Direct evidence for expression of type II flavoprotein subunit in human complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in aerobic respiration and the tricarboxylic acid cycle. We recently identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual and demonstrated mRNAs of these two isoforms, Type I Fp and Type II Fp, in skeletal muscle, liver, brain, heart, and kidney. Type I Fp was expressed at higher levels than Type II Fp in all cases. In the present study, the biochemical properties of Type II Fp-containing complex II in Raji cells predominantly expressing Type II Fp were investigated. Complex II having Type II Fp was separated from that having Type I Fp by isoelectric focusing in the presence of sucrose monolaurate. Together with the fact that succinate-ubiquinone reductase activity of mitochondria prepared from Raji cell was almost identical to that from human liver, these results clearly indicate the presence of two distinct isoforms of active complex II in human mitochondria.     

Crystal structure of mitochondrial respiratory membrane protein complex II

The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.     

Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host

The mitochondrial metabolic pathway of the parasitic nematode Ascaris suum changes dramatically during its life cycle, to adapt to changes in the environmental oxygen concentration. We previously showed that A. suum mitochondria express stage-specific isoforms of complex II (succinate-ubiquinone reductase: SQR/quinol-fumarate reductase: QFR). The flavoprotein (Fp) and small subunit of cytochrome b (CybS) in adult complex II differ from those of infective third stage larval (L3) complex II. However, there is no difference in the iron-sulfur cluster (Ip) or the large subunit of cytochrome b (CybL) between adult and L3 isoforms of complex II. In the present study, to clarify the changes that occur in the respiratory chain of A. suum larvae during their migration in the host, we examined enzymatic activity, quinone content and complex II subunit composition in mitochondria of lung stage L3 (LL3) A. suum larvae. LL3 mitochondria showed higher QFR activity ( approximately 160 nmol/min/mg) than mitochondria of A. suum at other stages (L3: approximately 80 nmol/min/mg; adult: approximately 70 nmol/min/mg). Ubiquinone content in LL3 mitochondria was more abundant than rhodoquinone ( approximately 1.8 nmol/mg versus approximately 0.9 nmol/mg). Interestingly, the results of two-dimensional bule-native/sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses showed that LL3 mitochondria contained larval Fp (Fp(L)) and adult Fp (Fp(A)) at a ratio of 1:0.56, and that most LL3 CybS subunits were of the adult form (CybS(A)). This clearly indicates that the rearrangement of complex II begins with a change in the isoform of the anchor CybS subunit, followed by a similar change in the Fp subunit.     

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.     

Direct evidence for two distinct forms of the flavoprotein subunit of human mitochondrial complex II (succinate-ubiquinone reductase)

Succinate-ubiquinone reductase (complex II) is an important enzyme complex in both the tricarboxylic acid cycle and aerobic respiration. A recent study showed that defects in human complex II are associated with cancers as well as mitochondrial diseases. Mutations in the four subunits of human complex II are associated with a wide spectrum of clinical presentations. Such tissue-specific clinical symptoms suggest the presence of multiple isoforms of the subunits, but subunit isoforms have not been previously reported. In the present study, we identified two distinct cDNAs for the human flavoprotein subunit (Fp) from a single individual, and demonstrated expression of these two isoforms in skeletal muscle, liver, brain, heart and kidney. Interestingly, one of the Fp isoforms was encoded as an intronless gene.     

MitoK ATP -dependent changes in mitochondrial volume and in complex II activity during ischemic and pharmacological preconditioning of langendorff-perfused rat heart

It has been proposed that activation of the mitochondrial ATP-sensitive potassium channel (mitoK_ATP) is part of signaling pathways triggering the cardioprotection afforded by ischemic preconditioning of the heart. This work was to analyze the mitochondrial function profile of Langendorff-perfused rat hearts during the different phases of various ischemia-reperfusion protocols. Specifically, skinned fibers of ischemic preconditioned hearts exhibit a decline in the succinate-supported respiration and complex II activity during ischemia, followed by a recovery during reperfusion. Meanwhile, the apparent affinity of respiration for ADP (which reflects the matrix volume expansion) is increased during preconditioning stimulus and, to a larger extent, during prolonged ischemia. This evolution pattern is mimicked by diazoxide and abolished by 5-hydroxydecanoate. It is concluded that opening the mitoK_ATP channel mediates the preservation of mitochondrial structure-function via a mitochondrial matrix shrinkage and a reversible inactivation of complex II during prolonged ischemic insult.     

Expression, purification, and characterization of human type II arginase

Human type II arginase, which is extrahepatic and mitochondrial in location, catalyzes the hydrolysis of arginine to form ornithine and urea. While type I arginases function in the net production of urea for excretion of excess nitrogen, type II arginases are believed to function primarily in the net production of ornithine, a precursor of polyamines, glutamate, and proline. Type II arginases may also regulate nitric oxide biosynthesis by modulating arginine availability for nitric oxide synthase. Recombinant human type II arginase was expressed in Escherichia coli and purified to apparent homogeneity. The Km of arginine for type II arginase is approximately 4.8 mM at physiological pH. Type II arginase exists primarily as a trimer, although higher order oligomers were observed. Borate is a noncompetitive inhibitor of the enzyme, with a Kis of 0.32 mM and a Kii of 0.3 mM. Ornithine, a product of the reaction catalyzed by arginase and a potent inhibitor of type I arginase, is a poor inhibitor of the type II isozyme. The findings presented here indicate that isozyme-selectivity exists between type I and type II arginases for binding of substrate and products, as well as inhibitors. Therefore, inhibitors with greater isozyme-selectivity for type II arginase may be identified and utilized for the therapeutic treatment of smooth muscle disorders, such as erectile dysfunction.     

Toward understanding the role of mitochondrial complex II in the intraerythrocytic stages of Plasmodium falciparum: Gene targeting of the Fp subunit

Malaria parasites in human hosts depend on glycolysis for most of their energy production, and the mitochondrion of the intraerythrocytic form is acristate. Although the genes for all tricarboxylic acid (TCA) cycle members are found in the parasite genome, the presence of a functional TCA cycle in the intraerythrocytic stage is still controversial. To elucidate the physiological role of Plasmodium falciparum mitochondrial complex II (succinate-ubiquinone reductase (SQR) or succinate dehydrogenase (SDH)) in the TCA cycle, the gene for the flavoprotein subunit (Fp) of the enzyme, pfsdha (P.falciparum gene for SDH subunit A, PlasmoDB ID: PF3D7_1034400) was disrupted. SDH is a well-known marker enzyme for mitochondria. In the pfsdha disruptants, Fp mRNA and polypeptides were decreased, and neither SQR nor SDH activity of complex II was detected. The suppression of complex II caused growth retardation of the intraerythrocytic forms, suggesting that complex II contributes to intraerythrocytic parasite growth, although it is not essential for survival. The growth retardation in the pfsdha disruptant was rescued by the addition of succinate, but not by fumarate. This indicates that complex II functions as a quinol-fumarate reductase (QFR) to form succinate from fumarate in the intraerythrocytic parasite.     

Mitochondrial complex II in the post-ischemic heart: oxidative injury and the role of protein S-glutathionylation

Mitochondrial superoxide (O2.) is an important mediator of ischemia/reperfusion (I/R) injury. The O2. generated in mitochondria also acts as a redox signal triggering cellular apoptosis. The enzyme succinate ubiquinone reductase (SQR or complex II) is one of the major mitochondrial components hosting regulatory thiols. Here the intrinsic protein S-glutathionylation (PrSSG) at the 70-kDa FAD-binding subunit of SQR was detected in rat heart and in isolated SQR using an anti-GSH monoclonal antibody. When rats were subjected to 30 min of coronary ligation followed by 24 h of reperfusion, the electron transfer activity (ETA) of SQR in post-ischemic myocardium was significantly decreased by 41.5 +/- 2.9%. The PrSSGs of SQR-70 kDa were partially or completely eliminated in post-ischemic myocardium obtained from in vivo regional I/R hearts or isolated global I/R hearts, respectively. These results were further confirmed by using isolated succinate cytochrome c reductase (complex II + complex III). In the presence of succinate, O2. was generated and oxidized the SQR portion of SCR, leading to a 60-70% decrease in its ETA. The gel band of the S-glutathionylated SQR 70-kDa polypeptide was cut out and digested with trypsin, and the digests were subjected to liquid chromatography/tandem mass spectrometry analysis. One cysteine residue, Cys(90), was involved in S-glutathionylation. These results indicate that the glutathione-binding domain, (77)AAFGLSEAGFNTACVTK(93) (where underline indicates Cys(90)), is susceptible to redox change induced by oxidative stress. Furthermore, in vitro S-glutathionylation of purified SQR resulted in enhanced SQR-derived electron transfer efficiency and decreased formation of the 70-kDa-derived protein thiyl radical induced by O2. . Thus, the decreasing S-glutathionylation and ETA in mitochondrial complex II are marked during myocardial ischemia/reperfusion. This redox-triggered impairment of complex II occurs in the post-ischemic heart and should be useful to identify disease pathogenesis related to reactive oxygen species-induced mitochondrial dysfunction.     

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.     

A reevaluation of the roles of hexokinase I and II in the heart

Hexokinase is responsible for glucose phosphorylation, a process fundamental to regulating glucose uptake. In some tissues, hexokinase translocates to the mitochondria, thereby increasing its efficiency and decreasing its susceptibility to product inhibition. It may also decrease free radical formation in the mitochondria and prevent apoptosis. Whether hexokinase translocation occurs in the heart is controversial; here, using immunogold labeling for the first time, we provide evidence for this process. Rat hearts (6 groups, n = 6/group), perfused with either glucose- or glucose + oleate (0.4 mmol/l)-containing buffer, were exposed to 30-min insulin stimulation, ischemia, or control perfusion. Hexokinase I (HK I) and hexokinase II (HK II) distributions were then determined. In glucose-perfused hearts, HK I-mitochondrial binding increased from 0.41 +/- 0.04 golds/mm in control hearts to 0.71 +/- 0.10 golds/mm after insulin and to 1.54 +/- 0.38 golds/mm after ischemia (P < 0.05). Similarly, HK II-mitochondrial binding increased from 0.16 +/- 0.02 to 0.53 +/- 0.08 golds/mm with insulin and 0.44 +/- 0.07 golds/mm after ischemia (P < 0.05). Under basal conditions, the fraction of HK I that was mitochondrial bound was five times greater than for HK II; insulin and ischemia caused a fourfold increase in HK II binding but only a doubling in HK I binding. Oleate decreased hexokinase-mitochondrial binding and abolished insulin-mediated translocation of HK I. Our data show that mitochondrial-hexokinase binding increases under insulin or ischemic stimulation and that this translocation is modified by oleate. These events are isoform specific, suggesting that HK I and HK II are independently regulated and implying that they perform different roles in cardiac glucose regulation.     

Classical and slow-binding inhibitors of human type II arginase

Arginases catalyze the hydrolysis of L-arginine to yield L-ornithine and urea. Recent studies indicate that arginases, both the type I and type II isozymes, participate in the regulation of nitric oxide production by modulating the availability of arginine for nitric oxide synthase. Due to the reciprocal regulation between arginase and nitric oxide synthase, arginase inhibitors have therapeutic potential in treating nitric oxide-dependent smooth muscle disorders, such as erectile dysfunction. We demonstrate the competitive inhibition of the mitochondrial human type II arginase by N(omega)-hydroxy-L-arginine, the intermediate in the reaction catalyzed by nitric oxide synthase, and its analogue N(omega)-hydroxy-nor-L-arginine, with K(i) values of 1.6 microM and 51 nM at pH 7.5, respectively. We also demonstrate the inhibition of human type II arginase by the boronic acid-based transition-state analogues 2(S)-amino-6-boronohexanoic acid (ABH) and S-(2-boronoethyl)-L-cysteine (BEC), which are known inhibitors of type I arginase. At pH 7.5, both ABH and BEC are classical, competitive inhibitors of human type II arginase with K(i) values of 0.25 and 0.31 microM, respectively. However, at pH 9.5, ABH and BEC are slow-binding inhibitors of the enzyme with K(i) values of 8.5 and 30 nM, respectively. The findings presented here indicate that the design of arginine analogues with uncharged, tetrahedral functional groups will lead to the development of more potent inhibitors of arginases at physiological pH.     

Cloning,expression, characterization,and interaction of two components of a human mitochondrial fatty acid synthase. Malonyltransferase and acyl carrier protein

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonyl-CoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonyl-CoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.     

Mitochondrial Adaptations to NaCl. Complex I Is Protected by Anti-Oxidants and Small Heat Shock Proteins, Whereas Complex II Is Protected by Proline and Betaine

High soil sodium (Na) is a common stress in natural and agricultural systems. Roots are usually the first tissues exposed to Na stress and Na stress-related impairment of mitochondrial function is likely to be particularly important in roots. However, neither the effects of NaCl on mitochondrial function, nor its protection by several potential adaptive mechanisms, have been well studied. This study investigated the effects of NaCl stress on maize (Zea mays) mitochondrial electron transport and its relative protection by osmoprotectants (proline, betaine, and sucrose), antioxidants (ascorbate, glutathione, and alpha-tocopherol), antioxidant enzymes (catalase and Cu/Zn-superoxide dismutase), and mitochondrial small heat shock proteins (sHsps). We demonstrate that Complex I electron transport is protected by antioxidants and sHsps, but not osmoprotectants, whereas Complex II is protected only by low concentrations of proline and betaine. These results indicate that NaCl stress damaged Complex I via oxidative stress and suggests that sHsps may protect Complex I as antioxidants, but NaCl damaged Complex II directly. This is the first study to demonstrate that NaCl stress differentially affects Complex I and II in plants and that protection of Complex I and II during NaCl stress is achieved by different mechanisms.     

Effect of oxygen on activation state of complex I and lack of oxaloacetate inhibition of complex II in Langendorff perfused rat heart

Two main entry points for electrons into the mitochondrial respiratory chain are NADH:ubiquinone oxidoreductase (complex I) and succinate:ubiquinone oxidoreductase (complex II). Metabolic regulation of these two respiratory complexes is not understood in detail. It has been suggested that the Krebs cycle metabolic intermediate oxaloacetate (OAA) inhibits complex II in vivo, whereas complex I undergoes a reversible active/de-active transition. In normoxic and anoxic hearts it has been shown that the proportion of complex I in the active and de-active states is different suggesting a possible mode of regulation of the enzyme by oxygen concentration. In the current studies rapid isolation of mitochondrial membranes in a state that preserves the activity of both complex I and complex II has been achieved using Langendorff perfused rat hearts. The findings indicate that the state of activation of complex I is controlled by the oxygen saturation in the perfusate. In addition, these studies show that complex II is fully active in the mitochondrion and not inhibited by OAA regardless of the oxygen concentration.     

Damage to mitochondrial complex I during cardiac ischemia reperfusion injury is reduced indirectly by anti-anginal drug ranolazine

Ranolazine, an anti-anginal drug, is a late Na(+) channel current blocker that is also believed to attenuate fatty acid oxidation and mitochondrial respiratory complex I activity, especially during ischemia. In this study, we investigated if ranolazine's protective effect against cardiac ischemia/reperfusion (IR) injury is mediated at the mitochondrial level and specifically if respiratory complex I (NADH Ubiquinone oxidoreductase) function is protected. We treated isolated and perfused guinea pig hearts with ranolazine just before 30 min ischemia and then isolated cardiac mitochondria at the end of 30 min ischemia and/or 30 min ischemia followed by 10 min reperfusion. We utilized spectrophotometric and histochemical techniques to assay complex I activity, Western blot analysis for complex I subunit NDUFA9, electron paramagnetic resonance for activity of complex I Fe-S clusters, enzyme linked immuno sorbent assay (ELISA) for determination of protein acetylation, native gel histochemical staining for respiratory supercomplex assemblies, and high pressure liquid chromatography for cardiolipin integrity; cardiac function was measured during IR. Ranolazine treated hearts showed higher complex I activity and greater detectable complex I protein levels compared to untreated IR hearts. Ranolazine treatment also led to more normalized electron transfer via Fe-S centers, supercomplex assembly and cardiolipin integrity. These improvements in complex I structure and function with ranolazine were associated with improved cardiac function after IR. However, these protective effects of ranolazine are not mediated by a direct action on mitochondria, but rather indirectly via cytosolic mechanisms that lead to less oxidation and better structural integrity of complex I.     

Mitochondrial Complex II Prevents Hypoxic but Not Calcium- and Proapoptotic Bcl-2 Protein-induced Mitochondrial Membrane Potential Loss

Mitochondrial membrane potential loss has severe bioenergetic consequences and contributes to many human diseases including myocardial infarction, stroke, cancer, and neurodegeneration. However, despite its prominence and importance in cellular energy production, the basic mechanism whereby the mitochondrial membrane potential is established remains unclear. Our studies elucidate that complex II-driven electron flow is the primary means by which the mitochondrial membrane is polarized under hypoxic conditions and that lack of the complex II substrate succinate resulted in reversible membrane potential loss that could be restored rapidly by succinate supplementation. Inhibition of mitochondrial complex I and F₀F₁-ATP synthase induced mitochondrial depolarization that was independent of the mitochondrial permeability transition pore, Bcl-2 (B-cell lymphoma 2) family proteins, or high amplitude swelling and could not be reversed by succinate. Importantly, succinate metabolism under hypoxic conditions restores membrane potential and ATP levels. Furthermore, a reliance on complex II-mediated electron flow allows cells from mitochondrial disease patients devoid of a functional complex I to maintain a mitochondrial membrane potential that conveys both a mitochondrial structure and the ability to sequester agonist-induced calcium similar to that of normal cells. This finding is important as it sets the stage for complex II functional preservation as an attractive therapy to maintain mitochondrial function during hypoxia.