Structure, function, and assembly of heme centers in mitochondrial respiratory complexes

The sequential flow of electrons in the respiratory chain, from a low reduction potential substrate to O(2), is mediated by protein-bound redox cofactors. In mitochondria, hemes-together with flavin, iron-sulfur, and copper cofactors-mediate this multi-electron transfer. Hemes, in three different forms, are used as a protein-bound prosthetic group in succinate dehydrogenase (complex II), in bc(1) complex (complex III) and in cytochrome c oxidase (complex IV). The exact function of heme b in complex II is still unclear, and lags behind in operational detail that is available for the hemes of complex III and IV. The two b hemes of complex III participate in the unique bifurcation of electron flow from the oxidation of ubiquinol, while heme c of the cytochrome c subunit, Cyt1, transfers these electrons to the peripheral cytochrome c. The unique heme a(3), with Cu(B), form a catalytic site in complex IV that binds and reduces molecular oxygen. In addition to providing catalytic and electron transfer operations, hemes also serve a critical role in the assembly of these respiratory complexes, which is just beginning to be understood. In the absence of heme, the assembly of complex II is impaired, especially in mammalian cells. In complex III, a covalent attachment of the heme to apo-Cyt1 is a prerequisite for the complete assembly of bc(1), whereas in complex IV, heme a is required for the proper folding of the Cox 1 subunit and subsequent assembly. In this review, we provide further details of the aforementioned processes with respect to the hemes of the mitochondrial respiratory complexes. This article is part of a Special Issue entitled: Cell Biology of Metals.     

Cytopathies involving mitochondrial complex II

Complex II (succinate-ubiquinone oxidoreductase) is the smallest complex in the respiratory chain and contains four nuclear-encoded subunits SdhA, SdhB, SdhC, and SdhD. It functions both as a respiratory chain component and an essential enzyme of the TCA cycle. Electrons derived from succinate can thus be directly transferred to the ubiquinone pool. Major insights into the workings of complex II have been provided by crystal structures of closely related bacterial enzymes, which have also been genetically manipulated to answer questions of structure-function not approachable using the mammalian system. This information, together with that accrued over the years on bovine complex II and by recent advances in understanding in vivo synthesis of the non-heme iron co-factors of the enzyme, is allowing better recognition of improper functioning of human complex II in diseased states. The discussion in this review is thus limited to cytopathies arising because the enzyme itself is defective or depleted by lack of iron-sulfur clusters. There is a clear dichotomy of effects. Enzyme depletion and mutations in SDHA compromise TCA activity and energy production, whereas mutations in SDHB, SDHC, and SDHD induce paraganglioma. SDHC and SDHD are the first tumor suppressor genes of mitochondrial proteins.     

Complex II phosphorylation is triggered by unbalanced redox homeostasis in cells lacking complex III

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.     

Abortive assembly of succinate-ubiquinone reductase (Complex II) in a ferrochelatase-deficient mutant of Escherichia coli

Heme molecules play important roles in electron transfer by redox proteins such as cytochromes. In addition, a structural role for heme in protein folding and the assembly of enzymes has been suggested. Previous results obtained using Escherichia coli hemA mutants, which are unable to synthesize 5-aminolevulinic acid, a precursor of porphyrins and hemes, have demonstrated a requirement for heme biosynthesis in the assembly of a functional succinate-ubiquinone reductase (SQR or complex II), which is a component of the aerobic respiratory chain. In the present study, in order to investigate the role of the heme in the assembly of E. coli SQR, we used a hemH (encodes ferrochelatase) mutant that lacks the ability to insert iron into the porphyrin ring. The hemH mutant failed to insert functional SQR into the cytoplasmic membrane, and the catalytic portion of SQR [the flavoprotein subunit (Fp) and the iron-sulfur protein subunit (Ip)] was localized in the cytoplasm of the cell. It is of interest to note that protoporphyrin IX accumulated in the mutant cells and inactivated the cytoplasmic succinate dehydrogenase (SDH) activity associated with the catalytic Fp-Ip complex. In contrast, SQR was assembled into the membrane of a heme-permeable hemH double mutant when hemin was present in the culture. Only a low level of SQR activity was found in the membrane when hemin was replaced by non-iron metalloporphyrins: Mn-, Co-, Ni-, Zn- and Cu-protoporphyrin IX, or protoporphyrin IX These results indicate that heme iron is indispensable for the functional assembly of SQR in the cytoplasmic membrane of E. coli, and provide a new insight into the biological role of heme in the molecular assembly of the multi-subunit enzyme complex.     

Synthesis of the proteins of complex III of the mitochondrial respiratory chain in heme-deficient cells

The presence of several proteins of complex III of the respiratory chain has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae lacking 5-aminolevulinic acid synthase and, hence, devoid of heme. The two 'core' proteins, apocytochrome b and the iron-sulfur protein, were observed in equal amounts in the heme-deficient and heme-sufficient cells with antiserum against complex III and the sensitive immuno-transfer technique. In addition, three other bands were detected with the complex III antiserum in the mitochondria from the cells lacking heme. One of these has a molecular weight similar to that reported for a precursor form of cytochrome c1. By contrast, when mitochondria from the heme-deficient cells were solubilized with mild detergents and treated with the complex III antiserum, almost no immunoprecipitation was obtained above that obtained with control serum. The presence of only one major labeled band with a molecular weight similar to subunit I was observed after gel electrophoresis. These results suggest that heme may be necessary for proper processing of the apoprotein of cytochrome c1 and for the assembly into the membrane of the subunits of complex III, rather than for the synthesis of the proteins.     

SDH mutations in cancer

The SDHA, SDHB, SDHC, SDHD genes encode the four subunits of succinate dehydrogenase (SDH; mitochondrial complex II), a mitochondrial enzyme involved in two essential energy-producing metabolic processes of the cell, the Krebs cycle and the electron transport chain. Germline loss-of-function mutations in any of the SDH genes or assembly factor (SDHAF2) cause hereditary paraganglioma/phaeochromocytoma syndrome (HPGL/PCC) through a mechanism which is largely unknown. Owing to the central function of SDH in cellular energy metabolism it is important to understand its role in tumor suppression. Here is reported an overview of genetics, clinical and molecular progress recently performed in understanding the basis of HPGL/PCC tumorigenesis.     

Absence of the psbH gene product destabilizes the Photosystem II complex and prevents association of the Photosystem II-X protein in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

PS II-H is a small hydrophobic protein that is universally present in the PS II core complex of cyanobacteria and plants. The role of PS II-H was studied by directed mutagenesis and biochemical analysis in the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1. The psbH disruptant could grow photoautotrophically; however, its growth was much slower than that of the wild type cell. Chromatography enabled the isolation of active oxygen-evolving PS II complexes from both the mutant and the wild type. The mutant yielded a relatively large amount of inactive PS II complex that lacked the following extrinsic proteins: the 33-kDa protein, the 12-kDa protein, and cytochrome c ₅₅₀ . There were differences between the psbH disruptant and the wild type in terms of the oxygen evolution activities of the cells, thylakoids, and PS II complexes. At high concentrations of 2,6-DCBQ, the activity was much lower in the mutant than in the wild type. Gel filtration chromatography of the PS II complexes showed that both active and inactive PS II complexes isolated from the mutant were mostly in the monomeric form, while the active PS II complex from the wild type was in the dimeric form. The polypeptide composition of both active and inactive PS II complexes from the mutant showed the absence of another small polypeptide, PS II-X. These results suggest that the PS II-H protein is essential for stable assembly of native dimeric PS II complex containing PS II-X.     

Mitochondrial targeting of vitamin E succinate enhances its pro-apoptotic and anti-cancer activity via mitochondrial complex II

Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 μM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 μM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.     

Mitochondrial respiration of complex II is not lower than that of complex I in mouse skeletal muscle

Skeletal muscle (SKM) requires a large amount of energy, which is produced mainly by mitochondria, for their daily functioning. Of the several mitochondrial complexes, it has been reported that the dysfunction of complex II is associated with several diseases, including myopathy. However, the degree to which complex II contributes to ATP production by mitochondria remains unknown. As complex II is not included in supercomplexes, which are formed to produce ATP efficiently, we hypothesized that complex II-linked respiration was lower than that of complex I. In addition, differences in the characteristics of complex I and II activity suggest that different factors might regulate their function. The isolated mitochondria from gastrocnemius muscle was used for mitochondrial respiration measurement and immunoblotting in male C57BL/6J mice. Student paired t-tests were performed to compare means between two groups. A univariate linear regression model was used to determine the correlation between mitochondrial respiration and proteins. Contrary to our hypothesis, complex II-linked respiration was not significantly less than complex I-linked respiration in SKM mitochondria (complex I vs complex II, 3402 vs 2840 pmol/[s × mg]). Complex I-linked respiration correlated with the amount of complex I incorporated in supercomplexes (r = 0.727, p < 0.05), but not with the total amount of complex I subunits. In contrast, complex II-linked respiration correlated with the total amount of complex II (r = 0.883, p < 0.05), but not with the amount of each complex II subunit. We conclude that both complex I and II play important roles in mitochondrial respiration and that the assembly of both supercomplexes and complex II is essential for the normal functioning of complex I and II in mouse SKM mitochondria.     

Nm23H2 facilitates coat protein complex II assembly and endoplasmic reticulum export in mammalian cells

The cytosolic coat protein complex II (COPII) mediates vesicle formation from the endoplasmic reticulum (ER) and is essential for ER-to-Golgi trafficking. The minimal machinery for COPII assembly is well established. However, additional factors may regulate the process in mammalian cells. Here, a morphological COPII assembly assay using purified COPII proteins and digitonin-permeabilized cells has been applied to demonstrate a role for a novel component of the COPII assembly pathway. The factor was purified and identified by mass spectrometry as Nm23H2, one of eight isoforms of nucleoside diphosphate kinase in mammalian cells. Importantly, recombinant Nm23H2, as well as a catalytically inactive version, promoted COPII assembly in vitro, suggesting a noncatalytic role for Nm23H2. Consistent with a function for Nm23H2 in ER export, Nm23H2 localized to a reticular network that also stained for the ER marker calnexin. Finally, an in vivo role for Nm23H2 in COPII assembly was confirmed by isoform-specific knockdown of Nm23H2 by using short interfering RNA. Knockdown of Nm23H2, but not its most closely related isoform Nm23H1, resulted in diminished COPII assembly at steady state and reduced kinetics of ER export. These results strongly suggest a previously unappreciated role for Nm23H2 in mammalian ER export.     

Effect of Electroconvulsive Shock on Mitochondrial Respiratory Chain in Rat Brain

It is well described that impairment of energy production has been implicated in the pathogenesis of a number of diseases. Although several advances have occurred over the past 20 years concerning the use and administration of electroconvulsive therapy (ECT) to minimize its side effects, little progress has been made in understanding its mechanism of action. In this work, our aim was to measure the activities of mitochondrial respiratory chain complexes II and IV and succinate dehydrogenase from rat brain after acute and chronic electroconvulsive shock (ECS). Our results showed that mitochondrial respiratory chain enzymes activities were increased after acute ECS in hippocampus, striatum and cortex of rats. Besides, we also demonstrated that complex II activity was increased after chronic ECS in cortex, while hippocampus and striatum were not affected. Succinate dehydrogenase, however, was inhibited after chronic ECS in striatum, activated in cortex and not affected in hippocampus. Finally, complex IV was not affected by chronic ECS in hippocampus, striatum and cortex. Our findings demonstrated that brain metabolism is altered by ECS.     

Heme deficiency selectively interrupts assembly of mitochondrial complex IV in human fibroblasts: revelance to aging

Heme deficiency was studied in young and old normal human fibroblasts (IMR90). Regardless of age, heme deficiency increased the steady-state level of oxidants and lipid peroxidation and sensitized the cells to fluctuations in intracellular Ca(2+). Heme deficiency selectively decreased the activity and protein content of mitochondrial complex IV (cytochrome c oxidase) by 95%, indicating a decrease in successful assembly. Complexes I-III and catalase remained intact under conditions of heme deficiency, whereas ferrochelatase was up-regulated. Complex IV is the only hemeprotein in the cell that contains heme a, which may account for its susceptibility. The rate of removal and assembly of complex IV declines with age. These findings are relevant to worldwide iron deficiency in women and children and to an age-related decline in complex IV in Alzheimer's disease patients.     

The tumor suppressor cybL,a component of the respiratory chain,mediates apoptosis induction

A genetic screen was established to clone apoptosis-inducing genes in a high-throughput format. It led to the isolation of several proapoptotic genes whose proteins are localized to mitochondria. One of the isolated genes is cytochrome bL (cybL also known as SDHC, CII-3, or QPs-1), a component of the respiratory chain complex II. It was further investigated because both cybL and another component of complex II, cybS, have recently been identified as tumor suppressor proteins, some of which act by controlling apoptosis. Our studies reveal that cell death induction by cybL expression is concomitant with a transient inhibition of complex II and the generation of reactive oxygen species. Importantly, cells that are constitutively deficient in cybL are resistant to a variety of proapoptotic cytostatic drugs and to the effects of the Fas receptor. Our results therefore identify complex II as a sensor for apoptosis induction and could explain the unexpected observation that complex II is inactivated in tumors.     

Molecular pathogenesis of tumorigenesis caused by succinate dehydrogenase defect

Succinate dehydrogenase (SDH), also named as complex II or succinate:quinone oxidoreductases (SQR) is a critical enzyme in bioenergetics and metabolism. This is because the enzyme is located at the intersection of oxidative phosphorylation and tricarboxylic acid cycle (TCA); the two major pathways involved in generating energy within cells. SDH is composed of 4 subunits and is assembled through a multi-step process with the aid of assembly factors. Not surprisingly malfunction of this enzyme has marked repercussions in metabolism leading to devastating tumors such as paraganglioma and pheochromocytoma. It is already known that mutations in the genes encoding subunits lead to tumorigenesis, but recent discoveries have indicated that mutations in the genes encoding the assembly factors also contribute to tumorigenesis. The mechanisms of pathogenesis of tumorigenesis have not been fully understood. However, a multitude of signaling pathways including succinate signaling was determined. We, here discuss how defective SDH may lead to tumor development at the molecular level and describe how yeast, as a model system, has contributed to understanding the molecular pathogenesis of tumorigenesis resulting from defective SDH.     

The role of the electron transport SDHC gene on lifespan and cancer

Much attention has been focused on the hypothesis that oxidative damage plays in cellular and organismal aging. It is known that oxygen is initially converted to superoxide anion (O2-), one of reactive oxygen species (ROS), by electron leaked from mainly complex III in the electron transport system present in mitochondria, where it is the major endogenous source of ROS. We have shown that a mutation in a subunit, cytochrome b large subunit (SDHC), of complex II, also results in increasing O2- production and therefore lead to apoptosis and precocious aging in Caenorhabditis elegans. Recently, individuals with an inherited propensity for vascularized head and neck tumors (i.e., paragangliomas) have been demonstrated to contain one of several mutations in complex II. To further explore the role of oxidative stress from mitochondria on apoptosis and cancer, we established a transgenic cell line with a point mutation at the ubiquinone binding region in the SDHC gene. As expected, this mutation increased O2- production from complex II and led to excess apoptosis. Moreover, a significant fraction of the surviving cells from the apoptosis were transformed, as evidenced by increased tumor formation after injection into mice. Oxidative stress results in the damage to the cellular components including mitochondria and, therefore leads to apoptosis. Furthermore, oxidative stress must cause mutations in DNA and leads to cancer. It is suggested that oxidative stress from mitochondria play an important role of both apoptosis, which leads to precocious aging, and cancer.     

Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts

Cytochrome c oxidase (COX) biogenesis requires COX10, which encodes a protoheme:heme O farnesyl transferase that participates in the biosynthesis of heme a. We created COX10 knockout mouse cells that lacked cytochrome aa3, were respiratory deficient, had no detectable complex IV activity, and were unable to assemble COX. Unexpectedly, the levels of respiratory complex I were markedly reduced in COX10 knockout clones. Pharmacological inhibition of COX did not affect the levels of complex I, and transduction of knockout cells with lentivirus expressing wild-type or mutant COX10 (retaining residual activity) restored complex I to normal levels. Pulse-chase experiments could not detect newly assembled complex I, suggesting that either COX is required for assembly of complex I or the latter is quickly degraded. These results suggest that in rapidly dividing cells, complex IV is required for complex I assembly or stability.     

Expression of a familial amyotrophic lateral sclerosis-associated mutant human superoxide dismutase in yeast leads to decreased mitochondrial electron transport

Strains of Saccharomyces cerevisiae that express either the wild type or the amyotrophic lateral sclerosis-associated mutant human copper-zinc superoxide dismutase (SOD1) proteins A4V and G93A, respectively, in a yeast SOD1-deficient parent strain were used to investigate the hypothesis that expression of a mutant SOD1 protein causes deficient mitochondrial electron transport as a possible mechanism for disease induction. Mitochondria isolated from the wild type SOD1-expressing yeast were identical to mitochondria from the parent strain in heme content and activities of complexes II, III, and IV. Mitochondria isolated from the A4V-expressing yeast had decreased rates of electron transport in complexes II+III, III, and IV and corresponding decreases in hemes b, c-c1, and a-a3 content compared to mitochondria from wild type human SOD1-expressing yeast. Mitochondria isolated from G93A-expressing yeast had decreased rates of electron transport in complex IV and probably in complex II with a corresponding decrease in heme a-a3 content. These results suggest that mutant SOD1-expression causes defective electron transport complex assembly and that the yeast system will provide an excellent model for the study of the mechanism of mutant SOD1-induced mitochondrial electron transport defects.     

The major light-harvesting complex of Photosystem II: aspects of its molecular and cell biology

The light-harvesting complex of photosystem II (LHC II) contains one major (LHC IIb) and at least three minor chlorophyll-protein components. The apoproteins of LHC IIb (LHCP) are encoded by nuclear genes and synthesized in the cytoplasm as a higher molecular weight precursor(s) (pLHCP). Several genes coding for pLHCP have been cloned from various higher plant species. The expression of these genes is dependent upon a variety of factors such as light, the developmental stage of the plastids and the plant. After its synthesis in the cytoplasm, pLHCP is imported into plastids, inserted into thylakoids, processed to its mature form, and assembled into LHC IIb. The pathway of assembly of LHC IIb in the thylakoid membranes is currently being investigated in several laboratories. We present a model that gives some details of the steps in the assembly process. Many of the steps involved in the synthesis and assembly are dependent on light and the stage of plastid development.Abbreviations PS Photosystem - LHC II Light-harvesting complex of PS II - LHCP Apoproteins of LHC IIb - pLHCP Precursor of LHCP - PAGE Polyacrylamide gel electrophoresis