Respiratory supercomplexes: structure,function and assembly

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.     

The multicollisional, obstructed, long-range diffusional nature of mitochondrial electron transport

Data are presented which reveal that ubiquinone (Q)-mediated electron transport is a multicollisional, obstructed, long-range diffusion process, where factors that affect the rate of lateral diffusion also affect the rate of electron transport. Based on fluorescence recovery after photobleaching measurements, it was concluded that Q-mediated electron transport occurs by the random collision of redox components which are independent lateral diffusants, each greater than 86% mobile and diffusing in a common pool. The diffusion process of Q-mediated electron transport is 1) multicollisional since the transfers of reducing equivalents between appropriate redox partners occur with less than 100% collision efficiency; 2) obstructed since its maximal rate as well as the rates of diffusion of all redox components involved vary as a function of the membrane protein density; and 3) long-range since the diffusion of all redox components is protein density-dependent, and the diffusion distance required for Q to catalyze the transfer of a reducing equivalent from Complex II to III must be, on average, greater than 37.6 nm. These findings and other theoretical treatments reveal that measurements of short-range diffusion (less than 10 nm), in which collisions between appropriate redox partners do not occur, on average, and which are not affected by membrane protein density, are irrelevant to the collisional process of electron transport. Thus, the data show that the maximum electron transport rate is dependent on both the diffusion rate and the concentration of the redox components. Sucrose was found to inhibit both the mobility of redox components as well as their electron transport rates. Data presented on the relationships between membrane viscosity, rates of lateral and rotational diffusion, and mobile fractions of redox components do not support rotationally immobile aggregates in the functional inner membrane. The high degree of unsaturated phospholipids and the absence of cholesterol in the bilayer of the native inner membrane reflect a requirement for a low resistance to motion of the redox components to compensate for the multicollisional, obstructive nature of their catalytically important collisions in this membrane. These findings support the Random Collision Model of electron transport in which the diffusion and concentration of redox components limit the maximum rate of electron transport.     

The multiplicity of dehydrogenases in the electron transport chain of plant mitochondria

The electron transport chain in mitochondria of different organisms contains a mixture of common and specialised components. The specialised enzymes form branches to the universal electron path, especially at the level of ubiquinone, and allow the chain to adjust to different cellular and metabolic requirements. In plants, specialised components have been known for a long time. However, recently, the known number of plant respiratory chain dehydrogenases has increased, including both components specific to plants and those with mammalian counterparts. This review will highlight the novel branches and their consequences for the understanding of electron transport and redundancy of electron paths.     

Transplasma membrane electron transport in plants

The presence of transplasma membrane electron transport in a variety of plant cells and tissues is reported. It is now agreed that this property of eukaryotic cells is of ubiquitous nature. Studies with highly purified plasma membranes have established the presence of electron transport enzymes. Two types of activities have been identified. One, termed Standard reductase, is of general occurrence. The other, inducible under iron deficiency and relatively more active, is Turbo reductase. However, the true nature of components participating in electron transport and their organization in the plasma membrane is not known. The electron transport is associated with proton release and uses intracellular NAD(P)H as substrate. The electron flow leads to changes in intracellular redox status, pH, and metabolic energy. The responsiveness of this system to growth hormones is also observed. These findings suggest a role for electron flow across the plasma membrane in cell growth and regulation of ion transport. Involvement of this system in many other cellular functions is also argued.     

Glycosphingolipids are required for sorting melanosomal proteins in the Golgi complex.

Although glycosphingolipids are ubiquitously expressed and essential for multicellular organisms, surprisingly little is known about their intracellular functions. To explore the role of glycosphingolipids in membrane transport, we used the glycosphingolipid-deficient GM95 mouse melanoma cell line. We found that GM95 cells do not make melanin pigment because tyrosinase, the first and rate-limiting enzyme in melanin synthesis, was not targeted to melanosomes but accumulated in the Golgi complex. However, tyrosinase-related protein 1 still reached melanosomal structures via the plasma membrane instead of the direct pathway from the Golgi. Delivery of lysosomal enzymes from the Golgi complex to endosomes was normal, suggesting that this pathway is not affected by the absence of glycosphingolipids. Loss of pigmentation was due to tyrosinase mislocalization, since transfection of tyrosinase with an extended transmembrane domain, which bypassed the transport block, restored pigmentation. Transfection of ceramide glucosyltransferase or addition of glucosylsphingosine restored tyrosinase transport and pigmentation. We conclude that protein transport from Golgi to melanosomes via the direct pathway requires glycosphingolipids.     

Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens

Methane-forming archaea are strictly anaerobic microbes and are essential for global carbon fluxes since they perform the terminal step in breakdown of organic matter in the absence of oxygen. Major part of methane produced in nature derives from the methyl group of acetate. Only members of the genera Methanosarcina and Methanosaeta are able to use this substrate for methane formation and growth. Since the free energy change coupled to methanogenesis from acetate is only − 36 kJ/mol CH₄, aceticlastic methanogens developed efficient energy-conserving systems to handle this thermodynamic limitation. The membrane bound electron transport system of aceticlastic methanogens is a complex branched respiratory chain that can accept electrons from hydrogen, reduced coenzyme F₄₂₀ or reduced ferredoxin. The terminal electron acceptor of this anaerobic respiration is a mixed disulfide composed of coenzyme M and coenzyme B. Reduced ferredoxin has an important function under aceticlastic growth conditions and novel and well-established membrane complexes oxidizing ferredoxin will be discussed in depth. Membrane bound electron transport is connected to energy conservation by proton or sodium ion translocating enzymes (F₄₂₀H₂ dehydrogenase, Rnf complex, Ech hydrogenase, methanophenazine-reducing hydrogenase and heterodisulfide reductase). The resulting electrochemical ion gradient constitutes the driving force for adenosine triphosphate synthesis. Methanogenesis, electron transport, and the structure of key enzymes are discussed in this review leading to a concept of how aceticlastic methanogens make a living. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

The ins and outs of sphingolipid synthesis

Sphingolipids are ubiquitous components of eukaryotic cell membranes, where they play important roles in intracellular signaling and in membrane structure. Even though the biochemical pathway of sphingolipid synthesis and its compartmentalization between the endoplasmic reticulum and Golgi apparatus have been known for many years, the molecular identity of the enzymes in this pathway has only recently been elucidated. Here, we summarize progress in the identification and characterization of the enzymes, the transport of ceramide from the endoplasmic reticulum to the Golgi apparatus, and discuss how regulating the synthesis of sphingolipids might impact upon their functions.     

Light Modulation of Enzyme Activity in Chloroplasts: Generation of Membrane-bound Vicinal-Dithiol Groups by Photosynthetic Electron Transport

Inhibitor experiments indicate that photosynthetic electron transport is required for light activation of the pea (Pisum sativum) leaf chloroplast enzymes NADP-linked glyceraldehyde-3-phosphate dehydrogenase, NADP-linked malic dehydrogenase, ribulose-5-phosphate kinase and sedoheptulose-1,7-diphosphate phosphatase, and for inactivation of glucose-6-phosphate dehydrogenase. Modulation of the activity of the dehydrogenases and kinase apparently involves a component preceding ferredoxin in the photosynthetic electron transport chain; activation of the phosphatase involves an electron transport component at the level of ferredoxin. Modulation of enzyme activity can be obtained in a broken chloroplast system consisting of membrane fragments and stromal extract. The capacity for light regulation in this system is reduced or eliminated when the membrane fraction is exposed to arsenite in the light or to sulfite in light or dark. Light-generated vicinal-dithiols seem therefore to be involved in modulation of the activity of the enzymes included in this study.     

The bioenergetics of methanogenesis

The reduction of CO2 or any other methanogenic substrate to methane serves the same function as the reduction of oxygen, nitrate or sulfate to more reduced products. These exergonic reactions are coupled to the production of usable energy generated through a charge separation and a protonmotive-force-driven ATPase. For the understanding of how methanogens derive energy from C-1 unit reduction one must study the biochemistry of the chemical reactions involved and how these are coupled to the production of a charge separation and subsequent electron transport phosphorylation. Data on methanogenesis by a variety of organisms indicates ubiquitous use of CH3-S-CoM as the final electron acceptor in the production of methane through the methyl CoM reductase and of 5-deazaflavin as a primary source of reducing equivalents. Three known enzymes serve as catalysts in the production of reduced 5-deazaflavin: hydrogenase, formate dehydrogenase and CO dehydrogenase. All three are potential candidates for proton pumps. In the organisms that must oxidize some of their substrate to obtain electrons for the reduction of another portion of the substrate to methane (e.g., those using formate, methanol or acetate), the latter two enzymes may operate in the oxidizing direction. CO2 is the most frequent substrate for methanogenesis but is the only substrate that obligately requires the presence of H2 and hydrogenase. Growth on methanol requires a B12-containing methanol-CoM methyl transferase and does not necessarily need any other methanogenic enzymes besides the methyl-CoM reductase system when hydrogenase is present. When bacteria grow on methanol alone it is not yet clear if they get their reducing equivalents from a reversal of methanogenic enzymes, thus oxidizing methyl groups to CO2. An alternative (since these and acetate-catabolizing methanogens possess cytochrome b) is electron transport and possible proton pumping via a cytochrome-containing electron transport chain. Several of the actual components of the methanogenic pathway from CO2 have been characterized. Methanofuran is apparently the first carbon-carrying cofactor in the pathway, forming carboxy-methanofuran. Formyl-FAF or formyl-methanopterin (YFC, a very rapidly labelled compound during 14C pulse labeling) has been implicated as an obligate intermediate in methanogenesis, since methanopterin or FAF is an essential component of the carbon dioxide reducing factor in dialyzed extract methanogenesis. FAF also carries the carbon at the methylene and methyl oxidation levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization of microsomal electron transport components from control,phenobarbital, and 3-methylcholanthrene treated mice: I. Distribution of electron transport components in ammonium-sulfate fractions from mouse liver microsomes

Using a modified ammonium sulfate fractionation procedure, seven major components were found in various submicrosomal fractions. Four of these proteins could be assigned to known components of the microsomal electron transport system, cytochromes P-450 and b₅, and the NADH- and NADPH-cytochrome c reductases. The similarity of this system to that seen in the separation of mitochondrial enzymes suggests that the lipo-protein complexes of these fractions are particulate in nature and represent functional subunits of the microsomal membrane.     

Lateral enzyme topology in the rough endoplasmic reticulum of rat liver

Summary Rough microsomes were subfractionated on the basis of different properties in order to investigate the nature and extent of the enzyme heterogeneity of these vesicles. A discontinuous gradient, containing monovalent cations allowed the separation of a ribosome-poor membrane fraction which was enriched in electron transport enzymes and relatively poor in phosphatases. Zonal centrifugation on a stabilizing gradient separated 3 fractions characterized by enrichment of electron transport enzymes, glucose-6-phosphatase and adenosinetriphosphatase, respectively. An essentially similar pattern was seen when ribosomes were removed with EDTA and the denuded vesicles subfractionated on a sucrose gradient. Rough microsomes from phenobarbitaltreated rats exhibited the same pattern both qualitatively and quantitatively. It appears that electron transport enzymes and two types of phosphatases are heterogeneously distributed among rough microsomal vesicles.This work has been supported by grants from the Swedish Medical Research Council. The authors wish to thank Mrs. Ulla-Britta Torndal for her valuable technical assistance     

N-Terminal Structure of Maize Ferredoxin:NADP+ Reductase Determines Recruitment into Different Thylakoid Membrane Complexes

To adapt to different light intensities, photosynthetic organisms manipulate the flow of electrons through several alternative pathways at the thylakoid membrane. The enzyme ferredoxin:NADP(+) reductase (FNR) has the potential to regulate this electron partitioning because it is integral to most of these electron cascades and can associate with several different membrane complexes. However, the factors controlling relative localization of FNR to different membrane complexes have not yet been established. Maize (Zea mays) contains three chloroplast FNR proteins with totally different membrane association, and we found that these proteins have variable distribution between cells conducting predominantly cyclic electron transport (bundle sheath) and linear electron transport (mesophyll). Here, the crystal structures of all three enzymes were solved, revealing major structural differences at the N-terminal domain and dimer interface. Expression in Arabidopsis thaliana of maize FNRs as chimeras and truncated proteins showed the N-terminal determines recruitment of FNR to different membrane complexes. In addition, the different maize FNR proteins localized to different thylakoid membrane complexes on expression in Arabidopsis, and analysis of chlorophyll fluorescence and photosystem I absorbance demonstrates the impact of FNR location on photosynthetic electron flow.     

Electron transport system associated with direct glucose oxidation in Gluconobacter oxydans

Summary The mode of electron transport associated with the dehydrogenase enzymes located on the cytoplasmic membrane inGluconobacter oxydans (ATCC 9937) has been postulated. High turnover of dehydrogenases under oxygen enrichment conditions is explained on the basis of a simplistic electron transport chain comprising cytochrome c553 (MW 23000) as a subunit of dehydrogenase and a cytochrome b562. The electron transport chain under low dissolved oxygen tension (DOT) is shown to comprise a number of cytochrome c species with very low midpoint potential difference.     

Investigation of the transverse topology of the microsomal membrane using combinations of proteases and the non-penetrating reagent diazobenzene sulfonate

Intact microsomal vesicles from rat liver were subjected to combined treatment with trypsin and an unspecific protease and were also examined after reaction with the chemical probe p-diazobenzene sulfonate. In addition, the latency of various enzymes in intact microsomal vesicles has been investigated. All microsomal electron transport enzymes studied, i.e. NADH-ferricyanide and cytochrome c reductases, cytochrome b₅, NADPH-cytochrome c reductase and cytochrome P-450, were either solubilized or inactivated by one or both treatments. The experimental data indicate that UDPglucuronyl-transferase is also localized at the outer surface of microsomes. In contrast, a number of hydrolytic enzymes are apparently located inside the permeability barrier of the membrane and presumably at the inner surface. Under conditions where the levels of electron transport enzyme activities or amounts are changed, such as in newborn rats and rats treated with phenobarbital or methylcholanthrene, the intramembranous position of these enzymes is the same as in control adult rats. This indicates that the enzyme molecules are not relocated after their insertion into the membrane.     

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems

Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.     

Mechanisms and functions of photosystem I-related alternative electron transport pathways in chloroplasts

This review analyzes various alternative pathways of chloroplast electron transport mediated by photoreactions of photosystem I (PSI) and unrelated to activity of photosystem II (PSII). The mechanisms and functional significance of the alternative pathways are considered. These pathways are complexly organized and comprise ferredoxin-dependent electron recycling around PSI, as well as electron donation to noncyclic chain in the region between PSII and PSI from reduced substances localized in the chloroplast stroma. For each of the alternative pathways, the origin of corresponding enzymes and their compartmentalization in the complex membrane system of the chloroplast are discussed. It is shown that operation of alternative electron transport pathways contributes to energy transduction and cell defense function, facilitates the absorption of inorganic carbon, and is significant for chloroplast respiration. Multiple mechanisms for regulation of alternative pathways have been revealed. It is concluded that PSI-related alternative electron transport pathways constitute an integral part of entire system of photosynthetic electron transport, this system being principally responsible for energy supply of phototrophic cells and whole plants.     

Identification of elastases associated with purified plasma membranes isolated from human monocytes and lymphocytes

Studies were carried out to understand the pathogenesis of amyloid formation and to localize the elastase-like enzymes postulated to be associated with the surface of human peripheral blood monocytes and lymphocytes. These enzymes are known to degrade serum amyloid A and amyloid A proteins. Pure plasma membrane preparations were obtained by allowing cells to attach to polyacrylamide beads, followed by their disruption. The purity of the membranes was monitored by electron microscopy and enzyme determinations. The extracted membrane enzymes which have molecular weights of 56000 and 30000, respectively, were inhibited by DFP, MeO-Suc-Ala-Ala-Pro-Val-CH2Cl, Ac-Pro-Phe-Arg-CH2Cl . HCl, and elastinal but were not inhibited by EDTA or epsilon-amino caproic acid, thus exhibiting the properties of elastases. These enzymes cleave serum amyloid A to amyloid protein A. In some individuals, cleavage stops at this point, while in others a second step occurs, resulting in complete protein degradation. This activity was comparable whether monocyte or lymphocyte plasma membranes were employed. Since lymphocyte dependent cytotoxicity has also been attributed to surface proteases, it is likely that a spectrum of membrane associated enzymes mediate important physiologic function of these mononuclear leukocytes.     

Localization of enzymes in specialized regions of the microsomal membrane

Microsomal vesicle were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADPH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patchers in the microsomal membrane.     

Localization of enzymes in specialized regions of the microsomal membrane.

Microsomal vesicles were centrifuged through sucrose density gradients containing deoxycholate. With 0.15% detergent electron transport enzymes and phosphatases could be separated. Increasing the deoxycholate concentration to 0.19% resulted in separation of the microsomal material into five bands containing (in order from the top of the gradient) adenosine monophosphatase, inosine diphosphatase and some glucose-6-phosphatase (band 1); NADH-linked (band 2) and NADH-linked (band 3) electron transport enzymes; and glucose-6-phosphatase (bands 4 and 5). It appears that enzymes are arranged in specialized patches in the microsomal membrane.