BBA - Bioenergetics (v.1787, #7)
Editorial Board (i).
Radical species, mitochondria and cardiac function by Bruno Tota; Richard D. Handy; Maria Carmela Cerra (773).
The cardioprotective effects elicited by p66Shc ablation demonstrate the crucial role of mitochondrial ROS formation in ischemia/reperfusion injury by Andrea Carpi; Roberta Menabò; Nina Kaludercic; PierGiuseppe Pelicci; Fabio Di Lisa; Marco Giorgio (774-780).
Although a major contribution to myocardial ischemia–reperfusion (I/R) injury is suggested to be provided by formation of reactive oxygen species (ROS) within mitochondria, sites and mechanisms are far from being elucidated. Besides a dysfunctional respiratory chain, other mitochondrial components, such as monoamine oxidase and p66Shc, might be involved in oxidative stress. In particular, p66Shc has been shown to catalyze the formation of H2O2.The relationship among p66Shc, ROS production and cardiac damage was investigated by comparing hearts from p66Shc knockout mice (p66Shc−/−) and wild-type (WT) littermates. Perfused hearts were subjected to 40 min of global ischemia followed by 15 min of reperfusion. Hearts devoid of p66Shc were significantly protected from I/R insult as shown by (i) reduced release of lactate dehydrogenase in the coronary effluent (25.7 ± 7.49% in p66Shc−/− vs. 39.58 ± 5.17% in WT); (ii) decreased oxidative stress as shown by a 63% decrease in malondialdehyde formation and 40 ± 8% decrease in tropomyosin oxidation. The degree of protection was independent of aging.The cardioprotective efficacy associated with p66Shc ablation was comparable with that afforded by other antioxidant interventions and could not be increased by antioxidant co-administration suggesting that p66Shc is downstream of other pathways involved in ROS formation. In addition, the absence of p66Shc did not affect the protection afforded by ischemic preconditioning.In conclusion, the absence of p66Shc reduces the susceptibility to reperfusion injury by preventing oxidative stress. The present findings provide solid and direct evidence that mitochondrial ROS formation catalyzed by p66Shc is causally related to reperfusion damage.
Keywords: Mitochondria; Oxidative stress; p66Shc; Monoamine oxidase;
Cardioprotection: A radical view by Claudia Penna; Daniele Mancardi; Raffaella Rastaldo; Pasquale Pagliaro (781-793).
A series of brief (a few minutes) ischemia/reperfusion cycles (ischemic preconditioning, IP) limits myocardial injury produced by a subsequent prolonged period of coronary artery occlusion and reperfusion. Postconditioning (PostC), which is a series of brief (a few seconds) reperfusion/ischemia cycles at reperfusion onset, attenuates also ischemia/reperfusion injury. In recent years the main idea has been that reactive oxygen species (ROS) play an essential, though double-edged, role in cardioprotection: they may participate in reperfusion injury or may play a role as signaling elements of protection in the pre-ischemic phase. It has been demonstrated that preconditioning triggering is redox-sensitive, using either ROS scavengers or ROS generators. We have shown that nitroxyl triggers preconditioning via pro-oxidative, and/or nitrosative stress-related mechanism(s). Several metabolites, including acetylcholine, bradykinin, opioids and phenylephrine, trigger preconditioning-like protection via a mitochondrial KATP-ROS-dependent mechanism. Intriguingly, and contradictory to the above mentioned theory of ROS as an obligatory part of reperfusion-induced damage, some studies suggest the possibility that some ROS at low concentrations could protect ischemic hearts against reperfusion injury. Yet, we demonstrated that ischemic PostC is also a cardioprotective phenomenon that requires the intervention of redox signaling to be protective. Emerging evidence suggests that in a preconditioning scenario a redox signal is required during the first few minutes of myocardial reperfusion following the index ischemic period. Intriguingly, the ROS signaling in the early reperfusion appear crucial to both preconditioning- and postconditioning-induced protection. Therefore, our and others' results suggest that the role of ROS in reperfusion may be reconsidered as they are not only deleterious.
Keywords: Cardioprotection; Ischemia/reperfusion; Mitochondria; Preconditioning; Postconditioning; ROS Signaling;
Postconditioning induces an anti-apoptotic effect and preserves mitochondrial integrity in isolated rat hearts by Claudia Penna; Maria-Giulia Perrelli; Stefania Raimondo; Francesca Tullio; Annalisa Merlino; Francesca Moro; Stefano Geuna; Daniele Mancardi; Pasquale Pagliaro (794-801).
Postconditioning (PostC) may limit mitochondrial damage and apoptotic signaling. We studied markers of apoptosis and mitochondrial protection in isolated rat hearts, which underwent a) perfusion without ischemia (Sham), b) 30-min ischemia (I) plus 2-hour reperfusion (R), or c) PostC protocol (5 intermittent cycles of 10-s reperfusion and 10-s ischemia immediately after the 30-min ischemia). Markers were studied in cytosolic (CF) and/or mitochondrial (MF) fractions. In CF, while pro-apoptotic factors (cytochrome c and caspase-3) were reduced, the anti-apoptotic markers (Bcl-2 and Pim-1) were increased by PostC, compared to the I/R group. Accordingly, phospho-GSK-3β and Bcl-2 levels increased in mitochondria of PostC group. Moreover, I/R reduced the level of mitochondrial structural protein (HSP-60) in MF and increased in CF, thus suggesting mitochondrial damage and HSP-60 release in cytosol, which were prevented by PostC. Electron microscopy confirmed that I/R markedly damaged cristae and mitochondrial membranes; damage was markedly reduced by PostC. Finally, total connexin-43 (Cx43) levels were reduced in the CF of the I/R group, whereas phospho-Cx43 level resulted in higher levels in the MF of the I/R group than the Sham group. PostC limited the I/R-induced increase of mitochondrial phospho-Cx43. Data suggest that PostC i) increases the levels of anti-apoptotic markers, including the cardioprotective kinase Pim-1, ii) decreases the pro-apoptotic markers, e.g. cytochrome c, iii) preserves the mitochondrial structure, and iv) limits the migration of phospho-Cx43 to mitochondria.
Keywords: Apoptosis; Cardioprotection; Ischemia/reperfusion; Mitochondria;
Reactive oxygen species as cardiovascular mediators: Lessons from endothelial-specific protein overexpression mouse models by Tatsiana Suvorava; Georg Kojda (802-810).
The term reactive oxygen species (ROS) summarizes several small chemical compounds such as superoxide, peroxynitrite, hydrogen peroxide and nitric oxide. The stoichiometry of the chemical reactions underlying generation and metabolism is subject of tight enzymatic regulation resulting in well balanced steady-state concentrations throughout the healthy body. ROS are short-lived and usually active at the site of production only, e.g. in vascular endothelial cells. Although an increase of vascular ROS-production is considered an important pathogenic factor in cardiovascular diseases, there is evidence for physiological or even beneficial effects as well. We have generated several transgenic mice using the Tie-2 promotor which expresses an enzyme of interest specifically in vascular endothelial cells. Here, we review some results obtained with mice carrying a Tie-2-driven overexpression of catalase or endothelial nitric oxide synthase (eNOS). Tie-2-catalase mice have a strongly reduced steady-state concentration of vascular hydrogen peroxide and show profound hypotension that is not dependent on the bioavailability of endothelial nitric oxide but is completely reversible by treatment with the catalase inhibitor aminotriazole. A similar hypotension was observed in transgenic mice with an endothelial-specific overexpression of eNOS but this hypotension is entirely dependent on vascular eNOS activity. These observations suggest a tonic effect of hydrogen peroxide on vascular smooth muscle. Further studies suggested that hydrogen peroxide promotes the exercise-induced increase of vascular eNOS expression and inhibits the release of endothelial progenitor cells induced by exercise training. In summary, our data support the concept of a dual role of ROS in the vascular system.
Keywords: Transgenic mice; Catalase; Endothelial NO-synthase (eNOS); Tie-2 promotor; Cardiovascular signaling; Exercise;
The role of nitric oxide and reactive oxygen species in the positive inotropic response to mechanical stretch in the mammalian myocardium by Yin Hua Zhang; Lewis Dingle; Rachel Hall; Barbara Casadei (811-817).
The endothelial nitric oxide synthase (eNOS) has been implicated in the rapid (Frank–Starling) and slow (Anrep) cardiac response to stretch. Our work and that of others have demonstrated that a neuronal nitric oxide synthase (nNOS) localized to the myocardium plays an important role in the regulation of cardiac function and calcium handling. However, the effect of nNOS on the myocardial response to stretch has yet to be investigated. Recent evidence suggests that the stretch-induced release of angiotensin II (Ang II) and endothelin 1 (ET-1) stimulates myocardial superoxide production from NADPH oxidases which, in turn, contributes to the Anrep effect. nNOS has also been shown to regulate the production of myocardial superoxide, suggesting that this isoform may influence the cardiac response to stretch or ET-1 by altering the NO-redox balance in the myocardium. Here we show that the increase in left ventricular (LV) myocyte shortening in response to the application of ET-1 (10 nM, 5 min) did not differ between nNOS−/− mice and their wild type littermates (nNOS+/+). Pre-incubating LV myocytes with the NADPH oxidase inhibitor, apocynin (100 μM, 30 min), reduced cell shortening in nNOS−/− myocytes only but prevented the positive inotropic effects of ET-1 in both groups. Superoxide production (O2 −) was enhanced in nNOS−/− myocytes compared to nNOS+/+; however, this difference was abolished by pre-incubation with apocynin. There was no detectable increase in O2 − production in ET-1 pre-treated LV myocytes. Inhibition of protein kinase C (chelerythrine, 1 μM) did not affect cell shortening in either group, however, protein kinase A inhibitor, PKI (2 μM), significantly reduced the positive inotropic effects of ET-1 in both nNOS+/+ and nNOS−/− myocytes. Taken together, our findings show that the positive inotropic effect of ET-1 in murine LV myocytes is independent of nNOS but requires NADPH oxidases and protein kinase A (PKA)-dependent signaling. These results may further our understanding of the signaling pathways involved in the myocardial inotropic response to stretch.
Keywords: Contraction; Endothelin-1; Neuronal nitric oxide synthase; Protein kinase A; Ventricular myocyte; NADPH oxidase;
Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP–PKG pathway activation by Daniela Pellegrino; Sruti Shiva; Tommaso Angelone; Mark T. Gladwin; Bruno Tota (818-827).
The ubiquitous anion nitrite (NO2 −) has recently emerged as an endocrine storage form of nitric oxide (NO) and a signalling molecule that mediates a number of biological responses. Although the role of NO in regulating cardiac function has been investigated in depth, the physiological signalling effects of nitrite on cardiac function have only recently been explored. We now show that remarkably low concentrations of nitrite (1 nM) significantly modulate cardiac contractility in isolated and perfused Langendorff rat heart. In particular, nitrite exhibits potent negative inotropic and lusitropic activities as evidenced by a decrease in left ventricular pressure and relaxation, respectively. Furthermore, we demonstrate that the nitrite-dependent effects are mediated by NO formation but independent of NO synthase (NOS) activity. Specifically, nitrite infusion in the Langendorff system produces NO and cGMP/PKG-dependent negative inotropism, as evidenced by the formation of cellular iron–nitrosyl complexes and inhibition of biological effect by NO scavengers and by PKG inhibitors. These data are consistent with the hypothesis that nitrite represents an eNOS-independent source of NO in the heart which modulates cardiac contractility through the NO–cGMP/PKG pathway. The observed high potency of nitrite supports a physiological function of nitrite as a source of cardiomyocyte NO and a fundamental signalling molecule in the heart.
Keywords: Nitrite; Nitric oxide; Heart; Langendorff rat heart; PKG; cGMP; NOS;
Response of blood vessels in vitro to hyperbaric oxygen (HBO): Modulation of VEGF and NO x release by external lactate or arginine by J. Yuan; R.D. Handy; A.J. Moody; P. Bryson (828-834).
Hyperbaric oxygen therapy (HBO) is suggested to promote angiogenesis during wound healing, but the mechanisms involved are not understood. This study used a novel isolated blood vessel preparation to explore the effects of air, normobaric oxygen or hyperbaric oxygen (2.2 ATA for 90 min) on the angiogenesis factor, vascular endothelial growth factor (VEGF), nitrite and nitrate (NO x ), lactate dehydrogenase (LDH) and lactate release from the tissue in normal Krebs Ringer, and the Ringer supplemented with either l-arginine, or 15 mM lactate to mimic a wound environment, or both (l-arginine + lactate). The in vitro blood vessel preparation remained viable during all experiments. There were no effects of HBO treatment on any of the parameters measured in normal Krebs Ringer, but some treatment-dependent effects were observed in supplemented Krebs Ringer. In the lactate supplemented Krebs Ringer, medium LDH levels increased in response to either normobaric oxygen (NBO) or HBO, compared to air alone. There were also small, but statistically significant increases in total glutathione due to HBO treatment, compared to NBO or air in the lactate supplemented medium, and in the combined supplement. There were no effects of HBO on NO x , changes in external medium lactate levels, or tissue VEGF in any of the Krebs Ringers tested. However, post treatment increases in VEGF were observed in the lactate supplemented medium, and for lactate release into the medium for the combined supplement. We conclude that HBO does not cause NO or VEGF production from the blood vessel in normal Krebs Ringer, but the data from supplemented medium show that the response of the tissue is subtly affected by the chemical environment around the blood vessel, and the tissue is more responsive to HBO when wound conditions are mimicked.
Keywords: Hyperbaric oxygen; Oxidative stress; Reactive oxidative species; Vascular endothelial growth factor; Nitric oxide; Angiogenesis;
The emergence of nitroxyl (HNO) as a pharmacological agent by Christopher H. Switzer; Wilmarie Flores-Santana; Daniele Mancardi; Sonia Donzelli; Debashree Basudhar; Lisa A. Ridnour; Katrina M. Miranda; Jon M. Fukuto; Nazareno Paolocci; David A. Wink (835-840).
Once a virtually unknown nitrogen oxide, nitroxyl (HNO) has emerged as a potential pharmacological agent. Recent advances in the understanding of the chemistry of HNO has led to the an understanding of HNO biochemistry which is vastly different from the known chemistry and biochemistry of nitric oxide (NO), the one-electron oxidation product of HNO. The cardiovascular roles of NO have been extensively studied, as NO is a key modulator of vascular tone and is involved in a number of vascular related pathologies. HNO displays unique cardiovascular properties and has been shown to have positive lusitropic and ionotropic effects in failing hearts without a chronotropic effect. Additionally, HNO causes a release of CGRP and modulates calcium channels such as ryanodine receptors. HNO has shown beneficial effects in ischemia reperfusion injury, as HNO treatment before ischemia-reperfusion reduces infarct size. In addition to the cardiovascular effects observed, HNO has shown initial promise in the realm of cancer therapy. HNO has been demonstrated to inhibit GAPDH, a key glycolytic enzyme. Due to the Warburg effect, inhibiting glycolysis is an attractive target for inhibiting tumor proliferation. Indeed, HNO has recently been shown to inhibit tumor proliferation in mouse xenografts. Additionally, HNO inhibits tumor angiogenesis and induces cancer cell apoptosis. The effects seen with HNO donors are quite different from NO donors and in some cases are opposite. The chemical nature of HNO explains how HNO and NO, although closely chemically related, act so differently in biochemical systems. This also gives insight into the potential molecular motifs that may be reactive towards HNO and opens up a novel field of pharmacological development.
Keywords: Nitroxyl; Nitric oxide; Heart failure; Ischemia reperfusion injury;
The role of nitrite in nitric oxide homeostasis: A comparative perspective by Frank B. Jensen (841-848).
Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O2 saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O2 saturations. The hemoglobin nitrite reductase activity depends on heme O2 affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia–reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.
Keywords: Nitrite; Nitric oxide; Hemoglobin; Hypoxia; Vasodilation; Cytoprotection;
Nitrite modulates contractility of teleost (Anguilla anguilla and Chionodraco hamatus, i.e. the Antarctic hemoglobinless icefish) and frog (Rana esculenta) hearts by M.C. Cerra; T. Angelone; M.L. Parisella; D. Pellegrino; B. Tota (849-855).
Being the largest form of intravascular and tissue storage of nitric oxide (NO) and a signalling molecule itself, the nitrite anion (NO2 −) has emerged as a key player in many biological processes. Since the heart is under an important NO-mediated autocrine–paracrine control, in mammals the cardiac effects of nitrite are under intensive investigation. In contrast, nothing is known in non-mammalian vertebrates. We evaluated nitrite influence on cardiac performance in the perfused beating heart of three different cold-blooded vertebrates, i.e. two teleost fishes, the temperate red-blooded Anguilla anguilla, the Antarctic stenotherm, hemoglobinless Chionodraco hamatus (icefish), and the frog Rana esculenta. We showed that, under basal conditions, in all animals nitrite influences cardiac mechanical performance, inducing negative inotropism in eel and frog, while being a positive inotrope in C. hamatus. In all species, these responses parallel the inotropic effects of authentic NO. We also demonstrated that the nitrite-dependent inotropic effects are i) dependent from NO synthase (NOS) activity in fish; ii) sensitive to NO scavenging in frog; iii) cGMP/PKG-dependent in both eel and frog. Results suggest that nitrite is an integral physiological source of NO and acts as a signalling molecule in lower vertebrate hearts, exerting relevant inotropic actions through different species-specific mechanisms.
Keywords: Nitrite; Nitric oxide; Working heart; Frog; Eel; Icefish; PKG; cGMP; NOS;
Is hydrogen sulfide a circulating “gasotransmitter” in vertebrate blood? by Kenneth R. Olson (856-863).
Hydrogen sulfide (H2S) is gaining acceptance as a signaling molecule and has been shown to elicit a variety of biological effects at concentrations between 10 and 1000 μmol/l. Dissolved H2S is a weak acid in equilibrium with HS− and S2− and under physiological conditions these species, collectively referred to as sulfide, exist in the approximate ratio of 20% H2S, 80% HS− and 0% S2−. Numerous analyses over the past 8 years have reported plasma or blood sulfide concentrations also in this range, typically between 30 and 300 μmol/l, thus supporting the biological studies. However, there is some question whether or not these concentrations are physiological. First, many of these values have been obtained from indirect methods using relatively harsh chemical conditions. Second, most studies conducted prior to 2000 failed to find blood sulfide in micromolar concentrations while others showed that radiolabeled 35S-sulfide is rapidly removed from blood and that mammals have a relatively high capacity to metabolize exogenously administered sulfide. Very recent studies using H2S gas-sensing electrodes to directly measure sulfide in plasma or blood, or HPLC analysis of head-space gas, have also indicated that sulfide does not circulate at micromolar levels and is rapidly consumed by blood or tissues. Third, micromolar concentrations of sulfide in blood or exhaled air should be, but are not, malodorous. Fourth, estimates of dietary sulfur necessary to sustain micromolar levels of plasma sulfide greatly exceed the daily intake. Collectively, these studies imply that many of the biological effects of sulfide are only achieved at supra-physiological concentrations and they question whether circulating sulfide is a physiologically relevant signaling molecule. This review examines the blood/plasma sulfide measurements that have been reported over the past 30 years from the perspective of the analytical methods used and the potential sources of error.
Keywords: Cardiovascular signaling; Sulfane sulfur; Acid-labile sulfur;
Physiological and pharmacological features of the novel gasotransmitter: Hydrogen sulfide by Daniele Mancardi; Claudia Penna; Annalisa Merlino; Piero Del Soldato; David A. Wink; Pasquale Pagliaro (864-872).
Hydrogen sulfide (H2S) has been known for hundreds of years because of its poisoning effect. Once the basal bio-production became evident its pathophysiological role started to be investigated in depth. H2S is a gas that can be formed by the action of two enzymes, cystathionine gamma-lyase and cystathionine beta-synthase, both involved in the metabolism of cysteine. It has several features in common with the other two well known “gasotransmitters” (nitric oxide and carbon monoxide) in the biological systems. These three gasses share some biological targets; however, they also have dissimilarities. For instance, the three gases target heme-proteins and open KATP channels; H2S as NO is an antioxidant, but in contrast to the latter molecule, H2S does not directly form radicals. In the last years H2S has been implicated in several physiological and pathophysiological processes such as long term synaptic potentiation, vasorelaxation, pro- and anti-inflammatory conditions, cardiac inotropism regulation, cardioprotection, and several other physiological mechanisms. We will focus on the biological role of H2S as a molecule able to trigger cell signaling. Our attention will be particularly devoted on the effects in cardiovascular system and in cardioprotection. We will also provide available information on H2S-donating drugs which have so far been tested in order to conjugate the beneficial effect of H2S with other pharmaceutical properties.
Keywords: Hydrogen sulfide; Cardioprotection; Gasotransmitter; Ischemic preconditioning; Nitric oxide;
Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco by Kunio Ido; Kentaro Ifuku; Yumiko Yamamoto; Seiko Ishihara; Akio Murakami; Keiji Takabe; Chikahiro Miyake; Fumihiko Sato (873-881).
The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)–PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone QA in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S2/QA −. These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.
Keywords: Extrinsic proteins; LHCII–PSII supercomplex; Nicotiana tabacum; Oxygen-evolving complex; Photosystem II; PsbP;
The S1 split signal of photosystem II; a tyrosine–manganese coupled interaction by Nicholas Cox; Felix M. Ho; Naray Pewnim; Ronald Steffen; Paul J. Smith; Kajsa G.V. Havelius; Joseph L. Hughes; Lesley Debono; Stenbjörn Styring; Elmars Krausz; Ron J. Pace (882-889).
Detailed optical and EPR analyses of states induced in dark-adapted PS II membranes by cryogenic illumination permit characterization and quantification of all pigment derived donors and acceptors, as well as optically silent (in the visible, near infrared) species which are EPR active. Near complete turnover formation of QA − is seen in all centers, but with variable efficiency, depending on the donor species. In minimally detergent-exposed PS II membranes, negligible (< 5%) oxidation of chlorophyll or carotenoid centers occurs for illumination temperatures 5–20 K. An optically silent electron donor to P680+ is observed with the same decay kinetics as the S1 split signal. Cryogenic donors to P680+ seen are: (i) transient (t 1/2 ∼ 150 s) tyrosine related species, including ‘split signals’ (∼ 15% total centers), (ii) reduced cytochrome b 559 (∼ 30–50% centers), and (iii) an organic donor, possibly an amino acid side chain, (∼ 30% centers).
Keywords: S1 split signal; Carotenoid; Chlorophyll; Cytochrome b 559; Pheophytin; Tyrosine; Exchange coupled interaction;
Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria by Clara Musicco; Valentina Capelli; Vito Pesce; Anna Maria Timperio; Menotti Calvani; Luigi Mosconi; Lello Zolla; Palmiro Cantatore; Maria Nicola Gadaleta (890-896).
Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H2O2 scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.
Keywords: Peroxiredoxin III; Aging; Rat liver mitochondria; Protein oxidation; Mass spectrometry; Two-dimensional electrophoresis;
Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. by Valeria Petronilli; Justina Šileikytė; Alessandra Zulian; Federica Dabbeni-Sala; Giulio Jori; Silvano Gobbo; Giuseppe Tognon; Peter Nikolov; Paolo Bernardi; Fernanda Ricchelli (897-904).
We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, 1O2) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.
Keywords: Mitochondria; Permeability transition pore; External thiols; Porphyrin; Photooxidation;
Identification of a specific fucoxanthin-chlorophyll protein in the light harvesting complex of photosystem I in the diatom Cyclotella meneghiniana by Thomas Veith; Jan Brauns; Wolfram Weisheit; Maria Mittag; Claudia Büchel (905-912).
Thylakoids of the diatom Cyclotella meneghiniana were separated by discontinuous gradient centrifugation into photosystem (PS) I, PSII, and fucoxanthin-chlorophyll protein (FCP) fractions. FCPs are homologue to light harvesting complexes of higher plants with similar function in e.g. brown algae and diatoms. Still, it is unclear if FCP complexes are specifically associated with either PSI or PSII, or if FCP complexes function as one antenna for both photosystems. However, a trimeric FCP complex, FCPa, and a higher FCP oligomer, FCPb, have been described for C. meneghiniana, already. In this study, biochemical and spectroscopical evidences are provided that reveal a different subset of associated Fcp polypeptides within the isolated photosystem complexes. Whereas the PSII associated Fcp antenna resembles FCPa since it contains Fcp2 and Fcp6, at least three different Fcp polypeptides are associated with PSI. By re-solubilisation and a further purification step Fcp polypeptides were partially removed from PSI and both fractions were analysed again by biochemical and spectroscopical means, as well as by HPLC. Thereby a protein related to Fcp4 and a so far undescribed 17 kDa Fcp were found to be strongly coupled to PSI, whereas presumably Fcp5, a subunit of the FCPb complex, is only loosely bound to the PSI core. Thus, an association of FCPb and PSI is assumed.
Keywords: Photosynthesis; Fluorescence; Fcp4; Light harvesting complex; Membrane protein; Diatom;
Effect of subunit IV on superoxide generation by Rhodobacter sphaeroides cytochrome bc 1 complex by Ying Yin; Shih-Chia Tso; Chang-An Yu; Linda Yu (913-919).
Previous studies indicate that the three-subunit cytochrome bc 1 core complex of Rhodobacter sphaeroides contains a fraction of the electron transfer activity of the wild-type enzyme. Addition of subunit IV to the core complex increases electron transfer activity to the same level as that of the wild-type complex. This activity increase may result from subunit IV preventing electron leakage, from the low potential electron transfer chain, and reaction with molecular oxygen, producing superoxide anion. This suggestion is based on the following observations: (1) the extent of cytochrome b reduction in the three-subunit core complex, by ubiquinol, in the presence of antimycin A, never reaches the same level as that in the wild-type complex; (2) the core complex produces 4 times as much superoxide anion as does the wild-type complex; and (3) when the core complex is reconstituted with subunit IVs having varying reconstitutive activities, the activity increase in reconstituted complexes correlates with superoxide production decrease and extent of cytochrome b reduction increase.
Keywords: Superoxide anion radical; Electron leakage; Supernumerary subunit; Cytochrome bc 1 complex; Protein–protein interaction;
Functional characterization of the thylakoid Ndh complex phosphorylation by site-directed mutations in the ndhF gene by Mercedes Martín; Helena T. Funk; Patricia H. Serrot; Peter Poltnigg; Bartolomé Sabater (920-928).
To investigate the phosphorylation of the NDH-F subunit of the thylakoid Ndh complex, we constructed three site-directed mutant transgenic tobaccos (Nicotiana tabacum) (T181A, T181S and T181D) in which the 541ACT543 triplet encoding the Thr-181 has been substituted by GCT, TCT or GAT encoding alanine, serine and aspartic acid, respectively. Western blots with phospho-threonine antibody detected the 73 kD NDH-F phosphorylated polypeptide in control but not in mutant tobaccos. Differences in Ndh activity, chlorophyll fluorescence and photosynthesis among mutants and control plant demonstrate the key role of the phosphorylation of conserved Thr-181 in the activity and function of the Ndh complex. The substitution of aspartic acid for threonine in T181D mimics the presumable activation effects of the threonine phosphorylation in Ndh activity, post-illumination increase of chlorophyll fluorescence and photosynthesis rapid responses to changing light intensities. A tentative role of the phosphorylation-activated Ndh complex is suggested to poise the redox level and, consequently, optimizing the rate of cyclic electron transport under field conditions.
Keywords: Ndh genes; Plastid transformation; Redox poising; Threonine phosphorylation;
The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana by Irina Grouneva; Torsten Jakob; Christian Wilhelm; Reimund Goss (929-938).
Intact cells of diatoms are characterized by a rapid diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid diatoxanthin epoxidation is dependent on the availability of the cofactor of diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.
Keywords: Alternative electron transport; Chlororespiration; Diatom; NPQ; Xanthophyll cycle;
Probing the pH sensitivity of R-phycoerythrin: Investigations of active conformational and functional variation by Lu-Ning Liu; Hai-Nan Su; Shi-Gan Yan; Si-Mi Shao; Bin-Bin Xie; Xiu-Lan Chen; Xi-Ying Zhang; Bai-Cheng Zhou; Yu-Zhong Zhang (939-946).
Crystal structures of phycobiliproteins have provided valuable information regarding the conformations and amino acid organizations of peptides and chromophores, and enable us to investigate their structural and functional relationships with respect to environmental variations. In this work, we explored the pH-induced conformational and functional dynamics of R-phycoerythrin (R-PE) by means of absorption, fluorescence and circular dichroism spectra, together with analysis of its crystal structure. R-PE presents stronger functional stability in the pH range of 3.5–10 compared to the structural stability. Beyond this range, pronounced functional and structural changes occur. Crystal structure analysis shows that the tertiary structure of R-PE is fixed by several key anchoring points of the protein. With this specific association, the fundamental structure of R-PE is stabilized to present physiological spectroscopic properties, while local variations in protein peptides are also allowed in response to environmental disturbances. The functional stability and relative structural sensitivity of R-PE allow environmental adaptation.
Keywords: Phycoerythrin; Stability; Tertiary structure; Spectra; Protein folding;
Modulation of mitochondrial K+ permeability and reactive oxygen species production by the p13 protein of human T-cell leukemia virus type 1 by Micol Silic-Benussi; Enrica Cannizzaro; Andrea Venerando; Ilaria Cavallari; Valeria Petronilli; Nicoletta La Rocca; Oriano Marin; Luigi Chieco-Bianchi; Fabio Di Lisa; Donna M. D'Agostino; Paolo Bernardi; Vincenzo Ciminale (947-954).
Human T-cell leukemia virus type-1 (HTLV-1) expresses an 87-amino acid protein named p13 that is targeted to the inner mitochondrial membrane. Previous studies showed that a synthetic peptide spanning an alpha helical domain of p13 alters mitochondrial membrane permeability to cations, resulting in swelling. The present study examined the effects of full-length p13 on isolated, energized mitochondria. Results demonstrated that p13 triggers an inward K+ current that leads to mitochondrial swelling and confers a crescent-like morphology distinct from that caused by opening of the permeability transition pore. p13 also induces depolarization, with a matching increase in respiratory chain activity, and augments production of reactive oxygen species (ROS). These effects require an intact alpha helical domain and strictly depend on the presence of K+ in the assay medium. The effects of p13 on ROS are mimicked by the K+ ionophore valinomycin, while the protonophore FCCP decreases ROS, indicating that depolarization induced by K+ vs. H+ currents has different effects on mitochondrial ROS production, possibly because of their opposite effects on matrix pH (alkalinization and acidification, respectively). The downstream consequences of p13-induced mitochondrial K+ permeability are likely to have an important influence on the redox state and turnover of HTLV-1-infected cells.
Keywords: K+ channel; Mitochondria; Permeability transition pore; Reactive oxygen species;
On chemomechanical coupling of the F1-ATPase molecular motor by Ping Xie (955-962).
F1-ATPase catalyzes ATP hydrolysis to drive the central γ-shaft rotating inside a hexameric cylinder composed of alternating α and β subunits. Experiments showed that the rotation of γ-shaft proceeds in steps of 120° and each 120°-rotation is composed of an 80° substep and a 40° substep. Here, based on the previously proposed models, an improved physical model for chemomechanical coupling of F1-ATPase is presented, with which the two-substep rotation is well explained. One substep is driven by the power stroke upon ATP binding, while the other one resulted from the passage of γ-shaft from previous to next adjacent β subunits via free diffusion. Using the model, the dynamics and kinetics of F1-ATPase, such as the rotating time of each substep, the dwell time at each pause and the rotation rate, are analytically studied. The theoretical results obtained with only three adjustable parameters reproduce the available experimental data well.
Keywords: F1-ATPase; Mechanochemical coupling mechanism; Rotary molecular motor; Hexameric motor protein;