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The mitochondrial electron transfer chain
... As previously revised in [12], it was Hatefi et al. [13] who first isolated from mitochondria four enzyme multi-subunit complexes that concur on the oxidation of NADH and succinate, namely NADH-Coenzyme Q reductase (Complex I, CI), succinate-Coenzyme Q reductase (Complex II, CII), ubiquinol-cytochrome c reductase (Complex III, CIII or cytochrome bc 1 Complex) and cytochrome c oxidase (Complex IV, CIV) [14]. ...
... The connection among these enzyme complexes is ensured by two mobile transporters of electrons, i.e., Coenzyme Q (CoQ, ubiquinone) and cytochrome c (cyt. c) [14]. The former is a lipophilic quinone incorporated in the lipid bilayer of the mtIM, while cyt. ...
... As we mentioned in the previous section, Hatefi et al. [13] accomplished the systematic resolution and reconstitution of four functional respiratory complexes from mitochondria, and proposed that the overall electron transfer from substrates to oxygen results from both intra-complex and inter-complex redox reactions: intra-complex electron transfer takes place in the "solid" state of redox components (e.g., flavins, FeS clusters, cytochromes) having fixed steric relation, whereas inter-complex electron transfer operates by rapid diffusion of the mobile components acting as co-substrates, i.e., CoQ and cyt. c [14]. In the following years, this proposal was confirmed by the kinetic analysis of Kröger and Klingenberg [25,26], leading Hackenbrock et al. [27] to postulate the Random Collision Model of Electron Transfer: ...
Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F1FO-ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F1FO-ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.
... The study of membranes was in its early stages. It should not be surprising that even the existence of the lipid bilayer was doubted by some researchers, since two models replaced it: the one for photosynthetic thylakoids (using lipoprotein subunits) proposed by Benson [43], and the other for mammalian mitochondria proposed by Green [44,45]. These models received special attention due to the recognition of the researchers who proposed them. ...
... Therefore, both Benson and Green were experts in highly specific and sophisticated membranes. The membranes of these organelles are not "typical"; they are in the upper range of protein-lipid ratios relative to PM [44,45]. The models of both authors are described below. ...
The plasma membrane (PM) has undergone important conceptual changes during the history of scientific research, although it is undoubtedly a cellular organelle that constitutes the first defining characteristic of cellular life. Throughout history, the contributions of countless scientists have been published, each one of them with an enriching contribution to the knowledge of the structure-location and function of each structural component of this organelle, as well as the interaction between these and other structures. The first published contributions on the plasmatic membrane were the transport through it followed by the description of the structure: lipid bilayer, associated proteins, carbohydrates bound to both macromolecules, association with the cytoskeleton and dynamics of these components.. The data obtained experimentally from each researcher were represented in graphic configurations, as a language that facilitates the understanding of cellular structures and processes. This paper presents a review of some of the concepts and models proposed about the plasma membrane, emphasizing the components, the structure, the interaction between them and the dynamics. The work is illustrated with resignified 3D diagrams to visualize the changes that occurred during the history of the study of this organelle. Schemes were redrawn in 3D from the original articles...
... Mitochondria produce the bulk of the energy used by almost all eukaryotic cells through oxidative phosphorylation (OXPHOS), which is composed of five membrane complexes (Complex I-V) [1,2]. Complex I-IV, defined as the electron transport chain [3][4][5], can pump protons from the mitochondrial matrix to the intermembrane space (IMS) and undertake the task of producing a proton concentration gradient [6,7]. ...
... The main function of mitochondria is to generate energy via the mitochondrial respiratory chain [2,3]. Therefore, we investigated whether the loss of Arabidopsis PHB3 affects the function of the mitochondrial respiratory complex. ...
Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits β and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.
... The ETC and the oxidative phosphorylation are constituted by a series of protein complexes whose normal function is to provide essential energy to the heart. The mitochondrial electron transport chain and the oxidative phosphorylation are the functional sequences of five major multisubunit complexes designated as complex I, complex II, ubiquinol-cytochrome c reductase (complex III), complex IV, and complex V; these enzyme complexes are connected by two mobile redox-active molecules, i.e., a lipophilic quinone, coenzyme Q (Co Q) or ubiquinone, and a hydrophilic heme protein, cytochrome c (cyt c) [115]. The ATP generation process in the heart requires an adequate supply of metabolites to complexes I and II from where electrons carried via Co Q to complex III and then via cyt c to complex IVand finally complex V to oxygen. ...
... Heart Fail Rev (2017) 22:[109][110][111][112][113][114][115][116][117][118][119][120][121] Content courtesy of Springer Nature, terms of use apply. Rights reserved. ...
The heart failure accounts for the highest mortality rate all over the world. The development of preventive therapeutic approaches is still in their infancy. Owing to the extremely high energy demand of the heart, the bioenergetics pathways need to respond efficiently based on substrate availability. The metabolic regulation of such heart bioenergetics is mediated by various rate limiting enzymes involved in energy metabolism. Although all the pertinent mechanisms are not clearly understood, the progressive decline in the activity of metabolic enzymes leading to diminished ATP production is known to cause progression of the heart failure. Therefore, metabolic therapy that can maintain the appropriate activities of metabolic enzymes can be a promising approach for the prevention and treatment of the heart failure. The flavonoids that constitute various human dietary ingredients also effectively offer a variety of health benefits. The flavonoids target a variety of metabolic enzymes and facilitate effective management of the equilibrium between production and utilization of energy in the heart. This review discusses the broad impact of metabolic enzymes in the heart functions and explains how the dysregulated enzyme activity causes the heart failure. In addition, the prospects of targeting dysregulated metabolic enzymes by developing flavonoid-based metabolic approaches are discussed.
... In addition, exogenously added CoQ was necessary to sustain CI activity in the purified I 1 III 2 SC (Letts et al., 2019), arguing against a tightly bound and segregated CoQ pool as a functional component of the SC. A large body of work from the late 1960s to the 1980s, resulted in the 'random collision model' to explain electron transfer between the respiratory chain complexes, and in the evidence that CoQ is present as an undifferentiated pool (Green and Tzagoloff, 1966;Kröger and Klingenberg, 1973a;Kröger and Klingenberg, 1973b;Hackenbrock et al., 1986;Chazotte and Hackenbrock, 1988). More recently, additional proof of the non-compartmentalized electronic routes from CI to CIII 2 and from complex II (CII) to CIII 2 , came from kinetic measurements in sub-mitochondrial particles. ...
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages compared with the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from Drosophila melanogaster tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenesis conditions, but not for the enhancement of respiratory chain catalysis.
... Therefore, changes in any of systems are associated with many diseased states. Moreover, mitochondrial ROS production is closely related to the mitochondrial ETC [36, 96,101,102]. ...
The advent of BCR-ABL tyrosine kinase inhibitors (TKIs) targeted therapy revolutionized the treatment of chronic myeloid leukemia (CML) patients. Mitochondria are the key organelles for the maintenance of myocardial tissue homeostasis. However, cardiotoxicity associated with BCR-ABL1 TKIs can directly or indirectly cause mitochondrial damage and dysfunction, playing a pivotal role in cardiomyocytes homeostatic system and putting the cancer survivors at higher risk. In this review, we summarize the cardiotoxicity caused by BCR-ABL1 TKIs and the underlying mechanisms, which contribute dominantly to the damage of mitochondrial structure and dysfunction: endoplasmic reticulum (ER) stress, mitochondrial stress, damage of myocardial cell mitochondrial respiratory chain, increased production of mitochondrial reactive oxygen species (ROS), and other kinases and other potential mechanisms of cardiotoxicity induced by BCR-ABL1 TKIs. Furthermore, detection and management of BCR-ABL1 TKIs will promote our rational use, and cardioprotection strategies based on mitochondria will improve our understanding of the cardiotoxicity from a mitochondrial perspective. Ultimately, we hope shed light on clinical decision-making. By integrate and learn from both research and practice, we will endeavor to minimize the mitochondria-mediated cardiotoxicity and reduce the adverse sequelae associated with BCR-ABL1 TKIs.
... In addition, exogenously added CoQ was necessary to sustain CI activity in the purified I 1 III 2 SC (3) , arguing against a tightly bound and segregated CoQ pool as a functional component of the SC. A large body of work from the late 1960's to the 1980's, resulted in the 'random collision model' to explain electron transfer between the respiratory chain complexes, and in the evidence that CoQ is present as an undifferentiated pool (19)- (23) . More recently, additional proof of the non-compartmentalized electronic routes from CI to CIII 2 and from complex II (CII) to CIII 2 , came from kinetic measurements in sub-mitochondrial particles. ...
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages to the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from insect tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenetic conditions, but not for the enhancement of respiratory chain catalysis.
... In addition, exogenously added CoQ was necessary to sustain CI activity in the purified I 1 III 2 SC (Letts et al., 2019), arguing against a tightly bound and segregated CoQ pool as a functional component of the SC. A large body of work from the late 1960s to the 1980s, resulted in the 'random collision model' to explain electron transfer between the respiratory chain complexes, and in the evidence that CoQ is present as an undifferentiated pool (Green and Tzagoloff, 1966;Kröger and Klingenberg, 1973a;Kröger and Klingenberg, 1973b;Hackenbrock et al., 1986;Chazotte and Hackenbrock, 1988). More recently, additional proof of the non-compartmentalized electronic routes from CI to CIII 2 and from complex II (CII) to CIII 2 , came from kinetic measurements in sub-mitochondrial particles. ...
Mammalian mitochondrial respiratory chain (MRC) complexes are able to associate into quaternary structures named supercomplexes (SCs), which normally coexist with non-bound individual complexes. The functional significance of SCs has not been fully clarified and the debate has been centered on whether or not they confer catalytic advantages to the non-bound individual complexes. Mitochondrial respiratory chain organization does not seem to be conserved in all organisms. In fact, and differently from mammalian species, mitochondria from insect tissues are characterized by low amounts of SCs, despite the high metabolic demands and MRC activity shown by these mitochondria. Here, we show that attenuating the biogenesis of individual respiratory chain complexes was accompanied by increased formation of stable SCs, which are missing in Drosophila melanogaster in physiological conditions. This phenomenon was not accompanied by an increase in mitochondrial respiratory activity. Therefore, we conclude that SC formation is necessary to stabilize the complexes in suboptimal biogenetic conditions, but not for the enhancement of respiratory chain catalysis.
... In this complex mechanism, mitochondria have a central role, representing the core of cellular respiration. Here, oxygen reacts with glucose to produce ATP (adenosine triphosphate), which serves as energy for the body [39]. In particular, oxygen molecules sit at the end of the electron transport chain, necessary for ATP production, as electron acceptors. ...
Hypoxia, even at non-lethal levels, is one of the most stressful events for all aerobic organisms as it significantly affects a wide spectrum of physiological functions and energy production. Aerobic organisms activate countless molecular responses directed to respond at cellular, tissue, organ, and whole-body levels to cope with oxygen shortage allowing survival, including enhanced neo-angiogenesis and systemic oxygen delivery. The benefits of hypoxia may be evoked without its detrimental consequences by exploiting the so-called normobaric oxygen paradox. The intermittent shift between hyperoxic-normoxic exposure, in addition to being safe and feasible, has been shown to enhance erythropoietin production and raise hemoglobin levels with numerous different potential applications in many fields of therapy as a new strategy for surgical preconditioning aimed at frail patients and prevention of postoperative anemia. This narrative review summarizes the physiological processes behind the proposed normobaric oxygen paradox, focusing on the latest scientific evidence and the potential applications for this strategy. Future possibilities for hyperoxic-normoxic exposure therapy include implementation as a synergistic strategy to improve a patient’s pre-surgical condition, a stimulating treatment in critically ill patients, preconditioning of athletes during physical preparation, and, in combination with surgery and conventional chemotherapy, to improve patients’ outcomes and quality of life.
... A solid model and the liquid model are two typical models for the organization of mitochondrial respiratory chain. The solid model (Figure 2A) assumes the complexes of the mitochondrial respiratory chain are anchored within a framework to ensure contact between each other with high catalytic activity [2,4]. However, with the purification of functional respiratory complexes and new understanding of the fluid nature of the mitochondrial energy transducing membrane, the liquid model ( Figure 2B) has gained acceptance [5,6]. ...
Mitochondrial oxidative phospho rylation, the center of cellular metabolism, is pivotal for the energy production in eukaryotes. Mitochondrial oxidative phosphorylation relies on the mitochondrial respiratory chain, which consists of four main enzyme complexes and two mobile electron carriers. Mitochondrial enzyme complexes also assemble into respiratory chain supercomplexes (SCs) through specific interactions. The SCs not only have respiratory functions but also improve the efficiency of electron transfer and reduce the production of reactive oxygen species (ROS). Impaired assembly of SCs is closely related to various diseases, especially neurodegenerative diseases. Therefore, SCs play important roles in improving the efficiency of the mitochondrial respiratory chain, as well as maintaining the homeostasis of cellular metabolism. Here, we review the structure, assembly, and functions of SCs, as well as the relationship between mitochondrial SCs and diseases.
... E The levels of intracellular ATP in shRNACs-1429 cells were significantly decreased compared to those in shRNA-NC cells (*P < 0.05) [14]. The electron transport chain is composed with four enzyme multi-subunit complexes, complex I (NADH-Coenzyme Q reductase), complex II (succinate-Coenzyme Q reductase), complex III (ubiquinol-cytochrome c reductase or cytochrome bc1 complex) and complex IV (cytochrome c oxidase) [15]. Complex I is the largest component of respiratory chain, transporting two electrons from NADH to reduce ubiquinone. ...
Background
Citrate Synthase ( Cs ) gene mutation (locus ahL4 ) has been found to play an important role in progressive hearing loss of A/J mice. HEI-OC1 cells have been widely used as an in vitro system to study cellular and molecular mechanisms related to hearing lose. We previously reported the increased apoptosis and the accumulation of reactive oxygen species in shRNA Cs -1429 cells, a Cs low-expressed cell model from HEI-OCI. The details of the mechanism of ROS production and apoptosis mediated by the abnormal expression of Cs needed to research furtherly.
Methods
iTRAQ proteomics was utilized to detect the differentially expressed proteins (DEPs) caused by low expression of Cs . The GO and KEGG pathways analysis were performed for annotation of the differentially expressed proteins. Protein–protein interaction network was constructed by STRING online database. Immunoblotting was utilized to confirm the protein levels of the the differentially expressed proteins.
Results
The differentially expressed proteins were significantly enriched in various signaling pathways mainly related to mitochondrial dysfunction diseases including Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, et al. Most noteworthy, the oxidative phosphorylation pathway was most significantly suppressed in the shRNA Cs -1429 cells,, in which a total of 10 differentially expressed proteins were enriched and were all downregulated by the abnormal expression of Cs. The downregulations of Ndufb5, Ndufv1 and Uqcrb were confirmed by immunoblotting. Meanwhile, the ATP levels of shRNA Cs -1429 cells were also reduced.
Conclusions
These results suggest that low level expression of Cs induces the inhibition of oxidative phosphorylation pathway, which is responsible for the high level production of reactive oxygen species and low level of ATP, leading to the apoptosis of cochlear cells. This study may provide new theories for understanding and therapy of progressive hearing loss.
... While the bioenergetic role of CoQ is clear, the structural organization of the respiratory chain and the mechanism by which electrons are transferred from reduced coenzymes to molecular oxygen are still a matter of debate. Following the observation that the concentration of ubiquinone was more than the concentration of the prosthetic groups in the redox enzymes, Green and Tzagoloff first introduced the idea of CoQ as a mobile electron carrier [38]. This hypothesis was supported by the pioneering work of Kroeger and Klingenberg [39], who derived a simple equation to describe the kinetic behavior of ubiquinone, concluding that all the quinones present in the inner mitochondrial membrane behave like a common pool and the rate of electron transfer depends only on the rate of input and output of electrons to the quinone's pool. ...
Coenzyme Q (CoQ) is a key component of the respiratory chain of all eukaryotic cells. Its function is closely related to mitochondrial respiration, where it acts as an electron transporter. However, the cellular functions of coenzyme Q are multiple: it is present in all cell membranes, limiting the toxic effect of free radicals, it is a component of LDL, it is involved in the aging process, and its deficiency is linked to several diseases. Recently, it has been proposed that coenzyme Q contributes to suppressing ferroptosis, a type of iron-dependent programmed cell death characterized by lipid peroxidation. In this review, we report the latest hypotheses and theories analyzing the multiple functions of coenzyme Q. The complete knowledge of the various cellular CoQ functions is essential to provide a rational basis for its possible therapeutic use, not only in diseases characterized by primary CoQ deficiency, but also in large number of diseases in which its secondary deficiency has been found.
... assembly process of respiratory complexes and supercomplexes. Atomic models of the electron transport chain components as inFigS 1,2: complexes I (Protein Data Bank (PDB) ID 6ZKC), II (PDB 1ZOY), III 2 (dimeric complex III; PDB ID 6Q9E), IV (PDB ID 5IY5) and V (PDB ID ...
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.
... The presence of soluble electron carriers in the electron transport system was proposed decades ago (Green and Tzagoloff, 1966). Ubiquinone transfers electrons from CI or CII to CIII, while cytochrome c (CytC) transfers electrons from CIII to CIV. ...
Mitochondrial respiratory complex subunits assemble in supercomplexes. Studies of supercomplexes have typically relied upon antibody-based quantification, often limited to a single subunit per respiratory complex. To provide a deeper insight into mitochondrial and supercomplex plasticity, we combine native electrophoresis and mass spectrometry to determine the supercomplexome of skeletal muscle from sedentary and exercise-trained mice. We quantify 422 mitochondrial proteins within 10 supercomplex bands in which we show the debated presence of complexes II and V. Exercise-induced mitochondrial biogenesis results in non-stoichiometric changes in subunits and incorporation into supercomplexes. We uncover the dynamics of supercomplex-related assembly proteins and mtDNA-encoded subunits after exercise. Furthermore, exercise affects the complexing of Lactb, an obesity-associated mitochondrial protein, and ubiquinone biosynthesis proteins. Knockdown of ubiquinone biosynthesis proteins leads to alterations in mitochondrial respiration. Our approach can be applied to broad biological systems. In this instance, comprehensively analyzing respiratory supercomplexes illuminates previously undetectable complexity in mitochondrial plasticity.
... The question of the organization of the respiratory chain has been debated since the pioneering studies aimed at identifying the molecular components and catalytic mechanisms [89,90]. Hackenbrock et al. (1986) described the "fluid" or "random collision model" of electron transfer, where each complex acts as an individual entity, CoQ and cytochrome c freely diffuse within the lipid bilayer, and electron transfer occurs during random and transient collision events [91]. ...
The mitochondrial respiratory chain encompasses four oligomeric enzymatic complexes (complex I, II, III and IV) which, together with the redox carrier ubiquinone and cytochrome c, catalyze electron transport coupled to proton extrusion from the inner membrane. The protonmotive force is utilized by complex V for ATP synthesis in the process of oxidative phosphorylation. Respiratory complexes are known to coexist in the membrane as single functional entities and as supramolecular aggregates or supercomplexes (SCs). Understanding the assembly features of SCs has relevant biomedical implications because defects in a single protein can derange the overall SC organization and compromise the energetic function, causing severe mitochondrial disorders. Here we describe in detail the main types of SCs, all characterized by the presence of complex III. We show that the genetic alterations that hinder the assembly of Complex III, not just the activity, cause a rearrangement of the architecture of the SC that can help to preserve a minimal energetic function. Finally, the major metabolic disturbances associated with severe SCs perturbation due to defective complex III are discussed along with interventions that may circumvent these deficiencies.
... In the classical model, mETC was described as the functional sequence of 4 multi-subunit enzymatic complexes, randomly distributed in the inner mitochondrial membrane, named NADHcoenzyme Q reductase (Complex I), succinate-coenzyme Q reductase (Complex II), ubiquinol-cytochrome c reductase (Complex III) and cytochrome c oxidase (Complex IV). In this model, the enzyme complexes are connected by two mobile electron carriers: coenzyme Q (CoQ) which is embedded in the inner membrane and takes electrons from complexes I or II to complex III, and cytochrome c (cyt c), located on the external surface of the inner membrane and carries electrons from complex III to complex IV [5]. This proposal was confirmed over the following years, leading to the postulation of the random collision model of electron transport by Hackenbrock [6]. ...
Electron transfer between respiratory complexes is an essential step for the efficiency of the mitochondrial oxidative phosphorylation. Until recently, it was stablished that ubiquinone and cytochrome c formed homogenous single pools in the inner mitochondrial membrane which were not influenced by the presence of respiratory supercomplexes. However, this idea was challenged by the fact that bottlenecks in electron transfer appeared after disruption of supercomplexes into their individual complexes. The postulation of the plasticity model embraced all these observations and concluded that complexes and supercomplexes co-exist and are dedicated to a spectrum of metabolic requirements. Here, we review the involvement of superassembly in complex I stability, the role of supercomplexes in ROS production and the segmentation of the CoQ and cyt c pools, together with their involvement in signaling and disease. Taking apparently conflicting literature we have built up a comprehensive model for the segmentation of CoQ and cyt c mediated by supercomplexes, discuss the current limitations and provide a prospect of the current knowledge in the field.
... In the cell respiratory system, for example, nicotinamide adenine dinucleotide hydrogen (NADH) shuttles electrons through the membrane, and three distinct complexes are involved in the electron transport chain in the mitochondria before reducing oxygen to water. 12,13 In photosynthesis, different quinones play similar roles between the reactive complexes involved. 14 In part, inspired by such systems, several chemical and electrochemical energy conversion or storage systems rely or are improved by the use of RMs (i.e., artificial photosynthesis, organic dye-sensitized solar cells, pseudocapacitors, and redox flow, lithium−sulfur, and metal−oxygen batteries). ...
The spin-spin interactions between unpaired electrons in organic (poly)radicals, especially nitroxides, are of largely inves-tigated and are of crucial importance for their applications in areas such as organic magnetism, molecular charge transfer or multiple spin labeling in structural biology. Recently, TEMPO and polymers functionalized with nitroxides have been described as successful redox mediators, however, the study of spin-spin interactions effect in such an area is absent. This communication deals with the preparation and study of a novel family of discrete polynitroxide molecules, with the same number of radical units but different arrangement to study how intramolecular spin-spin interactions affect on their electrochemical potential and their use as redox mediators.
... During the last 30 years, the structural organization of the respiratory complexes was intended to be understood in terms of two utmost paradigms: the "fluid" and the "solid" models. The "fluid" model postulates that all redox components are unconstrained diffusible particles with the electron carriers alternating in the middle of the gigantic complexes I-IV (1,2). This interpretation suggests that mitochondrial electron transfer is a diffusion-based stochastic collision process and that diffusion has an essential and regulating effect on electron transfer (3). ...
Several studies suggest that the assembly of mitochondrial respiratory complexes into structures known as supercomplexes (SCs) may increase the efficiency of the electron transport chain, reducing the rate of production of reactive oxygen species. Therefore, the study of the (dis)assembly of SCs may be relevant for the understanding of mitochondrial dysfunction reported in brain aging and major neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Here we briefly reviewed the biogenesis and structural properties of SCs, the impact of mtDNA mutations and mitochondrial dynamics on SCs assembly, the role of lipids on stabilization of SCs and the methodological limitations for the study of SCs. More specifically, we summarized what is known about mitochondrial dysfunction and SCs organization and activity in aging, AD and PD. We focused on the critical variables to take into account when postmortem tissues are used to study the (dis)assembly of SCs. Since few works have been performed to study SCs in AD and PD, the impact of SCs dysfunction on the alteration of brain energetics in these diseases remains poorly understood. The convergence of future progress in the study of SCs structure at high resolution and the refinement of animal models of AD and PD, as well as the use of iPSC-based and somatic cell-derived neurons, will be critical in understanding the biological relevance of the structural remodeling of SCs.
... Mitochondrial ATP synthesis is chemiosmotically coupled to the electron transfer chain (ETC), which is located in the mitochondrial inner membrane (MITCHELL 1966). The ETC involves four major membrane-bound complexes that mediate electron transfer from the substrates, NADH or succinate, to the terminal electron acceptor O2 (GREEN AND TZAGOLOFF 1966). ...
Complex I is the first enzyme involved in the mitochondrial electron transport chain. With more than 40 subunits of dual genetic origin, the biogenesis of complex I is highly intricate and poorly understood. We used Chlamydomonas reinhardtii as a model system to reveal factors involved in complex I biogenesis. Two insertional mutants, displaying a complex I assembly defect characterized by the accumulation of a 700 kDa subcomplex, were analyzed. Genetic analyses showed these mutations were allelic, with insertional mutations in the gene AMC1 ( Cre16.g688900 ) , encoding a low-complexity protein of unknown function. The complex I assembly and activity in the mutant was restored by complementation with the wild-type gene, confirming AMC1 is required for complex I biogenesis. The N-terminus of AMC1 targets a reporter protein to yeast mitochondria, implying that AMC1 resides and functions in the Chlamydomonas mitochondria. Accordingly, in both mutants, loss of AMC1 function results in decreased abundance of the mitochondrial nd4 transcript, which encodes the ND4 membrane subunit of complex I. Loss of ND4 in a mitochondrial nd4 mutant is characterized by a membrane arm assembly defect, similar to that exhibited by loss of AMC1. These results suggest AMC1 is required for the production of mitochondrially-encoded complex I subunits, specifically ND4. We discuss the possible modes of action of AMC1 in mitochondrial gene expression and complex I biogenesis .
... OXPHOS in mammals is carried out by five classes of protein complexes anchored on the inner membrane of mitochondria. These complexes are relatively independent in both structure and function, including complex I (NADH: ubiquinone oxidoreductase, CI), complex II (succinate:ubiquinone oxidoreductase, CII), complex III (cytochrome bc 1 complex, CIII), complex IV (cytochrome c oxidase, CIV), and complex V (ATP synthase, CV) (Wharton and Tzagoloff, 1962;Green and Tzagoloff, 1966;Hatefi, 1985;Papa et al., 2012;Zong et al., 2018a;Zong et al., 2018b;Gu et al., 2019). Complex I-IV are also called respiratory chain complexes, or electron transport chain (ETC) complexes, because they only participate in the process of electron transportation and oxygen consumption. ...
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.
... They are located at the interface between the environmental calorie supply and the energetic needs of cells in both normal and stressful conditions. In response to energy demands, various substrates are metabolized via several metabolic pathways to ultimately drive mitochondrial ATP synthesis by oxidative phosphorylation (Green and Tzagoloff, 1966). Mitochondrial energy metabolism is also linked to the generation of reactive oxygen species (ROS) as normal by-products. ...
Weaning is known to induce important nutritional and energetic stress in piglets. Low-birthweight ( LBW ) piglets, now frequently observed in swine production, are more likely to be affected. The weaning period is also associated with dysfunctional immune responses, uncontrolled inflammation and oxidative stress conditions that are recognized risk factors for infections and diseases. Mounting evidence indicates that mitochondria, the main cellular sources of energy in the form of adenosine 5′ triphosphate (ATP) and primary sites of reactive oxygen species production, are related to immunity, inflammation and bacterial pathogenesis. However, no information is currently available regarding the link between mitochondrial energy production and oxidative stress in weaned piglets. The objective of this study was to characterize markers of cellular and mitochondrial energy metabolism and oxidative status in both normal-birthweight ( NBW ) and LBW piglets throughout the peri-weaning period. To conduct the study, 30 multiparous sows were inseminated and litters were standardized to 12 piglets. All the piglets were weighted at day 1 and 120 piglets were selected and assigned to 1 of 2 experimental groups: NBW ( n = 60, mean weight of 1.73 ± 0.01 kg) and LBW piglets weighing less than 1.2 kg ( n = 60, 1.01 ± 0.01 kg). Then, 10 piglets from each group were selected at 14, 21 (weaning), 23, 25, 29 and 35 days of age to collect plasma and organ (liver, intestine and kidney) samples. Analysis revealed that ATP concentrations were lower in liver of piglets after weaning than during lactation ( P < 0.05) thus suggesting a significant impact of weaning stress on mitochondrial energy production. Oxidative damage to DNA (8-hydroxy-2′-deoxyguanosine, 8-OHdG ) and proteins (carbonyls) measured in plasma increased after weaning and this coincides with a rise in enzymatic antioxidant activity of glutathione peroxidase ( GPx ) and superoxide dismutase ( SOD ) ( P < 0.05). Mitochondrial activities of both GPx and SOD are also significantly higher ( P < 0.05) in kidney of piglets after weaning. Additionally, oxidative damage to macromolecules is more important in LBW piglets as measured concentrations of 8-OHdG and protein carbonyls are significantly higher ( P < 0.05) in plasma and liver samples, respectively, than for NBW piglets. These results provide novel information about the nature, intensity and duration of weaning stress by revealing that weaning induces mitochondrial dysfunction and cellular oxidative stress conditions which last for at least 2 weeks and more severely impact smaller piglets.
... Furthermore, abnormal mitochondria were observed, by electron microscopy in quadriceps of Coq8a -/mice, at late stages . As CoQ is required for electron transport from complex I and II to complex III in the respiratory chain (Green and Tzagoloff, 1966), we hypothesized that the mitochondrial respiration might be affected in Coq8a -/mice, in particular in cells with a high energy demand, like neurons and muscle cells. If mitochondrial respiration is altered, we speculate that this phenotype should arise early on, since low levels of COQ7 were observed in embryos and in 2,5 weeks old Coq8a -/mice. ...
ARCA2, a rare form of recessive ataxia, is characterized by early onset progressive ataxia, cerebellar atrophy and a mild Coenzyme Q10 deficiency. Most of the patients show additional neurological signs such as epilepsy and exercise intolerance. Mutations in the COQ8A gene lead to ARCA2. COQ8A is suggested as being an unorthodox protein kinase like, with a regulatory role in CoQ biosynthesis, in mammals. To better understand ARCA2, a constitutive Coq8a knock-out (KO) mouse model was generated, which recapitulates most of the patient’s symptoms. Here we report the use of cellular models and the affected tissues to uncover the molecular signature of COQ8A loss and CoQ deficit. Despite CoQ deficit in the muscle, no mitochondrial bioenergetics defect was uncovered. In parallel, we have identified, by RT-qPCR, a key set of genes that are dysregulated in cerebellum, very early on in the pathology. We are currently investigating these pathways to uncover the link with COQ8A function. Altogether, our experiments will shed light on the early molecular events that lead to ARCA2 and may help draw a link between COQ8A function, CoQ pools and the symptoms observed in patients.
... 48 The CoQ 10 modulates the permeability transition pore (PTP), a mitochondrial inner membrane conductance channel, being a potential mitochondrial inhibitor of apoptotic signal transduction. 49 Mencucci et al. 47 evaluated the potential protective effects of CoQ 10 at different concentrations, on human corneal cells (HCE) where the mitochondrial activity and survival were evaluated by means of 3-(4,5-dimethylthiazole-2-yl)2,5diphenyl-tetrazolium (MTT) reduction, and lactic dehydrogenase (LDH) release. Oxygen consumption and mitochondrial membrane potential were also evaluated. ...
The restoration of the skin barrier in acute and chronic wounds is controlled by several molecular mechanisms that synergistically regulate cell kinetics, enzymatic functions, and neurovascular activation. These pathways include genetic and epigenetic activation, which modulate physiological wound healing. Our review describes the genetic background of skin repair, namely transcription-independent diffusible damage signals, individual variability, epigenetic mechanism, controlled qualitative traits, post-translational mechanisms, antioxidants, nutrients, DNA modifications, bacteria activation, mitochondrial activity, and oxidative stress. The DNA background modulating skin restoration could be used to plan new diagnostics and therapeutics.
... Under physiological conditions, the mitochondria are maintained operative by a resident mitochondrial DNA in synergy with nuclear DNA, both regulating fusion and fission and mitophagy dynamics of the organelles [26] and, indeed, the expression and activity of the respiratory chain electron transfer (eT) complexes. These complexes, at the level of the inner mitochondrial membrane, either collide among themselves randomly [27], the hypothesis later on reconsidered by [28], or are organised in supramolecular structures of the individual complexes [29]. Relevant to cell bioenergetics, the structural stability of the supercomplexes and the functional performance of the respiratory chain both have been shown to be modulated by the mitochondrial membrane potential [30] and the protein complexes phosphorylation. ...
Cocaine abuse has long been known to cause morbidity and mortality due to its cardiovascular toxic effects. The pathogenesis of the cardiovascular toxicity of cocaine use has been largely reviewed, and the most recent data indicate a fundamental role of oxidative stress in cocaine-induced cardiovascular toxicity, indicating that mitochondrial dysfunction is involved in the mechanisms of oxidative stress. The comprehension of the mechanisms involving mitochondrial dysfunction could help in selecting the most appropriate mitochondria injury biological marker, such as superoxide dismutase-2 activity and glutathionylated hemoglobin. The potential use of modulators of oxidative stress (mitoubiquinone, the short-chain quinone idebenone, and allopurinol) in the treatment of cocaine cardiotoxic effects is also suggested to promote further investigations on these potential mitochondria-targeted antioxidant strategies.
... OXPHOS is adapted to hypoxia by remodeling the electron transport chain (ETC) as well as the activity of the TCA cycle. The mitochondrial respiratory chain was originally described as flavinand cytochromecontaining proteins in the inner mitochondrial matrix [1]. This model proposed the four major complexes, i.e. ...
Hypoxia triggers several mechanisms to adapt cells to a low oxygen environment. Mitochondria are major consumers of oxygen and a potential source of reactive oxygen species (ROS). In response to hypoxia they exchange or modify distinct subunits of the respiratory chain and adjust their metabolism, especially lowering the citric acid cycle. Intermediates of the citric acid cycle participate in regulating hypoxia inducible factors (HIF), the key mediators of adaptation to hypoxia. Here we summarize how hypoxia conditions mitochondria with consequences for ROS-production and the HIF-pathway.
... Totally 4 functional modules were purified and reconstructed by Hatefi et al., till 1962, termed CI-CIV. (Hatefi et al., 1962 From that time on, work by Green, Tzagoloff and Hackenbrock in the subsequent twenty years established the fluid model of the IMM organization, (Green and Tzagoloff 1966;Hochli and Hackenbrock 1976) where all redox components are independent diffusible particles with the small electron carriers shuttling between the huge respiratory complexes I-IV, hence electron transport is considered a multicollisional, obstructed and longrange diffusional process (Hackenbrock et al., 1986) Due to lack of structural information, the mechanism of protonpumping and electron-transfer within these complexes were largely unknown back then (Fig. 1). ...
Respirasome, a huge molecular machine that carries out cellular respiration, has gained growing attention since its discovery, because respiration is the most indispensable biological process in almost all living creatures. The concept of respirasome has renewed our understanding of the respiratory chain organization, and most recently, the structure of respirasome solved by Yang’s group from Tsinghua University (Gu et al. Nature 237(7622):639–643, 2016) firstly presented the detailed interactions within this huge molecular machine, and provided important information for drug design and screening. However, the study of cellular respiration went through a long history. Here, we briefly showed the detoured history of respiratory chain investigation, and then described the amazing structure of respirasome.
Hypoxia and ischemia damage sensitive organelles such as mitochondria, and mitochondrial dysfunction contributes to metabolic disorders in crustaceans under hypoxia. The mechanisms associated with ferroptosis in hypoxic disorders have not been determined in crustaceans. In particular, the early molecular events of mitochondrial dynamics in crustaceans require clarification. In this study, two evolutionarily conserved mitochondrial fission proteins, Drp1 and MTP18, were identified in oriental river prawn (Macrobrachium nipponense). In vitro, ferroptosis-mediated impairment of mitochondrial membrane potential was induced by hypoxia in oriental river prawn hemocytes. In hypoxia-induced hemocytes, activation of Drp1 by increased phosphorylation at S616 was identified. Drp1 mitochondrial translocation also increased, and mitochondrial fusion-related protein expression decreased in vivo. Altered mitochondrial fission-fusion dynamics have been linked to mitochondrial dysfunction, inducing a classic ferroptosis mechanism. Marf overexpression or Drp1 knockdown protected against mitochondrial dysfunction and ferroptotic cell death in vitro. Furthermore, hypoxia-induced mitochondrial fission was verified to be driven by Drp1/MTP18 interaction. Under hypoxia, MTP18 transcription was increased by the binding of activated HIF-1α to hypoxia response elements in its promoter. Conjointly, MTP18 knockdown resulted in less apoptosis and decreased prawn mortality in gill tissue in vitro, suggesting that adaptation to hypoxia involves a vital function by MTP18. In conclusion, we uncovered a conserved role of mitochondrial fission in hypoxia-induced ferroptotic cell death. Therefore, we suggest that specific modulation of MTP18/DRP1-mediated mitochondrial dynamics might be a potential therapeutic strategy in hypoxic stress-induced tissue injury in invertebrates.
Emerging challenges in marine environments include nanoplastics (NPs) pollution and coastal hypoxia. Although NPs toxicity in marine organisms is being increasingly documented, the complex interactions between coastal hypoxia and NPs remain largely unexplored. This study investigated the dual effects of polystyrene nanoplastics and different oxygen levels on redox homeostasis and bioenergetics in the marine model organism Mytilus galloprovincialis. Both NPs and hypoxia significantly disrupted redox homeostasis in mussels. Exposure to NPs alone increased electron transport chain activity, whereas exposure to hypoxia alone and co-exposure significantly reduced this activity. Metabolomic analysis showed that NPs primarily affected the pentose phosphate pathway (PPP), tricarboxylic acid (TCA) cycle, and amino acid metabolism; hypoxia exposure alone disrupted the TCA cycle, pyruvate metabolism, and glycolysis/gluconeogenesis, whereas combined exposure notably altered the TCA cycle, PPP, and sugar interconversion. This suggests that regulating these pathways would help mussels cope with the combined environmental stress. Furthermore, co-exposure severely disrupted redox homeostasis and energy metabolism in mussels, suggesting that hypoxia exacerbates NPs toxicity. We believe that these new findings would enhance our understanding of the compounded ecological risks posed by NPs in the context of climate change.
Men taking antioxidant vitamin E supplements have increased prostate cancer (PC) risk. However, whether pro-oxidants protect from PC remained unclear. In this work, we show that a pro-oxidant vitamin K precursor [menadione sodium bisulfite (MSB)] suppresses PC progression in mice, killing cells through an oxidative cell death: MSB antagonizes the essential class III phosphatidylinositol (PI) 3-kinase VPS34—the regulator of endosome identity and sorting—through oxidation of key cysteines, pointing to a redox checkpoint in sorting. Testing MSB in a myotubular myopathy model that is driven by loss of MTM1 —the phosphatase antagonist of VPS34—we show that dietary MSB improved muscle histology and function and extended life span. These findings enhance our understanding of pro-oxidant selectivity and show how definition of the pathways they impinge on can give rise to unexpected therapeutic opportunities.
RNA splicing dysregulation and the involvement of specific splicing factors are emerging as common factors in both obesity and metabolic disorders. The study provides compelling evidence that the absence of the splicing factor SRSF1 in mature adipocytes results in whitening of brown adipocyte tissue (BAT) and impaired thermogenesis, along with the inhibition of white adipose tissue browning in mice. Combining single‐nucleus RNA sequencing with transmission electron microscopy, it is observed that the transformation of BAT cell types is associated with dysfunctional mitochondria, and SRSF1 deficiency leads to degenerated and fragmented mitochondria within BAT. The results demonstrate that SRSF1 effectively binds to constitutive exon 6 of Ndufs3 pre‐mRNA and promotes its inclusion. Conversely, the deficiency of SRSF1 results in impaired splicing of Ndufs3, leading to reduced levels of functional proteins that are essential for mitochondrial complex I assembly and activity. Consequently, this deficiency disrupts mitochondrial integrity, ultimately compromising the thermogenic capacity of BAT. These findings illuminate a novel role for SRSF1 in influencing mitochondrial function and BAT thermogenesis through its regulation of Ndufs3 splicing within BAT.
This book describes the events of primary energy transduction in life processes. Life as we know it depends on pumping protons across membranes. New tools to study the protein complexes involved has led to recent intensified progress in the field.
Primary Energy Transduction in Biology focusses on recent structural results and new biophysical insights. These have been made possible by recent advances in high-resolution protein structures, in physical techniques to study reactions in real time, and in computational methods to study and refine both structures and their dynamics. Written and edited by leading experts, chapters discuss the latest key questions in cell respiration, photosynthesis, bioenergetics, proton transfer, electron transfer and membrane transport.
Biochemists, biophysicists and chemical biologists will find this book an essential resource for a complete understanding of the molecular machines of bioenergetics.
Energy-converting NADH:ubiquionone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism. It couples NADH oxidation and quinone reduction with proton translocation across the membrane, thus contributing to the formation of the proton motive force. Complex I deficiencies were found to be the most common source of human mitochondrial dysfunction, manifesting in a variety of neurodegenerative diseases. Seven core subunits of human complex I are encoded by mitochondrial DNA (mtDNA) that carry an unexpectedly large number of mutations in patient tissues. The biochemical consequences of these mutations are largely unknown due to the difficulty of experimental access to mitochondrial complex I. To understand the effects of the mutations, they should be introduced into complex I of Escherichia coli as a model system. It consists of 13 subunits, named NuoA to NuoN, and contains nine iron-sulfur clusters and one flavin mononucleotide as cofactors.
Mutations found in patients’ mtDNA were introduced into E. coli complex I and the resulting variants were biochemically characterized. Mutation V259AL disturbed the assembly of complex I leading to an inactive variant, whereas mutation F123LL caused the assembly of a stable but almost inactive complex. Inversion of seven base pairs in mtDNA resulted in a triple mutation. In E. coli the homologous mutation D213G/Q214K/P215VH led to the assembly of a stable but completely inactive variant. The biochemical effects on complex I caused by these mutations might contribute to their deleterious effects in humans. Only Position D213GH within the triple mutation is conserved. The single Mutation D213GH resulted in the assembly of a stable variant as well, but with the capability to catalyze redox-driven proton translocation, albeit at reduced levels. UV/vis-spectroscopic re-oxidation kinetics of the variant demonstrated that the cause of the diminished activity is the impaired formation of a catalytic intermediate of the energy converting process in the complex. From these data, a mechanism for energy coupling, which has not yet been fully elucidated, was proposed.
Via MD simulations and structural studies of complex I in different states of the catalytic cycle, key positions for several postulated mechanisms of proton translocation were proposed. The introduction of the mutations H322AM, H348AM, and H322A/H348AM confirmed a mechanistic proposal for their role in catalysis. Mutation E407KM was used to generate a symmetrical distribution of the central charged amino acids. The variant catalyzed redox-driven proton translocation, albeit with lower stoichiometry. This contradicts the hypothesis that charge asymmetry is essential for proton translocation and revealed the so-called "ND5-only" mechanism to be unlikely. The results from these four mutations in NuoM support the mechanistic proposal of a model in which dipole changes propagate in a wave-like manner from one end of the membrane arm to the other and back again, thus initiating the translocation of a proton via each of the four postulated proton channels.
Further point-mutations were introduced into subunits NuoA, H, J, K and CD to investigate the postulated functions of the respective amino acids. Possible clinically relevant mutations in these positions were introduced as well. The pathogenicity of the mutations could be explained via their drastic effects on E. coli complex I. First characterization of the additional variants helped to improve the understanding of the mechanistic processes at the interface between the peripheral arm and the membrane arm.
Previous work by our group described an interaction of complex I variant ΔNuoL with a monomer of LdcI, encoded by the gene cadA. The role of the LdcI in complex I assembly could not be clarified by characterization of complex I nor the ΔNuoL variant from different cadA-deletion strains and remains unclear. To identify the LdcI binding site on complex I by cryo-EM, the ΔNuoL variant with associated LdcI was purified. However, electron microscopic examination of negative-stained single particles could not identify the binding side. The Attempt to improve protein quality resulted in the loss of complex I-LdcI interaction.
A chaperone-like function for complex I and possible involvement in a repair mechanism associated with LdcI and the ΔNuoL variant were proposed for the proteins RavA and ViaA from E. coli. Characterization of complex I and the ΔNuoL variant from different ravA-viaA-deletion strains showed no specific effect of the deletions on the assembly or activity of the enzyme.
In this part, the research to develop novel RM for oxygen reduction reaction (ORR) capable of promoting the solution-phase formation of lithium peroxide (Li2O2) will be introduced. The new RM is derived from mimicking the biological redox mediation in cell respiration system, where vitamin K2 mediates the electron transfer from flavin mononucleotide to cytochrome b in the cell membrane. The redox potential of vitamin K2 is shown to satisfy the suitable ORR potential range in aprotic solvent, thereby enabling its functioning as a RM promoting the solution-based discharge. The use of vitamin K2 suppresses the growth of film-like Li2O2 even in ether electrolytes, which have been reported to drive surface-based discharge and early passivation of the electrode, thereby boosting the discharge capacity by ~30 times. The similarity of the redox mediation in the biological cell respiration and lithium–oxygen ‘cell’ inspires the exploration of redox active bio-organic molecules for high-performance RMs for lithium–oxygen batteries.
Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.
In this chapter we provide a review with a focus on the function of Coenzyme Q (CoQ, ubiquinone) in mitochondria. The notion of a mobile pool of CoQ in the lipid bilayer as the vehicle of electrons from respiratory complexes has somewhat changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising Complexes I and III; in such assembly the electron transfer is thought to be mediated by direct channelling, and we provide evidence for a kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound CoQ, besides being required for electron transfer from other dehydrogenases to Complex III. CoQ bound to Complex I and to Complex III is also involved in proton translocation; although the mechanism of the Q-cycle is well established for Complex III, the involvement of CoQ in proton translocation by Complex I is still debated. This review also briefly examines some additional roles of CoQ, such as the antioxidant effect of its reduced form and its postulated action at the transcriptional level.
Mitochondrial complex I, a proton-pumping NADH: ubiquinone oxidoreductase, is required for oxidative phosphorylation. However, the contribution of several human mutations to complex I deficiency is poorly understood. The unicellular alga Chlamydomonas reinhardtii was utilized to study complex I as, unlike in mammals, mutants with complete loss of the holoenzyme are viable. From a forward genetic screen for complex I-deficient insertional mutants, six mutants exhibiting complex I deficiency with assembly defects were isolated. Chlamydomonas mutants isolated from our screens, lacking the subunits NDUFV2 and NDUFB10, were used to reconstruct and analyze the effect of two human mutations in these subunit-encoding genes. The K209R substitution in NDUFV2, reported in Parkinson's disease patients, did not significantly affect the enzyme activity or assembly. The C107S substitution in the NDUFB10 subunit, reported in a case of fatal infantile cardiomyopathy, is part of a conserved C-(X)11-C motif. The cysteine substitutions, at either one or both positions, still allowed low levels of holoenzyme formation, indicating that this motif is crucial for complex I function but not strictly essential for assembly. We show that the algal mutants provide a simple and useful platform to delineate the consequences of patient mutations on complex I function.
Governing the fundamental reaction in lithium–oxygen batteries is vital to realizing their potentially high energy density. Here, novel oxygen reduction reaction (ORR) catalysts capable of mediating the lithium and oxygen reaction within a solution‐driven discharge, which promotes the solution‐phase formation of lithium peroxide (Li2O2), are reported, thus enhancing the discharge capacity. The new catalysts are derived from mimicking the biological redox mediation in the electron transport chain in Escherichia coli, where vitamin K2 mediates the oxidation of flavin mononucleotide and the reduction of cytochrome b in the cell membrane. The redox potential of vitamin K2 is demonstrated to coincide with the suitable ORR potential range of lithium–oxygen batteries in aprotic solvent, thereby enabling its successful functioning as a redox mediator (RM) triggering the solution‐based discharge. The use of vitamin K2 prevents the growth of film‐like Li2O2 even in an ether‐based electrolyte, which has been reported to induce surface‐driven discharge and early passivation of the electrode, thus boosting the discharge capacity by ≈30 times. The similarity of the redox mediation in the biological cell and lithium–oxygen “cell” inspires the exploration of redox active bio‐organic compounds for potential high‐performance RMs toward achieving high specific energies for lithium–oxygen batteries.
It was discovered over 60 years ago that the mitochondrial respiratory chain is constituted of a series of protein complexes imbedded in the inner mitochondrial membrane. Experimental evidence has more recently ascertained that the major respiratory complexes involved in energy conservation are assembled as supramolecular units (supercomplexes, SCs) in stoichiometric ratios. The functional role of SCs is less well defined, and still open to discussion. Several lines of evidence favour the concept that electron transfer from Complex I to Complex III operates by channelling of electrons through Coenzyme Q molecules bound to the SC I1III2IVn
, in contrast with the previously accepted hypothesis that the transfer of reducing equivalents from Complex I to Complex III occurs via random diffusion of the Coenzyme Q molecules in the lipid bilayer. On the contrary, electron transfer from Complex III to Complex IV seems to operate, at least in mammals, by random diffusion of cytochrome c molecules between the respiratory complexes even if assembled in SCs. Furthermore, another property provided by the supercomplex assembly is the control of generation of reactive oxygen species by Complex I, that might be important in the regulation of signal transduction from mitochondria. This review discusses physiological and pathological implications of the supercomplex assembly of the respiratory chain.
This book focuses on the use of various molecules with antioxidant properties in the treatment of major male genital tract disorders, especially male infertility, erectile dysfunction, and accessory gland infection. The coverage also includes discussion of pathophysiology, the molecular basis of male infertility, and the rationale for use of antioxidants, with particular attention to coenzyme Q10 and carnitine. Oxidative stress occurs when the production of reactive oxygen species, including free radicals, exceeds the body’s natural antioxidant defences, leading to cellular damage. Oxidative stress is present in about half of all infertile men, and reactive oxygen species can produce infertility both by damaging the sperm membrane, with consequences for sperm motility, and by altering the sperm DNA. There is consequently a clear rationale for the use of antioxidant treatments within andrology, and various in vitro and in vivo studies have indicated that many antioxidants indeed have beneficial impacts. In providing a detailed and up-to-date overview of the subject, this book will be of interest to both practitioners and researchers in andrology, endocrinology, and urology.
Human coenzyme Q (CoQ10) or ubiquinone is mainly known for its bioenergetic role as a proton and electron carrier in the inner mitochondrial membrane and is also an endogenous lipophilic antioxidant, ubiquitous in biological membranes. It is also present in plasma lipoproteins, where it plays a well-recognized antioxidant role. More recently coenzyme Q10 was also shown to affect gene expression by modulating the intracellular redox status. Its involvement in many cellular and extracellular functions suggests that its use as a food supplement could be beneficial in conditions associated with increased oxidative stress underlying different pathological conditions. In reproductive biology, CoQ10 has been shown to play a role in fertility of both males and females.
The notion of the compartmentalization of cellular functions, often by means of different organelles, arose from the convergence of two independent lines of investigation, one concentrating on cytological observations of cell inclusions and the other on biochemical studies of various metabolic pathways. The convergence occurred during the period of 1940–1960 when many important technical advances were made in the fractionation and isolation of reasonably homogeneous subcellular components. This meant that it became possible in many instances to assign specific functions to particular organelles or compartments of the cell. Today, every student of biology is aware of the fact that the mitochondrion is a specialized organelle whose primary functions are the conservation of oxidatively derived energy and its utilization for ATP synthesis. Some of the earlier developments that led to this recognition and other more recent highlights of studies of mitochondrial structure and function will be traced briefly in this chapter.
Evidence from several investigations demonstrates the existence of supramolecular units of Complex I, Complex III, and multiple copies of Complex IV in mitochondria and indicates that specific respiratory complexes may preferentially associate to form cytochrome-containing supercomplexes in the native membrane. There are now indications thatcardiolipin,a distinctive mitochondrial lipid, stabilizes the respiratory assemblies. The isolated supercomplexes are active with respect to both their component individual complexes and the entire respiratory function that relies on Coenzyme Q and cytochrome c as intermediate substrates. The latter finding argues against previous models of a random distribution of the respiratory complexes in mitochondria. The supercomplex organization is compatible with electron transfer, but experimental evidence is scant for an effective mechanism via substrate channeling compared to free diffusion of substrates in accordance with the random collision model. The finding that Complex I is almost totally associated in a supercomplex with Complex III seems to exclude a role for the ubiquinone pool in physiological electron transfer between these two complexes, whereas it is certainly required for electron transfer from Complex II or from other dehydrogenases to Complex III; likely, only a small fraction of Complex IV forms a functional supercomplex with channeling of cytochrome c. Nevertheless, the supercomplexes may physiologically exist in equilibrium with free complexes (plasticity model). The supercomplex organization appears to prevent excessive generation of reactive oxygen species from the respiratory chain; accordingly, many pathological conditions and the mitochondrial aging phenotype characterized by a loss of supercomplex assembly correlate with mitochondrial dysfunction and increased oxidative stress. Specific metabolic signals may also arise in the cell in response to a tuned production of reactive oxygen species as a consequence of the controlled dynamics of supercomplex assembling/disassembling at different physio-pathological conditions. The present review paper provides an updated and extensive discussion on the subject.
This chapter discusses the constraints of the mitochondrial membrane in determining and regulating electron transfer function. It focuses on the heme-containing components of the inner mitochondrial membrane, namely, the cytochromes. These cytochromes provide interesting intrinsic chromophores suitable for optical probing in situ. It provides an overview on physical techniques, which determine orientation, distances, structure, and motion of the electron carriers. It is generally accepted that the respiratory chain components undergo independent diffusion. The rotational and transverse motions of cytochrome c oxidase and cytochrome c are directly measured. The rotational motion of the ADP/ATP translocator has been established and the transverse diffusion of the cytochrome bc1 complex has been demonstrated. The chapter also explains that specificity is achieved by having specific interaction domains on the electron carrier for its redox partners. In this regard, much work has been done on cytochrome c to explain its interaction domains with the oxidase and the reductase
Purified preparations of cytochrome oxidase solubilized in bile acid exhibit random aggregations of subunit particles of about 50 and 100 A in diameter. When the bile acids are removed, the subunit particles organize into vesicular membranes. Removal of phospholipid from cytochrome oxidase eliminates the capacity of the subunits to organize into vesicles. This capacity is restored by adding back mitochondrial phospholipid to the lipid-depleted particles.
The electron microscopic structure of sectioned indirect flight muscle fibers of the blowfly Calliphora is described. Particular attention is paid to the organization of the sarcosomes (mitochondria) of this tissue, and this description is accompanied by an account of the appearance of these bodies in negatively stained preparations. In sectioned material, it has been shown that these sarcosomes are similar to other mitochondria in the disposition of the outer and inner limiting membranes, but that the cristae, confluent with the latter, are unusually regular, and form parallel plates, containing circular fenestrations forming cylindrical channels within the matrix. Negatively stained preparations of disrupted sarcosomes reveal that both the outer limiting membrane and the cristae membranes bear large numbers of small particles, similar in appearance to those described by Fernández-Morgán and others in various mitochondria. In Calliphora, these particles consist of a sub-spherical "head" and a cylindrical "stalk," and appear to be arranged on the mitochondrial membranes either randomly distributed, or collected into circular or elongated groups. Recent suggestions concerning the nature of these submitochondrial particles are discussed, and an attempt is made to correlate the aspects of organization of Calliphora sarcosomes, revealed by conventional sectioning of the "intact" structures, and by negative staining of sarcosomal derivatives.
A repeating particle associated with the cristae and the inner membrane of the external envelope has been recognized and characterized in beef heart mitochondria by correlated electron microscopic and biochemical studies. Many thousands (ca. 10(4) to 10(5)) of these particles, disposed in regular arrays, are present in a single mitochondrion. The repeating particle, called the elementary particle (EP), consists of three parts: (1) a spherical or polyhedral head piece (80 to 100 A in diameter); (2) a cylindrical stalk (about 50 A long and 30 to 40 A wide); and (3) a base piece (40 x 110 A). The base pieces of the elementary particles form an integral part of the outer dense layers of the cristae. The elementary particles can be seen in electron micrographs of mitochondria in situ, of isolated mitochondria, and of submitochondrial particles with a complete electron transfer chain. Negative staining with phosphotungstate is only one of several techniques that can be used for reproducible demonstration of the repeating particles and underlying subunit organization of mitochondrial membranes. A particulate unit containing a complete electron transfer chain can be isolated from beef heart mitochondria. The isolated unit approximates in size that of the elementary particle in situ. The molecular weight of the particle in situ is calculated to be 1.3 x 10(6). Evidence is presented for identifying the isolated unit with the elementary particle visualized in situ. The elementary particle of the mitochondrion is believed to be a prototype of a class of functional particles or macromolecular assemblies of similar size found in association with membranes generally.
Sonic treatment of mitochondria from beef heart results in a distribution of variously sized particles which can be fractionated by differential centrifugation. Electron microscopic observations of negatively stained fractions shows the presence of subunits, which are "knob-like" in appearance, attached to the mitochondrial membrane and membrane derivatives. Such subunits are not completely removed from the membrane by 5 minutes of sonic treatment. However, a fraction containing singular subunits of dimensions similar to those of units attached to the mitochondrial membrane has been observed. Enzymatic activities and cytochrome contents were determined for mitochondria and the fractionated, mitochondrial membrane derivatives. A concentration of enzyme activity and cytochrome content was found in fractions sedimented at 35,300 g and 79,420 g via differential centrifugation in 2x distilled water. The fraction with the highest enzymatic activities and cytochrome content, although rich in mitochondrial membrane derivatives, is deficient in membrane-bound subunits. The fraction with the lowest enzymatic activities and cytochrome content resembles detached mitochondrial subunits when examined in the electron microscope. The biochemical data and the electron microscopic observations suggest that the mitochondrial membrane and not the subunits are responsible for electron transport activity.
The isolation of the succinic-CoQ reductase from beef heart mitochondria is described. The flavine content of the preparation is 4.8 mμmoles/mg. protein, and all of the flavine is extracted by acid only after the preparation is treated with proteolytic enzymes. The preparation also contains non-heme iron, lipid, and protoheme, and the last mentioned is present in an amount equivalent to the flavine. Based on the flavine or heme content, the minimum molecular weight in terms of protein is 210,000.The heme present in the purified enzyme is not reduced by succinate, which makes its participation as an electron carrier in the reactions catalyzed by the enzyme very unlikely.A rapid spectrophotometric method for measuring the succinic-CoQ reductase activity of mitochondria and of the purified enzyme is described.
1.1. A subunit (ETPH) having nearly theoretical P/O ratios may be prepared from heavy mitochondria from beef heart when both Mg2+ and Mn2+ are included in the medium during the isolation procedure.2.2. Optimal concentration of the ions required during the preparation are 5 mM for Mg2+ and 10 mM for Mn2+.3.3. The addition of succinate (1 mM) improved the reliability of the preparative procedure, and minimized seasonal difficulties in the preparation of ETPH during summer months.4.4. Certain preparations of ETPH show increased lability of the phosphorylation mechanism, and deteriorate during the temperature equilibrium period of the assay, when shaken in the assay medium in the absence of substrate. This can be overcome by including substrate in the preliminary incubation and starting the assay reaction with ADP and the hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) trapping system.
A method is presented by which soluble cytochrome c1 was isolated form beef-heart mitochondria in relatively high yield. The purity of the final preparation, 27.2 μmoles of heme or iron per gram of protein, was considerably higher than previously reported.Chemical analysis of the purified cytochrome c1 disclosed that it was free from contamination by other cytochromes, flavin, non-heme iron and bile salts. Studies in the analytical ultracentrifuge and electrophoresis showed that the preparation was homogeneous. The molecular weight of cytochrome c1, calculated on the basis of the iron content of the purest preparation obtained, is 37 000.Enzymic reduction of the purified cytochrome c1 was obtained by the use of a wide variety of enzyme systems derived from beef heart mitochondria when succinate, DPNH or reduced Coenzyme Q was used as substrate. The sensitivity of this reaction to inhibition by antimycin A appeared to be determined by the characteristics of the enzyme system employed.Oxidation of the purified cytochrome c1 proceeded rapidly in the presence of cytochrome oxidase and cytochrome c, the rate being dependent upon the cytochrome c concentration. However, before interpreting this fact it should be noted that cytochromes c and c1 interacted when solutions of the two purified hemoproteins were mixed together.
Under the names myohæmatin and histohæmatin MacMunn (1884-1886) described a respiratory pigment, which he found in muscles and other tissues of representatives of almost all the orders of the animal kingdom. He found that this pigment, in the reduced state, gives a characteristic spectrum, with four absorption bands occupying the following positions: 615—593/567·5—561/554·5—546/532—511/. When oxidized, the pigment does not show absorption bands. In 1887 MacMunn described a method by which it can be extracted in a “modified form” from the muscles of birds and mammals. He found the pectoral muscle of a pigeon to be the most suitable material for the extraction of “myohæmatin,” in the belief that it was the sole colouring matter of the muscles in a pigeon bled to death. From this “modified myohæmatin” he also obtained other derivatives, such as acid hæmatin and hæmatoporphyrin, and he finally arrived at the conclusion that myo- and histohæmatin are respiratory pigments different and independent from hæmoglobin and its derivatives. In 1889 Levy carefully repeated MacMunn’s experiments in extracting myohæmatin from muscles of birds and mammals and obtained the substance described by MacMunn as “modified myohæmatin." But he regarded this substance as an ordinary hæmochromogen, derived from hæmoglobin.
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A mitochondrial preparation containing the complete electron transfer chain (EP1) as well as a preparation containing only one sector of the chain (Complex IV) were resolved into structural and catalytic protein fractions. The respective oxidation-reduction components of EP1 and Complex IV, as well as the lipid, were concentrated exclusively in the catalytic protein fraction. Integrated electron transfer activity appears to be unaffected by the removal of structural protein. Such losses in electron transfer activity as are sustained appear to be referable to the severity of the procedure for resolution rather than to the removal of structural protein. Under appropriate conditions structural protein will recombine with catalytic protein to “reconstitute” a particle that resembles the parent particle in respect to solubility, activity, spectral properties, and ultrastructure. The membrane-forming capability of the complexes is not affected by the removal of structural protein.
The preparation and properties of an electron transfer particle (ETP) obtained from beef heart mitochondria are described. ETP catalyzes the oxidation of 5.7 μmoles DPNH and 2 μmoles succinate per min per mg at 38°. This specific activity is 3 to 5 times greater than that of the original mitochondrial suspension. These two oxidative reactions are profoundly influenced by 1) concentration of phosphate, 2) the presence of metal complexing agents, and 3) the ionic strength of the medium.Preparations of ETP contain flavin, cytochromes a, b and c1, non-heme iron and copper in constant proportions as well as cytochrome c in variable quantity depending upon the conditions of isolation. Both flavin and hemes are reducible by either substrate and to the same complete extent. Preparations of ETP from which the bulk of cytochrome c has been removed show no requirement for added cytochrome c in the oxidation of DPNH by molecular oxygen, whereas a partial requirement for cytochrome c in the oxidation of succinate is demonstrable under some but not all conditions. The exposure of ETP to deoxycholate leads to a particle which can interact readily with external cytochrome c. Procedures which induce fragmentation of ETP are also described. The significance of the fact of the constant composition of ETP for the dynamics of electron transport is discussed.
Crude mitochondrial suspensions, prepared on a large scale from beef heart, have been fractionated by centrifugation in a buffered medium. Two principal fractions, heavy and light, have been isolated, and some of their properties with respect to the oxidation of citric acid cycle substrates and the accompanying phosphorylation have been studied. The heavy fraction, which resembles intact mitochondria much more closely than the light, catalyzes the oxidation of these substrates very efficiently with P/O ratios quite close to the assumed theoretical values. These preparations can be conveniently stored in the deep-freeze for long periods of time without considerable loss of activity.
When the heavy fraction of beef heart mitochondrial suspensions is exposed to sonic oscillation, a submitochondrial particle can be isolated which is indistinguishable from previously described ETP except that it is capable of coupling the oxidation of succinate and DPNH to phosphorylation —the maximum P/O ratios being respectively 1.2 and 2.0 ETP may be regarded as the least common denominator of both electron transport and oxidative phosphorylation. Whether phosphorylating or not, ETP is inactive in catalyzing citric cyclic oxidations. During its formation by sonication of mitochindria, some of the pyridinoprotein enzymes are completely lost while others are retained in part. The β-hydroxybutyric dehydrogenase is the only pyridinoprotein enzyme which is more concentrated in ETP than in the original mithocondrion. The presence of Mg++ during the separation of ETP from the sonicated suspension is essential for maximal phosphorylative activity.
The effects of acetone extraction on the electron-transport activities of beef-heart mitochondria have been investigated. Two standard preparations have been developed. Preparation I, made by extraction with acetone containing 4% water is devoid of CoQ, and requires the specific addition of CoQ to restore succinate oxidation with various electron acceptors. The restored succinoxidase activity is completely sensitive to antimycin A and KCN. Preparation II, made by extraction with acetone containing 10% water under specified conditions, has lost not only its complement of CoQ but also three-quarters of its lipid complement, and the combined addition of coenzyme Q plus unidentified mitochondrial lipids is required for restoration of succinoxidase activity. The physical state of the lipid which is added is a crucial factor in its maximum effectiveness. Particles extracted with acetone lose the capacity to oxidize DPNH by cytochrome c or oxygen, and this deficiency may be a consequence of the inability of the preparation to catalyze the oxidation of DPNH by CoQ. Comparable results were also obtained with rat-liver and beef-liver mitochondria.
The oxidoreduction properties of the components (flavoprotein, coenzyme Q, cytochrome b and cytochrome c1) involved in a highly active DPNH-cytochrome c reductase, purified from beef-heart mitochondria, have been studied. The components of the enzyme are rapidly reduced by DPNH and are oxidized in presence of catalytic amounts of cytochrome c plus cytochrome oxidase. The oxidoreduction changes of the electron carriers of the enzyme have also been studied in presence of Amytal and antimycin A, and the points of inhibition of these two compounds have been defined.As a result of these as well as other studies, a functional sequence for the participation of the above components in the DPNH oxidase system of mitochondria has been presented.
The combined electron-microscope and biochemical evidence regarding mitochondria and mitochondrial fractions, although still preliminary in character, indicates that all the functional enzymatic components of the electron-transport chain are compactly arranged in the "elementary particle," which may therefore be regarded as the ultimate unit of mitochondrial function. New data have also been obtained on the hydrated lipoprotein matrix, the recently isolated structural protein, solubilized pure lipid fractions, cytochromes, and other constituent elements of the respiratory-enzyme assemblies.
A protein has been isolated in large amounts from beef heart mitochondria which satisfies various criteria for a structural protein. The homogeneity of the isolated structural protein has been established by ultracentrifugal, electrophoretic, and end-group analysis. At neutral pH the structural protein forms a water-insoluble polymeric aggregate, but at pH 11, or in the presence of anionic detergents, a monomeric form of the protein can be demonstrated (m.w. 2 × 104-3 × 104). Cytochromes a, b, and c1 show analogous behavior with respect to the polymer-monomer transition. Structural protein forms one-to-one water-soluble complexes with each of the cytochromes, which are soluble in aqueous media at pH 7; the identity of these complexes has been rigorously established. The hydrophobic bond is the predominant type responsible both for the polymerization phenomenon and for complex formation between the monomeric species of the structural protein and of the cytochromes. Structural protein is capable of binding phospholipid; this property is shared by the three cytochromes. The interactions between structural protein and cytochromes and between structural protein and lipid have considerable relevance to the problem of mitochondrial organization.
The procedure described for the fractionation of mitochondria leads to the isolation of a particle, which uniquely contains all of the fixed components of electron transfer; the enzymic activity increases concomitantly with the purification of the particle. The rates of oxidation of DPNH and of succinate have been increased by a factor of 2.5 and the concentration of cytochrome a by a factor of 2.2. The specific concentrations of the other components have been increased to about the same degree. The isolated particle (designated the elementary particle) appears to be a physical and functional aggregate of the four complexes known collectively to constitute the electron transfer system; the theoretical molecular weight (on a protein basis) is 1.4 X 106; that of the particle described was calculated to be 2.1 X 106. Further purification (by removal of structural protein, etc.) led to an estimated minimal molecular weight of 1.4 X 106 but also decreased the enzymic activity.
The cytochrome composition of mitochondrial fractions which have been stripped of inner membrane subunits by exposure to high-frequency sound have been examined by lowtemperature spectroscopy. The ratio of cytochrome c to cytochrome a is not changed by the treatment, but the concentration of cytochrome per milligram of protein is increased and the concentrations of cytochromes c(1) and b change slightly. These cytochromes (c(1) and b) may be at least partly located in the subunits of the inner membrane, but the idea that all the respiratory components are located in single subunits of the mitochondrial cristae may be considered to be disproved.
A method is described for the formation of complexes between cytochrome c and phospholipids. These complexes are shown to approach certain stoichiometric proportions depending upon the conditions of formation. The major proteolipid complex formed from mixed phospholipids contains 22 gram atoms of phosphorus per 1 M of cytochrome c when the original cytochrome c is in excess of the phospholipid. When more mixed phospholipid is added the ratio approaches 32:1. Alcohol hastens the formation of the stoichiometric complex. Inhibition of complex formation by monovalent cations is proportional to ionic strength of the solution whereas di- and trivalent cations completely inhibit complex formation. The evidence indicates that mixed phospholipids produce a complex in which the amount of phosphorus bound to cytochrome c approaches the available number of charged sites on the cytochrome c.
This chapter describes hemoglobin and myoglobin. Hemoglobin and myoglobin are, among all proteins, ones that have been, and are, most actively studied; an enormous number of papers have been published over the past hundred years on all aspects of their properties and behavior. The study of these proteins has gone beyond the interest in their physiological role as oxygen carriers because they represent ideal models for investigating the properties of proteins in general, especially of enzymes. Correspondingly, current knowledge of the structure and function of hemoglobin and myoglobin is far greater than that available for any other protein. In spite of this, however, many questions still remain unsolved regarding the exact molecular mechanisms involved in the function of these proteins. Before discussing the properties of respiratory heme proteins, it is necessary to briefly describe some properties and reactions of their prosthetic group, mainly because the characteristic physiological functions of these proteins arise from the intrinsic reactivity of the heme.
The molecular weights of two enzyme complexes purified from beef-heart mitochondria have been determined by the light-scattering technique. The enzymes studied were coenzyme QH2-cytochrome c reductase (Complex III) and cytochrome c oxidase (cytochrome c:O2 oxidoreductase, EC 1.9.3.1) (Complex IV). The weight average molecular weight of Complex III from light-scattering data agrees closely with the minimal molecular weight estimated from the cytochrome c1 content. In the case of cytochrome oxidase, however, the molecular weight was about two times larger than the minimal molecular weight based on the cytochrome a content. This observation supports the view that there are two heme a prosthetic groups per molecule of cytochrome oxidase.
- Keilin