BBA - Bioenergetics (v.1757, #5-6)

The peripheral stalk of the mitochondrial ATP synthase by John E. Walker; Veronica Kane Dickson (286-296).
The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 Å to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic α3β3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 Å from the membrane surface and then penetrates into the α3β3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the δ-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b′ (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.
Keywords: ATP synthase; Mitochondria; Peripheral stalk; Structure; Function;

Structural and functional features of yeast V-ATPase subunit C by Omri Drory; Nathan Nelson (297-303).
V-ATPase is a multi-subunit membrane protein complex, it translocates protons across biological membranes, generating electrical and pH gradients which are used for varieties of cellular processes. V-ATPase is composed of two distinct sub-complexes: a membrane bound V0 sub-complex, composed of 6 different subunits, which is responsible for proton transport and a soluble cytosolic facing V1 sub-complex, composed of 8 different subunits which hydrolyse ATP. The two sub-complexes are held together via a flexible stator. One of the main features of eukaryotic V-ATPase is its ability to reversibly dissociate to its sub-complexes in response to changing cellular conditions, which arrest both proton translocation and ATP hydrolysis, suggesting a regulation function. Subunit C (vma5p in yeast) was shown by several biochemical, genetic and recent structural data to function as a flexible stator holding the two sectors of the complex together and regulating the reversible association/dissociation of the complex, partly via association with F-actin filaments. Structural features of subunit C that allow smooth energy conversion and interaction with actin and nucleotides are discussed.
Keywords: V-ATPase; Subunit C; 3D structure; Mechanism; Conformation;

Fo·F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP·Mg2+-induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem., 279, 12319–12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 × 10−6 M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo·F1-ATP synthase.
Keywords: Fo·F1-ATP-synthase; ATP hydrolysis; Paracoccus denitrificans;

Subunit movements in membrane-integrated EF0F1 during ATP synthesis detected by single-molecule spectroscopy by Boris Zimmermann; Manuel Diez; Michael Börsch; Peter Gräber (311-319).
The H+-ATPsynthase from E. coli was isolated and labelled at the γ- or ε-subunit with tetramethylrhodamine, and at the b-subunits with bisCy5. The double labelled enzymes were incorporated into liposomes. They showed ATP hydrolysis activity, and, after energization of the membrane by ΔpH and Δϕ, also ATP synthesis activity was observed. Fluorescence resonance energy transfer (FRET) was used to investigate the movements of either the γ-subunit or the ε-subunit relative to the b-subunits in single membrane-integrated enzymes. The results show that during catalysis, the γ–ε complex rotates stepwise relative to the b-subunit. The direction of rotation during ATP synthesis is opposite to that during ATP hydrolysis. The stepwise motion is characterized by dwell times (docking time of the γ–ε complex to one αβ pair) up to several hundred ms, followed by a rapid movement of the γ- and ε-subunit to the next αβ pair within 0.2 ms. The same FRET levels (i.e., the same γ–b and ε–b distances) are observed during proton transport-coupled ATP hydrolysis and ATP synthesis, indicating that the reaction proceeds via the same intermediates in both directions. Under non-catalytic conditions, i.e., in the absence of ATP or without energization also, three FRET levels are found, however, the distances differ from those under catalytic conditions. We conclude that this reflects a movement of the ε-subunit during active/inactive transition.
Keywords: Single-molecule spectroscopy; Fluorescence resonance energy transfer; EF0F1; Fluorescence labelling; Subunit rotation; ATPsynthase;

Modulation of proton pumping efficiency in bacterial ATP synthases by Paola Turina; Alberto Rebecchi; Manuela D'Alessandro; Sofie Anefors; B. Andrea Melandri (320-325).
The ATP synthase in chromatophores of Rhodobacter caspulatus can effectively generate a transmembrane pH difference coupled to the hydrolysis of ATP. The rate of hydrolysis was rather insensitive to the depletion of ADP in the assay medium by an ATP regenerating system (phospho-enol-pyruvate (PEP) and pyruvate kinase (PK)). The steady state values of ΔpH were however drastically reduced as a consequence of ADP depletion. The clamped concentrations of ADP obtained using different PK activities in the assay medium could be calculated and an apparent K d≈0.5 μM was estimated. The extent of proton uptake was also strongly dependent on the addition of phosphate to the assay medium. The K d for this effect was about 70 μM. Analogous experiments were performed in membrane fragment from Escherichia coli. In this case, however, the hydrolysis rate was strongly inhibited by P i, added up to 3 mM. Inhibition by P i was nearly completely suppressed following depletion of ADP. The K d's for the ADP and P i were in the micromolar range and submillimolar range, respectively, and were mutually dependent from the concentration of the other ligand. Contrary to hydrolysis, the pumping of protons was rather insensitive to changes in the concentrations of the two ligands. At intermediate concentrations, proton pumping was actually stimulated, while the hydrolysis was inhibited. It is concluded that, in these two bacterial organisms, ADP and phosphate induce a functional state of the ATP synthase competent for a tightly coupled proton pumping, while the depletion of either one of these two ligands favors an inefficient (slipping) functional state. The switch between these states can probably be related to a structural change in the C-terminal α-helical hairpin of the ε-subunit, from an extended conformation, in which ATP hydrolysis is tightly coupled to proton pumping, to a retracted one, in which ATP hydrolysis and proton pumping are loosely coupled.
Keywords: ATP synthase; Proton pumping efficiency; Epsilon subunit; ADP binding; P i binding; Proton slipping;

The role of subunit epsilon in the catalysis and regulation of FOF1-ATP synthase by Boris A. Feniouk; Toshiharu Suzuki; Masasuke Yoshida (326-338).
The regulation of ATP synthase activity is complex and involves several distinct mechanisms. In bacteria and chloroplasts, subunit epsilon plays an important role in this regulation, (i) affecting the efficiency of coupling, (ii) influencing the catalytic pathway, and (iii) selectively inhibiting ATP hydrolysis activity. Several experimental studies indicate that the regulation is achieved through large conformational transitions of the α-helical C-terminal domain of subunit epsilon that occur in response to membrane energization, change in ATP/ADP ratio or addition of inhibitors. This review summarizes the experimental data obtained on different organisms that clarify some basic features as well as some molecular details of this regulatory mechanism. Multiple functions of subunit epsilon, its role in the difference between the catalytic pathways of ATP synthesis and hydrolysis and its influence on the inhibition of ATP hydrolysis by ADP are also discussed.
Keywords: ATP synthase; Regulation; Epsilon; Inhibition; ADP; Protonmotive force;

At least two features of the crystal structures of the cytochrome b 6 f complex from the thermophilic cyanobacterium, Mastigocladus laminosus and a green alga, Chlamydomonas reinhardtii, have implications for the pathways and mechanism of charge (electron/proton) transfer in the complex: (i) The narrow 11 × 12 Å portal between the p-side of the quinone exchange cavity and p-side plastoquinone/quinol binding niche, through which all Q/QH2 must pass, is smaller in the b 6 f than in the bc 1 complex because of its partial occlusion by the phytyl chain of the one bound chlorophyll a molecule in the b 6 f complex. Thus, the pathway for trans-membrane passage of the lipophilic quinone is even more labyrinthine in the b 6 f than in the bc 1 complex. (ii) A unique covalently bound heme, heme c n, in close proximity to the n-side b heme, is present in the b 6 f complex. The b 6 f structure implies that a Q cycle mechanism must be modified to include heme c n as an intermediate between heme b n and plastoquinone bound at a different site than in the bc 1 complex. In addition, it is likely that the heme b nc n couple participates in photosytem I-linked cyclic electron transport that requires ferredoxin and the ferredoxin: NADP+ reductase. This pathway through the n-side of the b 6 f complex could overlap with the n-side of the Q cycle pathway. Thus, either regulation is required at the level of the redox state of the hemes that would allow them to be shared by the two pathways, and/or the two different pathways are segregated in the membrane.
Keywords: Carbon fixation; Cytochrome b 6 f, bc 1 complexes; Cyclic electron transport; Heme c n; Q cycle;

A functional hybrid between the cytochrome bc 1 complex and its physiological membrane-anchored electron acceptor cytochrome c y in Rhodobacter capsulatus by Dong-Woo Lee; Yavuz Ozturk; Aygun Mamedova; Artur Osyczka; Jason W. Cooley; Fevzi Daldal (346-352).
The membrane integral ubihydroquinone (QH2): cytochrome (cyt) c oxidoreductase (or the cyt bc 1 complex) and its physiological electron acceptor, the membrane-anchored cytochrome c y (cyt c y), are discrete components of photosynthetic and respiratory electron transport chains of purple non-sulfur, facultative phototrophic bacteria of Rhodobacter species. In Rhodobacter capsulatus, it has been observed previously that, depending on the growth condition, absence of the cyt bc 1 complex is often correlated with a similar lack of cyt c y (Jenney, F. E., et al. (1994) Biochemistry 33, 2496–2502), as if these two membrane integral components form a non-transient larger structure. To probe whether such a structural super complex can exist in photosynthetic or respiratory membranes, we attempted to genetically fuse cyt c y to the cyt bc 1 complex. Here, we report successful production, and initial characterization, of a functional cyt bc 1-c y fusion complex that supports photosynthetic growth of an appropriate R. capsulatus mutant strain. The three-subunit cyt bc 1-c y fusion complex has an unprecedented bis-heme cyt c 1-c y subunit instead of the native mono-heme cyt c 1, is efficiently matured and assembled, and can sustain cyclic electron transfer in situ. The remarkable ability of R. capsulatus cells to produce a cyt bc 1-c y fusion complex supports the notion that structural super complexes between photosynthetic or respiratory components occur to ensure efficient cellular energy production.
Keywords: Photosynthetic and respiratory electron transfer; Membrane proteins supercomplexes; Cytochrome bc 1 complex; Electron carrier cytochrome c y; Rhodobacter capsulatus;

Recent X-ray structures determined for the Photosystem II (PSII) core complex isolated from cyanobacteria have provided important information for understanding the functionality of this photosynthetic enzyme including its water splitting activity. As yet, no high-resolution structure is available for PSII of plants or eukaryotes in general. However, crystal structures have been determined for some components of plant PSII which together with the cyanobacterial structure can be used to interpret lower resolution structures of plant PSII derived from electron cryomicroscopy (cryo-EM). Here, we utilise the published X-ray structures of a cyanobacterial PSII core, Light Harvesting Complex II (LHCII), PsbP and PsbQ proteins to construct a model of the plant LHCII–PSII supercomplex using a 17 Å resolution 3D electron density map of the spinach supercomplex determined by cryo-EM and single particle analysis. In so doing, we tentatively identify the relative positioning of the chlorophylls within the supercomplex and consider energy transfer pathways between the different subunits. The modelling has also allowed density to be assigned to the three extrinsic proteins of plant PSII, PsbO, PsbP and PsbQ associated with the water splitting centre and concluded that although the position of PsbO is the same as in cyanobacteria, PsbP and PsbQ are located in different positions to the cyanobacterial extrinsic PsbU and PsbV proteins.
Keywords: Photosynthesis; Photosystem II supercomplex; Electron microscopy; Single particle analysis; 3D structure;

Cyclic electron flow in C3 plants by Pierre Joliot; Anne Joliot (362-368).
This paper summarized our present view on the mechanism of cyclic electron flow in C3 plants. We propose that cyclic and linear pathways are in competition for the reoxidation of the soluble primary PSI acceptor, Ferredoxin (Fd), that freely diffuses in the stromal compartment. In the linear mode, Fd binds ferredoxin-NADP-reductase and electrons are transferred to NADP+ and then to the Benson and Calvin cycle. In the cyclic mode, Fd binds a site localized on the stromal side of the cytochrome b6f complex and electrons are transferred to P700 via a mechanism derived from the Q-cycle. In dark-adapted leaves, the cyclic flow operates at maximum rate, owing to the partial inactivation of the Benson and Calvin cycle. For increasing time of illumination, the activation of the Benson and Calvin cycle, and thus, that of the linear flow, is associated with a subsequent decrease in the rate of the cyclic flow. Under steady-state conditions of illumination, the contribution of cyclic flow to PSI turnover increases as a function of the light intensity (from 0 to ∼50% for weak to saturating light, respectively). Lack of CO2 is associated with an increase in the efficiency of the cyclic flow. ATP concentration could be one of the parameters that control the transition between linear and cyclic modes.
Keywords: Cyclic and Linear electron flows; Photosystem I; cytochrome b6f;

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition by Andrei G. Yakovlev; Tatiana A. Shkuropatova; Luidmila G. Vasilieva; Anatoli Ya. Shkuropatov; Peter Gast; Vladimir A. Shuvalov (369-379).
Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as ΦB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (ΦB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than ∼50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA ) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+BA . Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P* → P+BA in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the ΦB molecule. In the range of 740–795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and ΦB, the absorption difference spectra collected for mutant RCs at 30–50 fs resemble the difference spectrum of the P+ΦB charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471–1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of ΦB in the B-branch within about 40 fs, which is earlier by ∼80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.
Keywords: Femtosecond spectroscopy; Nuclear wave packet; Purple bacterial reaction center; Charge separation; Mutation; Pigment replacement;

The regulatory role of mitochondria in capacitative calcium entry by Jerzy Duszyński; Rafał Kozieł; Wojciech Brutkowski; Joanna Szczepanowska; Krzysztof Zabłocki (380-387).
Capacitative regulation of calcium entry is a major mechanism of Ca2+ influx into electrically non-excitable cells, but it also operates in some excitable ones. It participates in the refilling of intracellular calcium stores and in the generation of Ca2+ signals in excited cells. The mechanism which couples depletion of intracellular calcium stores located in the endoplasmic reticulum with opening of store-operated calcium channels in the plasma membrane is not clearly understood. Mitochondria located in close proximity to Ca2+ channels are exposed to high Ca2+ concentration, and therefore, they are able to accumulate this cation effectively. This decreases local Ca2+ concentration and thereby affects calcium-dependent processes, such as depletion and refilling of the intracellular calcium stores and opening of the store-operated channels. Finally, mitochondria modulate the intensity and the duration of calcium signals induced by extracellular stimuli. Ca2+ uptake by mitochondria requires these organelles to be in the energized state. On the other hand, Ca2+ flux into mitochondria stimulates energy metabolism. To sum up, mitochondria couple cellular metabolism with calcium homeostasis and signaling.
Keywords: Store-operated channel; Mitochondria; Capacitative calcium entry; Endoplasmic reticulum; Plasma membrane;

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase by Kristina Faxén; Lina Salomonsson; Pia Ädelroth; Peter Brzezinski (388-394).
Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of ≤250 μM to the outside of the vesicles did not alter the transition rates between intermediates P R (P 3 ) →  F 3  →  O 4 . However, proton pumping was impaired specifically during the P 3  →  F 3 , but not during the F 3  →  O 4 transition at Zn2+ concentrations of ≤ 25 μM. Furthermore, proton pumping during the P 3  →  F 3 transition was typically impaired with the “as isolated” CytcO, which was found to contain Zn2+ ions at μM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P 3  →  F 3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a K I of ∼ 1 μM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P 3  →  F 3 transition.
Keywords: Electron transfer; Proton transfer; Cytochrome aa3; Zn2+; Electrochemical proton gradient; Quinol oxidase; Rhodobacter sphaeroides;

Reaction mechanism of bovine heart cytochrome c oxidase by Shinya Yoshikawa; Kazumasa Muramoto; Kyoko Shinzawa-Itoh; Hiroshi Aoyama; Tomitake Tsukihara; Takashi Ogura; Kunitoshi Shimokata; Yukie Katayama; Hideo Shimada (395-400).
The 1.9 Å resolution X-ray structure of the O2 reduction site of bovine heart cytochrome c oxidase in the fully reduced state indicates trigonal planar coordination of CuB by three histidine residues. One of the three histidine residues has a covalent link to a tyrosine residue to ensure retention of the tyrosine at the O2 reduction site. These moieties facilitate a four electron reduction of O2, and prevent formation of active oxygen species. The combination of a redox-coupled conformational change of an aspartate residue (Asp51) located near the intermembrane surface of the enzyme molecule and the existence of a hydrogen bond network connecting Asp51 to the matrix surface suggest that the proton-pumping process is mediated at Asp51. Mutation analyses using a gene expression system of the Asp51-containing enzyme subunit yield results in support of the proposal that Asp51 plays a critical role in the proton pumping process.
Keywords: Cytochrome c oxidase; Heme protein; Membrane protein; Proton pump; O2 reduction; Electron transfer; X-ray crystallography; Resonance Raman spectroscopy; Site-directed mutagenesis;

Elementary steps of proton translocation in the catalytic cycle of cytochrome oxidase by Michael I. Verkhovsky; Ilya Belevich; Dmitry A. Bloch; Mårten Wikström (401-407).
Proton translocation in the catalytic cycle of cytochrome c oxidase (CcO) proceeds sequentially in a four-stroke manner. Every electron donated by cytochrome c drives the enzyme from one of four relatively stable intermediates to another, and each of these transitions is coupled to proton translocation across the membrane, and to uptake of another proton for production of water in the catalytic site. Using cytochrome c oxidase from Paracoccus denitrificans we have studied the kinetics of electron transfer and electric potential generation during several such transitions, two of which are reported here. The extent of electric potential generation during initial electron equilibration between CuA and heme a confirms that this reaction is not kinetically linked to vectorial proton transfer, whereas oxidation of heme a is kinetically coupled to the main proton translocation events during functioning of the proton pump. We find that the rates and amplitudes in multiphase heme a oxidation are different in the O H  →  E H and P M  →  F steps of the catalytic cycle, and that this is reflected in the kinetics of electric potential generation. We discuss this difference in terms of different driving forces and relate our results, and data from the literature, to proposed mechanisms of proton pumping in cytochrome c oxidase.
Keywords: Bioenergetics; Cytochrome c oxidase; Proton pump; Electron transfer; Oxygen reduction;

On the localized coupling of respiration and phosphorylation in mitochondria by Lev S. Yaguzhinsky; Vladimir I. Yurkov; Inna P. Krasinskaya (408-414).
This paper is an overview of the theoretical and experimental studies performed in our laboratory to answer the question whether there exist conditions where the hypothetical mechanism of the localized coupling of respiration and phosphorylation postulated by R. Williams in 1961 operates. These studies were undertaken to verify the earlier suggestion that mitochondria may exist in two structural and functional states. Correspondingly, there are two operation modes of oxidative phosphorylation, one of which corresponds to the Williams' mechanism of localized coupling and the other, to the Mitchell's mechanism of delocalized coupling. The paper considers the principle of the energy conservation of oxidative reactions in mitochondrial membranes in the form of the thermodynamic potential of hydrogen ions (Δμ sol) lacking, in part, the solvation shell. We present experimental evidence for the existence of the mechanism of localized coupling and describes the conditions favorable for its implementation. The experiments described in this paper show that the aforementioned models for proton coupling are not necessarily alternative. A conclusion is made that, depending on the particular conditions, either localized or delocalized coupling mechanisms of oxidative phosphorylation may come into operation.
Keywords: Membrane; Mitochondria; Localized coupling; Oxidative phosphorylation;

Proton in the well and through the desolvation barrier by Armen Y. Mulkidjanian (415-427).
The concept of the membrane proton well was suggested by Peter Mitchell to account for the energetic equivalence of the chemical (ΔpH) and electrical (Δψ) components of the proton-motive force. The proton well was defined as a proton-conducting crevice passing down into the membrane dielectric and able to accumulate protons in response to the generation either of Δψ or of ΔpH. In this review, the concept of proton well is contrasted to the desolvation penalty of > 500 meV for transferring protons into the membrane core. The magnitude of the desolvation penalty argues against deep proton wells in the energy-transducing enzymes. The shallow ΔpH- and Δψ-sensitive proton traps, mechanistically linked to the functional groups in the membrane interior, seem more realistic. In such constructs, the draw of a trapped proton into the membrane core can happen at the expense of some exergonic reaction, e.g., release of another proton from the membrane into the aqueous phase. It is argued that the proton transfer in the ATP synthase and the cytochrome bc complex could proceed in this way.
Keywords: Proton transfer; Solvation energy; Chemiosmotic theory; Membrane transport; Bacterial flagellum; FOF1-type ATP synthase; Cytochrome bc 1 complex; Rhodobacter capsulatus;

Functional and structural data are reviewed which provide evidence that proton pumping in cytochrome c oxidase is associated with extended allosteric cooperativity involving the four redox centers in the enzyme . Data are also summarized showing that the H+/e stoichiometry for proton pumping in the cytochrome span of the mitochondrial respiratory chain is flexible. The ΔpH component of the bulk-phase membrane electrochemical proton gradient exerts a decoupling effect on the proton pump of both the bc1 complex and cytochrome c oxidase. A slip in the pumping efficiency of the latter is also caused by high electron pressure. The mechanistic and physiological implications of proton-pump slips are examined. The easiness with which bulk phase ΔpH causes, at least above a threshold level, decoupling of proton pumping indicates that for active oxidative phosphorylation efficient protonic coupling between redox complexes and ATP synthase takes place at the membrane surface, likely in cristae, without significant formation of delocalized ΔμH+. A role of slips in modulating oxygen free radical production by the respiratory chain and the mitochondrial pathway of apoptosis is discussed.
Keywords: Respiratory chain; Proton pumps; Cytochrome c oxidase; bc1 complex;

A key component in cellular bioenergetics is the ATP synthase. The enzyme from archaea represents a new class of ATPases, the A1AO ATP synthases. They are composed of two domains that function as a pair of rotary motors connected by a central and peripheral stalk(s). The structure of the chemically-driven motor (A1) was solved by small angle X-ray scattering in solution, and the structure of the first A1AO ATP synthases (from methanoarchaea) was obtained recently by single particle analyses. These studies revealed novel structural features such as a second peripheral stalk and a collar-like structure. Interestingly, the membrane-embedded electrically-driven motor (AO) is very different in archaea with sometimes novel, exceptional subunit composition.
Keywords: Archaea; Bioenergetics; A1AO ATP synthase; Evolution;

A new look at UCP 1 by Richard K. Porter (446-448).
There has been a resurgence of interest in mitochondrial uncoupling protein 1 due to a desire to understand the regulation of the prominent role it plays in control of metabolic flux in brown adipose tissue and non-shivering thermogenesis, combined with the fact that UCP 1 acts as a paradigm for other novel less abundant uncoupling proteins. In this manuscript, we review the recent evidence for detection, purification, identification and function of UCP 1 in thymus mitochondria. In addition, we review the two proposed mechanisms for fatty acid dependent UCP 1 activity, namely (a) the flippase (flip-flop) model and (b) the cofactor/activation model, and the implication for these models of recent data showing that glucose-O-ω-palmitate cannot facilitate UCP 1 activity.
Keywords: Uncoupling protein-1; Thymus; Thymocytes; Mitochondria; Oxygen consumption, Glucose-O-ω-palmitate;

Uncoupling proteins: A role in protection against reactive oxygen species—or not? by Barbara Cannon; Irina G. Shabalina; Tatiana V. Kramarova; Natasa Petrovic; Jan Nedergaard (449-458).
A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the “novel” UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.

The physiological regulation of uncoupling proteins by David G. Nicholls (459-466).
Despite the enormous interest in the roles of novel uncoupling proteins, there is still great uncertainty as to their mechanism and regulation. The regulation of the architypal uncoupling protein 1 from brown adipose tissue was elucidated more than 20 years ago. This review suggests that a number of the approaches and criteria developed for the study of UCP1 could with profit be applied to current investigations of the novel UCPs.
Keywords: Uncoupling protein; Mitochondria; Purine nucleotide; Fatty acid; Thermogenesis; Membrane potential; Proton conductance;

Certain aspects of uncoupling due to mitochondrial uncoupling proteins in vitro and in vivo by Andrea Dlasková; Tomáš Špaček; Eva Škobisová; Jitka Šantorová; Petr Ježek (467-473).
Thermogenic uncoupling has been proven only for UCP1 in brown adipose tissue. All other isoforms of UCPs are potentially acting in suppression of mitochondrial reactive oxygen species (ROS) production. In this contribution we show that BAT mitochondria can be uncoupled by lauric acid in the range of ∼100 nM when endogenous fatty acids are combusted by carnitine cycle and β-oxidation is properly separated from the uncoupling effect. Respiration increased up to 3 times when related to the lowest fatty acid content (BSA present plus carnitine cycle). We also illustrated that any effect leading to more coupled states leads to enhanced H2O2 generation and any effect resulting in uncoupling gives reduced H2O2 generation in BAT mitochondria. Finally, we report doubling of plant UCP transcript in cells as well as amount of protein detected by 3H-GTP-binding sites in mitochondria of shoots and roots of maize seedlings subjected to the salt stress.
Keywords: Fatty acid-induced uncoupling; Brown adipose tissue mitochondria; Uncoupling protein-1; Carnitine cycle; Maize root and shoot mitochondria;

A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1 by Valeri Beck; Martin Jabůrek; Eamon P. Breen; Richard K. Porter; Petr Ježek; Elena E. Pohl (474-479).
Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (∼107) into a single bilayer. Despite a comparatively large membrane area (up to 300 μm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to ∼200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.
Keywords: Gramicidin; Oleic acid; Arachidonic acid; Brown adipose tissue; Human recombinant protein; Artificial membranes;

Mitochondrial UCPs: New insights into regulation and impact by Francis E. Sluse; Wieslawa Jarmuszkiewicz; Rachel Navet; Pierre Douette; Gregory Mathy; Claudine M. Sluse-Goffart (480-485).
Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.
Keywords: Mitochondria; Uncoupling protein; Metabolic regulation; Mitoproteome;

Fenofibrate activates the biochemical pathways and the de novo expression of genes related to lipid handling and uncoupling protein-3 functions in liver of normal rats by Elena Silvestri; Pieter de Lange; Maria Moreno; Assunta Lombardi; Maurizio Ragni; Anna Feola; Luigi Schiavo; Fernando Goglia; Antonia Lanni (486-495).
Fibrates (anti-hyperlipidemic agents) enhance the mRNA expression of uncoupling protein 2 (UCP2) in the liver and that of uncoupling protein 3 (UCP3) in skeletal muscle in standard-diet-fed rats and induce a de novo expression of UCP3 (mRNA and protein) in the liver of high-fat-fed rats. Here, we report that in the liver of normal rats, fenofibrate induces a de novo expression of UCP3 and a 6-fold increase in UCP2 mRNA, whereas UCP2 protein was not detectable. Indeed, we evidenced an ORF in UCP2 exon 2 potentially able to inhibit the expression of the protein. Fenofibrate increases the expression and activity of hepatic enzymes and cofactors involved in lipid handling and UCP3 activity and, as is the case for UCP3, induces other muscle-specific genes (e.g., Carnitine palmitoyl transferase 1b and Ubiquinone biosynthesis protein COQ7 homolog). In addition, we demonstrated that in mitochondria from fenofibrate-treated rats a palmitoyl-carnitine-induced GDP-sensitive uncoupling takes place, involving UCP3 rather than other uncouplers (i.e., UCP2 and Adenine Nucleotide Translocase). Thus, the liver of fenofibrate-treated standard-diet- fed rat is a useful model for investigations of the biochemical functions of UCP3 and allowed us to demonstrate that fenofibrate programs a gene-expression pattern able to modulate lipid handling and UCP3 activation.
Keywords: Fenofibrate; Uncoupling protein; Adenine nucleotide translocase; Mitochondrial thioesterase I; Mitochondrial uncoupling; Liver;

Caloric restriction (CR) decreases aging rate and mitochondrial ROS (MitROS) production and oxidative stress in rat postmitotic tissues. Low levels of these parameters are also typical traits of long-lived mammals and birds. However, it is not known what dietary components are responsible for these changes during CR. It was recently observed that 40% protein restriction without strong CR also decreases MitROS generation and oxidative stress. This is interesting because protein restriction also increases maximum longevity (although to a lower extent than CR) and is a much more practicable intervention for humans than CR. Moreover, it was recently found that 80% methionine restriction substituting it for l-glutamate in the diet also decreases MitROS generation in rat liver. Thus, methionine restriction seems to be responsible for the decrease in ROS production observed in caloric restriction. This is interesting because it is known that exactly that procedure of methionine restriction also increases maximum longevity. Moreover, recent data show that methionine levels in tissue proteins negatively correlate with maximum longevity in mammals and birds. All these suggest that lowering of methionine levels is involved in the control of mitochondrial oxidative stress and vertebrate longevity by at least two different mechanisms: decreasing the sensitivity of proteins to oxidative damage, and lowering of the rate of ROS generation at mitochondria.
Keywords: Mitochondria; Methionine restriction; Caloric restriction; Free radical; Aging; DNA damage;

Mitochondrial ROS-induced ROS release: An update and review by Dmitry B. Zorov; Magdalena Juhaszova; Steven J. Sollott (509-517).
Unstable mitochondrial membrane potential and redox transitions can occur following insults including ischemia/reperfusion injury and toxin exposure, with negative consequences for mitochondrial integrity and cellular survival. These transitions can involve mechanisms such as the recently described process, “Reactive Oxygen Species (ROS)-induced ROS-release” (RIRR), and be generated by circuits where the mitochondrial permeability transition (MPT) pore and the inner membrane anion channel (IMAC) are involved. The exposure to excessive oxidative stress results in an increase in ROS reaching a threshold level that triggers the opening of one of the requisite mitochondrial channels. In turn, this leads to the simultaneous collapse of the mitochondrial membrane potential and a transient increased ROS generation by the electron transfer chain. Generated ROS can be released into cytosol and trigger RIRR in neighboring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production leading to potentially significant mitochondrial and cellular injury. This review and update considers a variety of RIRR mechanisms (involving MPT, IMAC and episodes of mitochondrial transient hyperpolarization). RIRR could be a general cell biology phenomenon relevant to the processes of programmed mitochondrial destruction and cell death, and may contribute to other mechanisms of post-ischemic pathologies, including arrhythmias.
Keywords: Oxidative stress; Membrane potential; Permeability transition pore; Redox; Cardiac myocytes; Oscillation;

Effect of oxidative stress on dynamics of mitochondrial reticulum by O.Yu. Pletjushkina; K.G. Lyamzaev; E.N. Popova; O.K. Nepryakhina; O.Yu. Ivanova; L.V. Domnina; B.V. Chernyak; V.P. Skulachev (518-524).
Fission of the mitochondrial reticulum (the thread–grain transition) and following gathering of mitochondria in the perinuclear area are induced by oxidative stress. It is shown that inhibitors of the respiratory chain (piericidin and myxothiazol) cause fission of mitochondria in HeLa cells and fibroblasts, whereas a mitochondria-targeted antioxidant (MitoQ) inhibits this effect. Hydrogen peroxide also induced the fission, which was stimulated by the inhibitors of respiration and suppressed by MitoQ. In untreated cells, the mitochondrial reticulum consisted of numerous electrically-independent fragments. Prolonged treatment with MitoQ resulted in drastic increase in size and decrease in number of these fragments. Local photodamage of mitochondria caused immediate depolarization of a large fraction of the mitochondrial network in MitoQ-treated cells. Our data indicate that the thread–grain transition of mitochondria depends on production of reactive oxygen species (ROS) in initial segments of the respiratory chain and is a necessary step in the process of elimination of mitochondria (mitoptosis).
Keywords: Metochondrial reticulum; Fission; Fusion; Metochondrial inhibitors; Reactive oxygen species (ROS); Antioxidants;

Production of reactive oxygen species in mitochondria of HeLa cells under oxidative stress by Boris V. Chernyak; Denis S. Izyumov; Konstantin G. Lyamzaev; Alina A. Pashkovskaya; Olga Y. Pletjushkina; Yuri N. Antonenko; Dmitrii V. Sakharov; Karel W.A. Wirtz; Vladimir P. Skulachev (525-534).
Mitochondria can be a source of reactive oxygen species (ROS) and a target of oxidative damage during oxidative stress. In this connection, the effect of photodynamic treatment (PDT) with Mitotracker Red (MR) as a mitochondria-targeted photosensitizer has been studied in HeLa cells. It is shown that MR produces both singlet oxygen and superoxide anion upon photoactivation and causes photoinactivation of gramicidin channels in a model system (planar lipid bilayer). Mitochondria-targeted antioxidant (MitoQ) inhibits this effect. In living cells, MR-mediated PDT initiates a delayed (“dark”) accumulation of ROS, which is accelerated by inhibitors of the respiratory chain (piericidin, rotenone and myxothiazol) and inhibited by MitoQ and diphenyleneiodonium (an inhibitor of flavin enzymes), indicating that flavin of Complex I is involved in the ROS production. PDT causes necrosis that is prevented by MitoQ. Treatment of the cell with hydrogen peroxide causes accumulation of ROS, and the effects of inhibitors and MitoQ are similar to that described for the PDT model. Apoptosis caused by H2O2 is augmented by the inhibitors of respiration and suppressed by MitoQ. It is concluded that the initial segments of the respiratory chain can be an important source of ROS, which are targeted to mitochondria, determining the fate of the cell subjected to oxidative stress.
Keywords: Mitochondria; Reactive oxygen species; Oxidative stress; Photo dynamic treatment;

Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol by Alberto Boveris; Laura B. Valdez; Tamara Zaobornyj; Juanita Bustamante (535-542).
Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0–6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7–2.0 times for NO and 4–16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24–53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.
Keywords: Mitochondrial membrane potential; mtNOS; NO release; H2O2 release; Voltage-dependent enzyme activity;

Heart failure is a complex syndrome of numerous dysfunctional components which converge to cause chronic progressive failure of ventricular contractile function and maintenance of cardiac output demand. The aim of this brief review is to highlight some of the mounting evidence indicating that augmented superoxide, related reactive oxygen species and other free radicals contribute to the oxidative stress evident during the progression of heart failure. While much of the source of increased reactive oxygen species is mitochondrial, there are other intracellular sources, which together are highly reactive with functional and structural cellular lipids and proteins. Bioenergetic defects limiting ATP synthesis in the failing myocardium relate not only to post-translational modification of electron transport respiratory chain proteins but also to perturbation of Krebs Cycle enzyme-dependent synthesis of NADH. Accumulation of pathological levels of lipid peroxides relate to dysfunction in the intrinsic capacity to clear and renew dysfunctional proteins. This review also features key limitations of human heart failure studies and potential clinical therapies that target the elevated oxidative stress that is a hallmark of human heart failure.
Keywords: Heart failure; Reactive oxygen species; Mitochondria; Krebs cycle enzyme; Antioxidant; Cardiolipin; Omega-3 PUFA;

Generation of superoxide by the mitochondrial Complex I by Vera G. Grivennikova; Andrei D. Vinogradov (553-561).
Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2 ·−) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2 ·− production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 μM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent K i's close to their K m's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.
Keywords: Superoxide generation; NADH:ubiquinone reductase; Complex I; Respiratory chain (Bovine heart submitochondrial particles);

We found that reversible inactivation of mitochondrial complex I by S-nitroso-N-acetyl-d,l-penicillamine (SNAP) in isolated rat heart mitochondria resulted in a three-fold increase in H2O2 production, when mitochondria were respiring on pyruvate and malate, (but not when respiring on succinate or in the absence of added respiratory substrate). The inactivation of complex I and the increased H2O2 production were present in mitochondria washed free of SNAP or NO, but were partially reversed by light or dithiothreitol, treatments known to reverse S-nitrosation. Specific inhibition of complex I with rotenone increased H2O2 production to a similar extent as that caused by SNAP. The results suggest that S-nitrosation of complex I can reversibly increase oxidant production by mitochondria, which is potentially important in cell signalling and/or pathology.
Keywords: Mitochondria; Reactive oxygen species; Nitric oxide;

HIFα prolyl hydroxylases (PHDs) are a family of enzymes that regulate protein levels of the α subunit of the hypoxia inducible transcription factor (HIF) under different oxygen levels. PHDs catalyse the conversion of a prolyl residue, molecular oxygen and α-ketoglutarate to hydroxy-prolyl, carbon dioxide and succinate in a reaction dependent on ferrous iron and ascorbate as cofactors. Recently it was shown that pseudo-hypoxia, HIF induction under normoxic conditions, is an important feature of tumours generated as a consequence of inactivation of the mitochondrial tumour suppressor ‘succinate dehydrogenase’ (SDH). Two models have been proposed to describe the link between SDH inhibition and HIF activation. Both models suggest that a mitochondrial-generated signal leads to the inhibition of PHDs in the cytosol, however, the models differ in the nature of the proposed messenger. The first model postulates that mitochondrial-generated hydrogen peroxide mediates signal transduction while the second model implicates succinate as the molecular messenger which leaves the mitochondrion and inhibits PHDs in the cytosol. Here we show that pseudo-hypoxia can be observed in SDH-suppressed cells in the absence of oxidative stress and in the presence of effective antioxidant treatment.
Keywords: Mitochondria; Cancer; Reactive oxygen species; Succinate dehydrogenase;

Melatonin as antioxidant, geroprotector and anticarcinogen by Vladimir N. Anisimov; Irina G. Popovich; Mark A. Zabezhinski; Sergey V. Anisimov; Georgy M. Vesnushkin; Irina A. Vinogradova (573-589).
The effect of the pineal indole hormone melatonin on the life span of mice, rats and fruit flies has been studied using various approaches. It has been observed that in female CBA, SHR, SAM and transgenic HER-2/neu mice long-term administration of melatonin was followed by an increase in the mean life span. In rats, melatonin treatment increased survival of male and female rats. In D. melanogaster, supplementation of melatonin to nutrient medium during developmental stages produced contradictory results, but and increase in the longevity of fruit flies has been observed when melatonin was added to food throughout the life span. In mice and rats, melatonin is a potent antioxidant both in vitro and in vivo. Melatonin alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at high concentration in the COMET assay. Melatonin has inhibited mutagenesis and clastogenic effect of a number of indirect chemical mutagens. Melatonin inhibits the development of spontaneous and 7-12-dimethlbenz(a)anthracene (DMBA)- or N-nitrosomethylurea-induced mammary carcinogenesis in rodents; colon carcinogenesis induced by 1,2-dimethylhydrazine in rats, N-diethylnitrosamine-induced hepatocarcinogenesis in rats, DMBA-induced carcinogenesis of the uterine cervix and vagina in mice; benzo(a)pyrene-induced soft tissue carcinogenesis and lung carcinogenesis induced by urethan in mice. To identify molecular events regulated by melatonin, gene expression profiles were studied in the heart and brain of melatonin-treated CBA mice using cDNA gene expression arrays (15,247 and 16,897 cDNA clone sets, respectively). It was shown that genes controlling the cell cycle, cell/organism defense, protein expression and transport are the primary effectors for melatonin. Melatonin also increased the expression of some mitochondrial genes (16S, cytochrome c oxidases 1 and 3 (COX1 and COX3), and NADH dehydrogenases 1 and 4 (ND1 and ND4)), which agrees with its ability to inhibit free radical processes. Of great interest is the effect of melatonin upon the expression of a large number of genes related to calcium exchange, such as Cul5, Dcamkl1 and Kcnn4; a significant effect of melatonin on the expression of some oncogenesis-related genes was also detected. Thus, we believe that melatonin may be used for the prevention of premature aging and carcinogenesis.
Keywords: Melatonin; Free radicals; Gene activity; Mutagenesis; Life span; Longevity; Tumorigenesis; Mouse; Rat; Fruit fly;

Properties of the permeability transition in VDAC1 −/− mitochondria by Alexandra Krauskopf; Ove Eriksson; William J. Craigen; Michael A. Forte; Paolo Bernardi (590-595).
Opening of the permeability transition pore (PTP), a high-conductance mitochondrial channel, causes mitochondrial dysfunction with Ca2+ deregulation, ATP depletion, release of pyridine nucleotides and of mitochondrial apoptogenic proteins. Despite major efforts, the molecular nature of the PTP remains elusive. A compound library screening led to the identification of a novel high affinity PTP inhibitor (Ro 68-3400), which labeled a ∼32 kDa protein that was identified as isoform 1 of the voltage-dependent anion channel (VDAC1) [A.M. Cesura, E. Pinard, R. Schubenel, V. Goetschy, A. Friedlein, H. Langen, P. Polcic, M.A. Forte, P. Bernardi, J.A. Kemp, The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore. J. Biol. Chem. 278 (2003) 49812–49818]. In order to assess the role of VDAC1 in PTP formation and activity, we have studied the properties of mitochondria from VDAC1 −/− mice. The basic properties of the PTP in VDAC1 −/− mitochondria were indistinguishable from those of strain-matched mitochondria from wild-type CD1 mice, including inhibition by Ro 68-3400, which labeled identical proteins of 32 kDa in both wild-type and VDAC1 −/− mitochondria. The labeled protein could be separated from all VDAC isoforms. While these results do not allow to exclude that VDAC is part of the PTP, they suggest that VDAC is not the target for PTP inhibition by Ro 68-3400.
Keywords: Mitochondria; Permeability transition; VDAC1;

Mitochondrial DNA mutations cause resistance to opening of the permeability transition pore by Justin L. Mott; Dekui Zhang; Shin-Wen Chang; H. Peter Zassenhaus (596-603).
The age-related accumulation of mitochondrial DNA mutations has the potential to impair organ function and contribute to disease. In support of this hypothesis, accelerated mitochondrial mutagenesis is pathogenic in the mouse heart, and there is an increase in myocyte apoptosis. The current study sought to identify functional alterations in cell death signaling via mitochondria. Of particular interest is the mitochondrial permeability transition pore, opening of which can initiate cell death, while pore inhibition is protective. Here, we show that mitochondria from transgenic mice that develop mitochondrial DNA mutations have a marked inhibition of calcium-induced pore opening. Temporally, inhibited pore opening coincides with disease. Pore inhibition also correlates with an increase in Bcl-2 protein integrated into the mitochondrial membrane. We hypothesized that pore inhibition was mediated by mitochondrial Bcl-2. To test this hypothesis, we treated isolated mitochondria with Bcl-2 antagonistic peptides (derived from the BH3 domain of Bax or Bid). These peptides released the inhibition to pore opening. The data are consistent with a Bcl-2-mediated inhibition of pore opening. Thus, mitochondrial DNA mutations induce an adaptive–protective response in the heart that inhibits opening of the mitochondrial permeability pore.
Keywords: Apoptosis; mtDNA; Bcl-2; Mitochondria; Calcium; Permeability transition pore;

Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina by Séverine Lorin; Eric Dufour; Annie Sainsard-Chanet (604-610).
The filamentous fungus Podospora anserina has a limited lifespan. In this organism, aging is systematically associated to mitochondrial DNA instability. We recently provided evidence that the respiratory function is a key determinant of its lifespan. Loss of function of the cytochrome pathway leads to the compensatory induction of an alternative oxidase, to a decreased production of reactive oxygen species and to a striking increase in lifespan. These changes are associated to the stabilization of the mitochondrial DNA. Here we review and discuss the links between these different parameters and their implication in the control of lifespan. Since we demonstrated the central role of mitochondrial metabolism in aging, the same relationship has been evidenced in several model systems from yeast to mice, confirming the usefulness of simple organisms as P. anserina for studying lifespan regulation.
Keywords: Mitochondrion; Aging; Respiratory chain; Alternative oxidase; ROS; Mitochondrial DNA stability; Podospora anserina;

Mitochondrial DNA and ageing by Aleksandra Trifunovic (611-617).
The accumulation of mitochondrial DNA mutations has been proposed as a potential mechanism in the physiological processes of ageing and age-related disease. Although mitochondria have long been anticipated as a perpetrator of ageing, there was little experimental evidence to link these changes directly with the cellular pathology of ageing. Recently, considerable progress in understanding basic mitochondrial genetics and in identifying acquired mtDNA mutations in ageing has been made. Furthermore, the creation of mtDNA-mutator mice has provided the first direct evidence that accelerating the mtDNA mutation rate can result in premature ageing, consistent with the view that loss of mitochondrial function is a major causal factor in ageing. This review will, therefore, focus on recent developments in ageing research related to the role played by mtDNA.
Keywords: Mitochondria; Ageing; mtDNA mutations; mtDNA deletion;

To support the rapid growth of tumors, the cell can respond by increasing the number of mitochondria, in a concerted biosynthesis of mitochondrial constituents (nuclear and mitochondria encoded). Increased transcription, availability and stability of oxidative phosphorylation mRNAs, without increasing mitochondria number could also lead to more rapid energy-yielding effects. Mitochondria biogenesis and de novo formation of respiratory chain components imply coordination of nuclear and mt gene transcription. The mitochondrial mass is regulated by a number of physiopathological conditions. In response to external stimuli, mitochondria biogenesis is dependent on an orchestrated crosstalk between the nuclear and the mitochondrial genomes. Based on the higher incidence of glycolysis over oxidative phosphorylation in cancer tissues, we studied by differential proteomics the energy metabolism pathway of matched samples of normal and cancer tissue. Our results indicated that oxidative phosphorylation in cancer cells seemed altered because there is an unbalanced coordination between nuclear- and mitochondria-encoded mitochondrial proteins.
Keywords: Mitochondria; Bioenergetics; Proteomics; Cancer; Differential expression;

Mitochondrial DNA copy number is regulated by cellular proliferation: A role for Ras and p66Shc by Mirella Trinei; Ina Berniakovich; Pier Giuseppe Pelicci; Marco Giorgio (624-630).
The abundance of mitochondria is regulated by biogenesis and division. These processes are controlled by cellular factors, given that, for example, mitochondria have to replicate their DNA prior to cell division. However, the mechanisms that allow a synchronization of cell proliferation with mitochondrial genome replication are still obscure. We report here our investigations on the role of proliferation and the contribution of Ras and p66Shc in the regulation of mitochondrial DNA copy number. Ras proteins mediate a variety of receptor-transduced mitogenic signals and appear to play an essential role in the cellular response to growth factors. P66Shc is a genetic determinant of life span in mammals and has been implicated in the regulation of receptor signaling and various mitochondrial functions. First, we confirmed previous reports showing that mitochondrial DNA is replicated during a specific phase of the cell cycle (the pre-S phase) and provided novel evidences that this process is regulated by mitogenic growth factors. Second, we showed that mitochondrial DNA replication is activated following Ras-induced cellular hyper-proliferation. Finally, we showed that p66Shc expression induces mitochondrial DNA replication, both in vitro and in vivo. We suggest that mitochondria are target of intracellular signaling pathways leading to proliferation, involving Ras and p66Shc, which might function to integrate cellular bio-energetic requirements and the inheritance of mitochondrial DNA in a cell cycle-dependent manner.
Keywords: DNA; Ras signaling; p66Shc and aging;

MMI1 (YKL056c, TMA19), the yeast orthologue of the translationally controlled tumor protein (TCTP) has apoptotic functions and interacts with both microtubules and mitochondria by Mark Rinnerthaler; Stefanie Jarolim; Gino Heeren; Elfriede Palle; Simona Perju; Harald Klinger; Edith Bogengruber; Frank Madeo; Ralf J. Braun; Lore Breitenbach-Koller; Michael Breitenbach; Peter Laun (631-638).
The yeast orthologue of mammalian TCTP is here proposed to be named Mmi1p (microtubule and mitochondria interacting protein). This protein displays about 50% amino acid sequence identity with its most distantly related orthologs in higher organisms and therefore probably belongs to a small class of yeast proteins which have housekeeping but so far incompletely known functions needed for every eukaryotic cell. Previous investigations of the protein in both higher cells and yeast revealed that it is highly expressed during active growth, but transcriptionally down-regulated in several kinds of stress situations including starvation stress. In human cells, TCTP presumably has anti-apoptotic functions as it binds to Bcl-XL in vivo. TCTP of higher cells was also shown to interact with the translational machinery. It has acquired an additional function in the mammalian immune system, as it is identical with the histamine releasing factor. Here, we show that in S. cerevisiae induction of apoptosis by mild oxidative stress, replicative ageing or mutation of cdc48 leads to translocation of Mmi1p from the cytoplasm to the mitochondria. Mmi1p is stably but reversibly attached to the outer surface of the mitochondria and can be removed by digestion with proteinase K. Glutathionylation of Mmi1p, which is also induced by oxidants, is not a prerequisite or signal for translocation as shown by replacing the only cysteine of Mmi1p by serine. Mmi1p probably interacts with yeast microtubules as deletion of the gene confers sensitivity to benomyl. Conversely, the deletion mutant displays resistance to hydrogen peroxide stress and shows a small but significant elongation of the mother cell-specific lifespan. Our results so far indicate that Mmi1p is one of the few proteins establishing a functional link between microtubules and mitochondria which may be needed for correct localization of mitochondria during cell division.
Keywords: TCTP; Mitochondria; Microtubules; Apoptosis; Oxidative stress; Ageing, heat shock;

Multiple pathways of cytochrome c release from mitochondria in apoptosis by Vladimir Gogvadze; Sten Orrenius; Boris Zhivotovsky (639-647).
Release of cytochrome c from mitochondria is a key initiative step in the apoptotic process, although the mechanisms regulating permeabilization of the outer mitochondrial membrane and the release of intermembrane space proteins remain controversial. Here, we discuss possible scenarios of the outer membrane permeabilization. The mechanisms by which the intermembrane space proteins are released from mitochondria depend presumably on cell type and on the nature of the apoptotic stimulus. The variety of mechanisms that can lead to outer membrane permeabilization might explain diversities in the response of mitochondria to numerous apoptotic stimuli in different types of cells.
Keywords: Apoptosis; Mitochondria; Cytochrome c; Bcl-2 family; Permeability transition;

Apoptotic interactions of cytochrome c: Redox flirting with anionic phospholipids within and outside of mitochondria by H. Bayir; B. Fadeel; M.J. Palladino; E. Witasp; I.V. Kurnikov; Y.Y. Tyurina; V.A. Tyurin; A.A. Amoscato; J. Jiang; P.M. Kochanek; S.T. DeKosky; J.S. Greenberger; A.A. Shvedova; V.E. Kagan (648-659).
Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some – as yet unidentified – role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.
Keywords: Cytochrome c; Phospholipid; Mitochondrion;

Expression of an expanded polyglutamine domain in yeast causes death with apoptotic markers by Sviatoslav Sokolov; Andrey Pozniakovsky; Natalia Bocharova; Dmitry Knorre; Fedor Severin (660-666).
Huntington's disease is caused by specific mutations in huntingtin protein. Expansion of a polyglutamine (polyQ) repeat of huntingtin leads to protein aggregation in neurons followed by cell death with apoptotic markers. The connection between the aggregation and the degeneration of neurons is poorly understood. Here, we show that the physiological consequences of expanded polyQ domain expression in yeast are similar to those in neurons. In particular, expression of expanded polyQ in yeast causes apoptotic changes in mitochondria, caspase activation, nuclear DNA fragmentation and death. Similar to neurons, at the late stages of expression the expanded polyQ accumulates in the nuclei and seems to affect the cell cycle of yeast. Interestingly, nuclear localization of the aggregates is dependent on functional caspase Yca1. We speculate that the aggregates in the nuclei disturb the cell cycle and thus contribute to the development of the cell death process in both systems. Our data show that expression of the polyQ construct in yeast can be used to model patho-physiological effects of polyQ expansion in neurons.
Keywords: Huntingtin; Polyglutamine; Yeast; Mitochondria; Apoptosis;

The objective of this study was to evaluate mitochondrial alterations in a cell-based model of myocardial ischemia/reperfusion (I/R) injury. Using GFP-biosensors and fluorescence deconvolution microscopy, we investigated mitochondrial morphology in relation to Bax and Bid activation in the HL-1 cardiac cell line. Mitochondria underwent extensive fragmentation during ischemia. Bax translocation from cytosol to mitochondria was initiated during ischemia and proceeded during reperfusion. However, Bax translocation was not sufficient to induce cell death or mitochondrial dysfunction. Bid processing was caspase-8 dependent, and Bid translocation to mitochondria occurred after Bax translocation and clustering, and minutes before cell death. Clustering of Bax into distinct regions on mitochondria could be prevented by CsA, an inhibitor of the mitochondrial permeability transition pore, and also by SB203580, an inhibitor of p38 MAPK. Surprisingly, mitochondrial fragmentation which occurred during ischemia and before Bax translocation could be reversed by the addition of the p38 inhibitor SB203580 at reperfusion. Taken together, these results implicate p38 MAPK in the mitochondrial remodeling response to I/R that facilitates Bax recruitment to mitochondria.
Keywords: Ischemia; Reperfusion; Cell death; Mitochondria; Bid; Bax;

Assaying the probabilities of obtaining maternally inherited heteroplasmy as the basis for modeling OXPHOS diseases in animals by Mikhail G. Bass; Vassilina A. Sokolova; Maria E. Kustova; Elena V. Grachyova; Oksana V. Kidgotko; Alexander V. Sorokin; Vadim B. Vasilyev (679-685).
Gross alterations in cell energy metabolism underlie manifestations of hereditary OXPHOS (oxidative phosphorylation) diseases, many of which depend on proportion of mutant mitochondrial DNA (mtDNA) in tissues. An animal model of OXPHOS disease with maternal inheritance of mitochondrial heteroplasmy might help understanding the peculiarities of abnormal mtDNA distribution and its effect on pre- and postnatal development. Previously we obtained mice that carry human mtDNA in some tissues. It co-existed with murine mtDNA (heteroplasmy) and was transmitted maternally to the progeny of animals developed from zygotes injected with human mitochondria. To analyze the probability of obtaining heteroplasmic mice we increased the number of experiments with early embryos and obtained more specimens from F1. About 33% of zygotes injected with human mtDNA developed into post-implantation embryos (7th–13th days). Lower amount of such developed into neonate mice (ca. 21%). Among post-implantation embryos and in generations F0 and F1 percentages of human mtDNA-carriers were ca. 14–16%. Such percentages are sufficient for modeling maternally inherited heteroplasmy in small animal groups. More data are needed to understand the regularities of anomalous mtDNA distribution among cells and tissues and whether heart and muscles frequently carrying human mtDNA in our experiments are particularly susceptible to heteroplasmy.
Keywords: Energy metabolism; OXPHOS diseases modeling; Human mtDNA; mtDNA transfer; Transgenic mice; Maternal inheritance;

Mitochondrial subpopulations and heterogeneity revealed by confocal imaging: Possible physiological role? by Andrey V. Kuznetsov; Jakob Troppmair; Robert Sucher; Martin Hermann; Valdur Saks; Raimund Margreiter (686-691).
Heterogeneity of mitochondria has been reported for a number of various cell types. Distinct mitochondrial subpopulations may be present in the cell and may be differently involved in physiological and pathological processes. However, the origin and physiological roles of mitochondrial heterogeneity are still unknown. In mice skeletal muscle, a much higher oxidized state of subsarcolemmal mitochondria as compared with intermyofibrillar mitochondria has been demonstrated. Using confocal imaging technique, we present similar phenomenon for rat soleus and gastrocnemius muscles, where higher oxidative state of mitochondrial flavoproteins correlates also with elevated mitochondrial calcium. Moreover, subsarcolemmal mitochondria demonstrate distinct arrangement and organization. In HL-1 cardiomyocytes, long thread mitochondria and small grain mitochondria are observed irrespective of a particular cellular region, showing also heterogeneous membrane potential and ROS production. Possible physiological roles of intracellular mitochondrial heterogeneity and specializations are discussed.
Keywords: Mitochondria; Mitochondrial heterogeneity; Skeletal muscles; HL-1 cardiomyocytes; Confocal imaging;

Mitochondria are crucial organelles for life and death of the cell. They are prominent players in energy conversion and integrated signaling pathways including regulation of Ca2+ signals and apoptosis. Their functional versatility is matched by their morphological plasticity and by their high mobility, allowing their transport at specialized cellular sites. This transport occurs by interactions with a variety of cytoskeletal proteins that also have the ability to influence shape and function of the organelle. A growing body of evidence suggests that mitochondria use cytoskeletal proteins as tracks for their movement; in turn, mitochondrial morphology and function is regulated via mostly uncharacterized pathways, by the cytoskeleton.
Keywords: Mitochondria; Fusion; Fission; Cytoskeleton; Motility; Kinesin; Dynein; Trichoplein;

Anti-HIV drugs and the mitochondria by Marcello Pinti; Paolo Salomoni; Andrea Cossarizza (700-707).
Several drugs are currently used that can significantly prolong the course of the infection with the human immunodeficiency virus (HIV), the cause of the acquired immunodeficiency syndrome (AIDS). Among these drugs, the nucleosidic inhibitors of viral reverse transcriptase can alter mitochondrial (mt) function by inhibiting the mitochondrial DNA polymerase gamma (the enzyme responsible for the replication of mtDNA). Decreased mtDNA content provokes a diminished synthesis of respiratory chain enzymes, leading to alterations in mt function. These are in turn responsible for a variety of side effects frequently observed in HIV+ patients, that range from hyperlactatemia and lactic acidosis to lipodystrophy, a pathology characterized by accumulation of visceral fat, breast adiposity, cervical fat-pads, hyperlipidemia, insulin resistance and fat wasting in face and limbs. In this paper, data concerning the effects of different compounds on mitochondria, their role in the pathogenesis of lipodystrophy, and problems related to studies on the mt toxicity of antiviral drugs are reviewed and thoroughly discussed.
Keywords: Mitochondria; HIV; AIDS; Mitochondrial DNA; Antiretroviral therapy; HAART;

Possibility of transkingdom gene therapy for Complex I diseases by Takao Yagi; Byoung Boo Seo; Eiko Nakamaru-Ogiso; Mathieu Marella; Jennifer Barber-Singh; Tetsuo Yamashita; Akemi Matsuno-Yagi (708-714).
Defects of complex I are involved in many human mitochondrial diseases, and therefore we have proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into mammalian cell lines. The expressed NDI1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species induced by rotenone. It was further shown that the NDI1 protein can be functionally expressed in tissues such as skeletal muscles and the brain of rodents, which scarcely induced an inflammatory response. The use of NDI1 as a potential molecular therapy for complex I-deficient diseases is briefly discussed, including the proposed animal model.
Keywords: NADH dehydrogenase; Complex I; Gene therapy; Mitochondrial disease; Parkinson's disease; Adeno-associated virus;

Mitochondrial potassium channels: From pharmacology to function by Adam Szewczyk; Jolanta Skalska; Marta Głąb; Bogusz Kulawiak; Dominika Malińska; Izabela Koszela-Piotrowska; Wolfram S. Kunz (715-720).
Mitochondrial potassium channels, such as ATP-regulated or large conductance Ca2+-activated and voltage gated channels were implicated in cytoprotective phenomenon in different tissues. Basic effects of these channels activity include changes in mitochondrial matrix volume, mitochondrial respiration and membrane potential, and generation of reactive oxygen species. In this paper, we describe the pharmacological properties of mitochondrial potassium channels and their modulation by channel inhibitors and potassium channel openers. We also discuss potential side effects of these substances.
Keywords: Mitochondria; Potassium channels; Potassium channel openers; Sulfonylurea; Diazoxide;

Ever since its discovery in 1953 by N. O. Kaplan and coworkers, the physiological role of the proton-translocating transhydrogenase has generally been assumed to be that of generating mitochondrial NADPH. Mitochondrial NADPH can be used in a number of important reactions/processes, e.g., biosynthesis, maintenance of GSH, apoptosis, aging etc. This assumed role has found some support in bacteria but not in higher eukaryotes, a situation which changed dramatically with two recent but separate findings, both using transhydrogenase knockouts, in the nematode C. elegans and the mouse strain C57BL/6J. The latter, which is due to a spontaneous deletion mutation in the Nnt gene, was serendipitously found during investigations of the diabetic properties of these mice. The implications of these findings for the overall role of transhydrogenase in cell metabolism and disease are discussed.
Keywords: Transhydrogenase; Mitochondria; NADPH; ROS; Oxidative stress; Diabetes;