative stress, and during pathogenicity. While the bulk of the protein forms transmembrane helices, a periplasmic domain, the Q-loop, is expected to be involved in binding and oxidation of (ubi)quinol. According to the length of the Q-loop, bd oxidases are classified into the S (short)- and the L (long)-subfamilies. Here, we show that either shortening the Q-loop of the Escherichia coli oxidase from the L-subfamily or replacing it by one from the S-subfamily leads to the production of labile and inactive variants, indicating a role for the extended Q-loop in the stability of the enzyme.
Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Sequência de Aminoácidos , Grupo dos Citocromos b/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Estabilidade Enzimática/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Oxirredução , Oxirredutases/genética , Ubiquinona/análogos & derivados , Ubiquinona/química , Ubiquinona/metabolismoRESUMO
Respirasome, as a vital part of the oxidative phosphorylation system, undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons. Copious research has been carried out on this lynchpin of respiration. From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I1III2IV1, scientists have gradually uncovered the mysterious veil of the electron transport chain (ETC). With the discovery of the mammalian respiratory mega complex I2III2IV2, a new perspective emerges in the research field of the ETC. Behind these advances glitters the light of the revolution in both theory and technology. Here, we give a short review about how scientists 'see' the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.
Assuntos
Respiração Celular , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Animais , Microscopia Crioeletrônica , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Humanos , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Modelos Moleculares , Fosforilação Oxidativa , PrótonsRESUMO
Terminal oxidases of aerobic respiratory chains catalyze the transfer of electrons from the respiratory substrate, cytochrome c or quinol, to O2 with the formation of two H2O molecules. There are two known families of these membrane oxidoreductases: heme-copper oxidase superfamily and bd-type oxidase family (cytochromes bd) found in prokaryotes only. The redox reaction catalyzed by these enzymes is coupled to the generation of proton motive force used by the cell to synthesize ATP and to perform other useful work. Due to the presence of the proton pump, heme-copper oxidases create the membrane potential with a greater energy efficiency than cytochromes bd. The latter, however, play an important physiological role that enables bacteria, including pathogenic ones, to survive and reproduce under adverse environmental conditions. This review discusses the features of organization and molecular mechanisms of functioning of terminal oxidases from these two families in the light of recent experimental data.
Assuntos
Cobre/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Heme/química , Biocatálise , Transporte de Elétrons , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Escherichia coli/metabolismo , Geobacillus/metabolismo , Óxido Nítrico/química , Óxido Nítrico/metabolismo , OxirreduçãoRESUMO
Cytochrome bd oxidases are terminal reductases of bacterial and archaeal respiratory chains. The enzyme couples the oxidation of ubiquinol or menaquinol with the reduction of dioxygen to water, thus contributing to the generation of the protonmotive force. Here, we determine the structure of the Escherichia coli bd oxidase treated with the specific inhibitor aurachin by cryo-electron microscopy (cryo-EM). The major subunits CydA and CydB are related by a pseudo two fold symmetry. The heme b and d cofactors are found in CydA, while ubiquinone-8 is bound at the homologous positions in CydB to stabilize its structure. The architecture of the E. coli enzyme is highly similar to that of Geobacillus thermodenitrificans, however, the positions of heme b595 and d are interchanged, and a common oxygen channel is blocked by a fourth subunit and substituted by a more narrow, alternative channel. Thus, with the same overall fold, the homologous enzymes exhibit a different mechanism.
Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Oxirredutases/química , Oxirredutases/metabolismo , Homologia de Sequência de Aminoácidos , Grupo dos Citocromos b/ultraestrutura , Complexo de Proteínas da Cadeia de Transporte de Elétrons/ultraestrutura , Proteínas de Escherichia coli/ultraestrutura , Geobacillus/enzimologia , Heme/química , Heme/metabolismo , Modelos Moleculares , Oxirredutases/ultraestrutura , Oxigênio/metabolismo , Prótons , Especificidade por Substrato , Ubiquinona/química , Ubiquinona/metabolismo , ÁguaRESUMO
Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site.