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1.

Background

Reactive oxygen species (ROS) are among the main determinants of cellular damage during ischemia and reperfusion. There is also ample evidence that mitochondrial ROS production is involved in signaling during ischemic and pharmacological preconditioning. In a previous study we analyzed the mitochondrial effects of the efficient preconditioning drug diazoxide and found that it increased the mitochondrial oxidation of the ROS-sensitive fluorescent dye 2′,7′-dichlorodihydrofluorescein (H2DCF) but had no direct impact on the H2O2 production of submitochondrial particles (SMP) or intact rat heart mitochondria (RHM).

Methods

H2O2 generation of bovine SMP and tightly coupled RHM was monitored under different conditions using the amplex red/horseradish peroxidase assay in response to diazoxide and a number of inhibitors.

Results

We show that diazoxide reduces ROS production by mitochondrial complex I under conditions of reverse electron transfer in tightly coupled RHM, but stimulates mitochondrial ROS production at the Qo site of complex III under conditions of oxidant-induced reduction; this stimulation is greatly enhanced by uncoupling. These opposing effects can both be explained by inhibition of complex II by diazoxide. 5-Hydroxydecanoate had no effect, and the results were essentially identical in the presence of Na+ or K+ excluding a role for putative mitochondrial KATP-channels.

General significance

A straightforward rationale is presented to mechanistically explain the ambivalent effects of diazoxide reported in the literature. Depending on the metabolic state and the membrane potential of mitochondria, diazoxide-mediated inhibition of complex II promotes transient generation of signaling ROS at complex III (during preconditioning) or attenuates the production of deleterious ROS at complex I (during ischemia and reperfusion).  相似文献   

2.
Lipoperoxidative damage to the respiratory chain proteins may account for disruption in mitochondrial electron transport chain (ETC) function and could lead to an augment in the production of reactive oxygen species (ROS). To test this hypothesis, we investigated the effects of lipoperoxidation on ETC function and cytochromes spectra of Saccharomyces cerevisiae mitochondria. We compared the effects of Fe2+ treatment on mitochondria isolated from yeast with native (lipoperoxidation-resistant) and modified (lipoperoxidation-sensitive) fatty acid composition. Augmented sensitivity to oxidative stress was observed in the complex III-complex IV segment of the ETC. Lipoperoxidation did not alter the cytochromes content. Under lipoperoxidative conditions, cytochrome c reduction by succinate was almost totally eliminated by superoxide dismutase and stigmatellin. Our results suggest that lipoperoxidation impairs electron transfer mainly at cytochrome b in complex III, which leads to increased resistance to antimycin A and ROS generation due to an electron leak at the level of the QO site of complex III.  相似文献   

3.
Available data indicate that superoxide anion (O2•− ) is released from mitochondria, but apart from VDAC (voltage dependent anion channel), the proteins involved in its transport across the mitochondrial outer membrane still remain elusive. Using mitochondria of the yeast Saccharomyces cerevisiae mutant depleted of VDAC (Δpor1 mutant) and the isogenic wild type, we studied the role of the TOM complex (translocase of the outer membrane) in the efflux of O2•− from the mitochondria. We found that blocking the TOM complex with the fusion protein pb2-DHFR decreased O2•− release, particularly in the case of Δpor1 mitochondria. We also observed that the effect of the TOM complex blockage on O2•− release from mitochondria coincided with the levels of O2•− release as well as with levels of Tom40 expression in the mitochondria. Thus, we conclude that the TOM complex participates in O2•− release from mitochondria.  相似文献   

4.
The mitochondrial generation of reactive oxygen species (ROS) plays a central role in many cell signaling pathways, but debate still surrounds its regulation by factors, such as substrate availability, [O2] and metabolic state. Previously, we showed that in isolated mitochondria respiring on succinate, ROS generation was a hyperbolic function of [O2]. In the current study, we used a wide variety of substrates and inhibitors to probe the O2 sensitivity of mitochondrial ROS generation under different metabolic conditions. From such data, the apparent Km for O2 of putative ROS-generating sites within mitochondria was estimated as follows: 0.2, 0.9, 2.0, and 5.0 μm O2 for the complex I flavin site, complex I electron backflow, complex III QO site, and electron transfer flavoprotein quinone oxidoreductase of β-oxidation, respectively. Differential effects of respiratory inhibitors on ROS generation were also observed at varying [O2]. Based on these data, we hypothesize that at physiological [O2], complex I is a significant source of ROS, whereas the electron transfer flavoprotein quinone oxidoreductase may only contribute to ROS generation at very high [O2]. Furthermore, we suggest that previous discrepancies in the assignment of effects of inhibitors on ROS may be due to differences in experimental [O2]. Finally, the data set (see supplemental material) may be useful in the mathematical modeling of mitochondrial metabolism.The production of reactive oxygen species (ROS)2 by mitochondria has been implicated in numerous disease states, including but not limited to sepsis, solid state tumor survival, and diabetes (1). In addition, mitochondrial ROS (mtROS) play key roles in cell signaling (reviewed in Refs. 2 and 3). There exist within mitochondria several sites for the generation of ROS, with the most widely studied being complexes I and III of the electron transport chain (ETC). However, there is currently some debate regarding the relative contribution of these complexes to overall ROS production (49) and the factors that may alter this distribution. One such factor considered herein is [O2]. Estimates of physiological [O2] within tissues (i.e. interstitial [O2]) range from 37 down to 6 μm at 5–40 μm away from a blood vessel (10). More recently, EPR oximetry has estimated tissue [O2] to be in the 12–60 μm range (11). In addition, elegant studies with hepatocytes have shown that O2 gradients exist within cells, such that an extracellular [O2] of 6–10 μm yields an [O2] of ∼5 μm close to the plasma membrane, dropping to 1–2 μm close to mitochondria deep within the cell (12). In cardiomyocytes, at an extracellular [O2] of 29 μm, intracellular [O2] varied in the range 6–25 μm (13). Clearly, different tissues consume O2 at different rates, so these gradients can vary considerably between tissue and cell types.By definition, the generation of reactive oxygen species by any mechanism, is an O2-dependent process. However, measurements in intact cells have indicated that mtROS generation increases at lower O2 levels (1–5% O2) (14). Proponents of an increase in mtROS in response to hypoxia suggest that under such conditions, reduction of the ETC results in increased leakage of electrons to O2 at the QO site of complex III (14). Such a model posits that increased hypoxic ROS is a mitochondria-autonomous signaling mechanism (i.e. it is an inherent property of the mitochondrial ETC). Therefore, mtROS generation should increase in hypoxia regardless of the experimental system being studied, including isolated mitochondria. In contrast to this hypothesis, we and others have demonstrated that ROS generation by mitochondria is a positive function of [O2] across a wide range of values (0.1–1000 μm O2) (1518), suggesting that signaling mechanisms external to mitochondria may be required to facilitate the increased hypoxic mtROS production observed in cells.One limitation of our previous work (15) was that only a single respiratory condition was studied, namely succinate as respiratory substrate (feeding electrons into complex II) plus rotenone to inhibit backflow of electrons through complex I (5, 7). The possibility exists that under different metabolic conditions, which may lead to differential redox states between the cytochromes in the ETC (19, 20), ROS generation may exhibit a different response to [O2]. Thus, in the current study, we examined the response of mtROS generation to [O2] under 11 different conditions, using a variety of respiratory substrates and inhibitors (for a thorough review of electron entry points to the ETC under various substrate/inhibitor combinations, see Ref. 21). Fig. 1 shows a schematic of the mitochondrial ETC, highlighting sites of electron entry resulting from various substrates, binding sites of inhibitors, and major sites of ROS generation. Fig. 2 shows the specific details of each experimental condition, indicating the predicted sites of ROS generation resulting from the use of each substrate/inhibitor combination. The legend to Fig. 2 provides an explanation of each condition.Open in a separate windowFIGURE 1.Mitochondrial pathways of electron flow resulting from the substrates and inhibitors used in this study. Substrates used were glutamate/malate (which generates NADH via the tricarboxylic acid cycle, feeding into complex I), succinate (which feeds electrons directly into complex II), and palmitoyl-carnitine (which feeds electrons into the ETC via acyl-CoA dehydrogenase as well as through the β-oxidation pathway). (For a more thorough explanation, refer to Ref. 21.) Inhibitors used were rotenone (which inhibits at the downstream Q binding site of complex I (9)), malonate (a competitive inhibitor of complex II (25, 26)), and antimycin A (a complex III inhibitor that prevents electron flow to the QI site of complex III, thus stabilizing QH˙ at the QO site (6, 28)).Open in a separate windowFIGURE 2.Pathways of electron flow for the substrate/inhibitor combinations used in conditions A–L. Each panel includes the respective maximal respiration rate (VO2 max; nmol of O2/min/mg of protein) measured under each condition. A, glutamate/malate/malonate. Electrons enter through complex I, whereas electron entry at complex II is inhibited by malonate. ROS generation occurs at the FMN site of complex I as well as the QO site of complex III. B, glutamate/malate/malonate/rotenone. Electrons enter through complex I. Electron passage through complex I is inhibited by rotenone binding at the downstream Q site, resulting in maximal ROS production at the FMN site of complex I. ROS production at the QO site of complex III is prevented due to no electrons reaching the complex from either complexes I or II, both of which are inhibited. C, glutamate/malate/malonate/antimycin A. Electrons enter through complex I only, since complex II is blocked. Flow of electrons is inhibited by the complex III inhibitor antimycin A, resulting in ROS production at the QO site of complex III, as well as the FMN site of complex I. D, succinate. Electrons enter at complex II. ROS is generated by the flow of electrons though the QO site of complex III as well as the backflow of electrons through complex I. E, succinate/rotenone. Electrons enter at complex II, and ROS is generated at the QO site of complex III, because rotenone is present to inhibit backflow of electrons through complex I. F, succinate/antimycin A. Electrons enter through complex II. ROS is generated at both complex I via backflow and complex III QO, with an increased rate at the latter due to inhibition by antimycin A. G, succinate/rotenone/antimycin A. Electrons enter through complex II. Backflow of electrons through complex I is inhibited by rotenone, whereas ROS generation at complex III QO is augmented due to the presence of antimycin A. H, glutamate/malate/succinate. Electrons enter at both complexes I and II. ROS is generated from the complex I FMN site and the complex III QO site. J, glutamate/malate/succinate/antimycin A. Electrons enter at complexes I and II. ROS generation occurs at the complex I FMN and is augmented at the complex III QO site by antimycin A. K, palmitoyl-carnitine. Electrons enter at the ETFQOR. ROS is generated at the ETFQOR as well as complex I via backflow and at the complex III QO site. L, palmitoyl-carnitine/rotenone. Electron entry is at the ETFQOR. ROS is generated at the ETFQOR as well as at the complex III QO site, whereas ROS due to complex I backflow is blocked by rotenone. Glu, glutamate; Mal, malate; Suc, succinate; PC, palmitoyl-carnitine; Rot, rotenone; AntiA, antimycin A; Malon, malonate.The results of these studies indicated that although ROS generation under all experimental conditions exhibited the same overall response to [O2] (i.e. hyperbolic, with decreased ROS at low [O2]), the apparent Km for O2 varied widely between metabolic states.  相似文献   

5.
Complex I is responsible for most of the mitochondrial H2O2 release, low during the oxidation of the NAD linked substrates and high during succinate oxidation, via reverse electron flow. This H2O2 production appear physiological since it occurs at submillimolar concentrations of succinate also in the presence of NAD substrates in heart (present work) and rat brain mitochondria (Zoccarato et al., Biochem J, 406:125–129, 2007). Long chain fatty acyl-CoAs, but not fatty acids, act as strong inhibitors of succinate dependent H2O2 release. The inhibitory effect of acyl-CoAs is independent of their oxidation, being relieved by carnitine and unaffected or potentiated by malonyl-CoA. The inhibition appears to depend on the unbound form since the acyl-CoA effect decreases at BSA concentrations higher than 2 mg/ml; it is not dependent on ΔpH or Δp and could depend on the inhibition of reverse electron transfer at complex I, since palmitoyl-CoA inhibits the succinate dependent NAD(P) or acetoacetate reduction.  相似文献   

6.
The biological effects of ultraviolet radiation (UV), such as DNA damage, mutagenesis, cellular aging, and carcinogenesis, are in part mediated by reactive oxygen species (ROS). The major intracellular ROS intermediate is hydrogen peroxide, which is synthesized from superoxide anion (O2) and further metabolized into the highly reactive hydroxyl radical. In this study, we examined the involvement of mitochondria in the UV‐induced H2O2 accumulation in a keratinocyte cell line HaCaT. Respiratory chain blockers (cyanide‐p‐trifluoromethoxy‐phenylhydrazone and oligomycin) and the complex II inhibitor (theonyltrifluoroacetone) prevented H2O2 accumulation after UV. Antimycin A that inhibits electron flow from mitochondrial complex III to complex IV increased the UV‐induced H2O2 synthesis. The same effect was seen after incubation with rotenone, which blocks electron flow from NADH‐reductase (complex I) to ubiquinone. UV irradiation did not affect mitochondrial transmembrane potential (ΔΨm). These data indicate that UV‐induced ROS are produced at complex III via complex II (succinate‐Q‐reductase). J. Cell. Biochem. 80:216–222, 2000. © 2000 Wiley‐Liss, Inc.  相似文献   

7.
Chlamydomonas reinhardtii mutants D1-R323H, D1-R323D, and D1-R323L showed elevated chlorophyll fluorescence yields, which increased with decline of oxygen evolving capacity. The extra step K ascribed to the disturbance of electron transport at the donor side of PS II was observed in OJIP kinetics measured in mutants with a PEA fluorometer. Fluorescence decay kinetics were recorded and analyzed in a pseudo-wild type (pWt) and in mutants of C. reinhardtii with a Becker and Hickl single photon counting system in pico- to nanosecond time range. The kinetics curves were fitted by three exponentials. The first one (rapid, with lifetime about 300 ps) reflects energy migration from antenna complex to the reaction center (RC) of photosystem II (PS II); the second component (600–700 ps) has been assigned to an electron transfer from P680 to QA, while the third one (slow, 3 ns) assumingly originates from charge recombination in the radical pair [P680+• Pheo−•] and/or from antenna complexes energetically disconnected from RC II. Mutants showed reduced contribution of the first component, whereas the yield of the second component increased due to slowing down of the electron transport to QA. The mutant D1-R323L with completely inactive oxygen evolving complex did not reveal rapid component at all, while its kinetics was approximated by two slow components with lifetimes of about 2 and 3 ns. These may be due to two reasons: a) disconnection between antennae complexes and RC II, and b) recombination in a radical pair [P680+• Pheo−•] under restricted electron transport to QA. The data obtained suggest that disturbance of oxygen evolving function in mutants may induce an upshift of the midpoint redox potential of QA/QA couple causing limitation of electron transport at the acceptor side of PS II.  相似文献   

8.
Chemical mechanisms of antioxidant and electron donating function of the hypothalamic proline-rich polypeptides have been clarified on the molecular level. The antioxidant-chelating property of Galarmin and Gx-NH2 was established by their capability to inhibit copper(II) dichloride catalyzed H2O2 decomposition, thus preventing formation of HO and HOO radicals. The antiradical activity of Galarmin and Gx-NH2 was determined by their ability to react with 2,2-diphenyl-1-picrylhydrazyl radical applying differential pulse voltammetry and UV–Vis spectrophotometry methods. Galarmin manifest antiradical activity towards 2,2-diphenyl-1-picrylhydrazyl radical, depending on the existence of phenolic OH group in tyrosine residue at the end of the molecule. The presence of antiradical activity and reduction properties of Galarmin are confirmed by the existence of an oxidation specific peak in voltammograms made by differential pulse voltammetry at E  = 0.795 V vs. Ag/Ag+ aq.  相似文献   

9.
Quinones such as ubiquinone are the lipid soluble electron and proton carriers in the membranes of mitochondria, chloroplasts and oxygenic bacteria. Quinones undergo controlled redox reactions bound to specific sites in integral membrane proteins such as the cytochrome bc1 oxidoreductase. The quinone reactions in bacterial photosynthesis are amongst the best characterized, presenting a model to understand how proteins modulate cofactor chemistry. The free energy of ubiquinone redox reactions in aqueous solution and in the QA and QB sites of the bacterial photosynthetic reaction centers (RCs) are compared. In the primary QA site ubiquinone is reduced only to the anionic semiquinone (Q•−) while in the secondary QB site the product is the doubly reduced, doubly protonated quinol (QH2). The ways in which the protein modifies the relative energy of each reduced and protonated intermediate are described. For example, the protein stabilizes Q•− while destabilizing Q= relative to aqueous solution through electrostatic interactions. In addition, kinetic and thermodynamic mechanisms for stabilizing the intermediate semiquinones are compared. Evidence for the protein sequestering anionic compounds by slowing both on and off rates as well as by binding the anion more tightly is reviewed.  相似文献   

10.
H+-FOF1-ATP synthase (F-ATPase, F-type ATPase, FOF1 complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.  相似文献   

11.
This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+]o) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas. (Mol Cell Biochem 269: 165–173, 2005)  相似文献   

12.
Apoptosis-inducing factor (AIF)-deficient harlequin (Hq) mice undergo neurodegeneration associated with a 40–50% reduction in complex I level and activity. We tested the hypothesis that AIF and complex I regulate reactive oxygen species (ROS) production by brain mitochondria. Isolated Hq brain mitochondria oxidizing complex I substrates displayed no difference compared to wild type (WT) in basal ROS production, H2O2 removal, or ROS production stimulated by complex I inhibitors rotenone or 1-methyl-4-phenylpyridinium. In contrast, ROS production caused by reverse electron transfer to complex I was attenuated by ~50% in Hq mitochondria oxidizing the complex II substrate succinate. Basal and rotenone-stimulated rates of H2O2 release from in situ mitochondria did not differ between Hq and WT synaptosomes metabolizing glucose, nor did the level of in vivo oxidative protein carbonyl modifications detected in synaptosomes, brain mitochondria, or homogenates. Our results suggest that AIF does not directly modulate ROS release from brain mitochondria. In addition, they demonstrate that in contrast to ROS produced by mitochondria oxidizing succinate, ROS release from in situ synaptosomal mitochondria or from isolated brain mitochondria oxidizing complex I substrates is not proportional to the amount of complex I. These findings raise the important possibility that complex I contributes less to physiological ROS production by brain mitochondria than previously suggested.  相似文献   

13.
Reactive oxygen species (ROS), namely superoxide radical (O2 ) and hydrogen peroxide (H2O2) are generated when plant tissues endure a variety of environmental stresses, including light stress. The extremely short life times of ROS makes the study of their production in planta very difficult. The use of ROS-specific tracer dyes, 3-3′ diaminobenzidine and nitroblue tetrazolium, together with high-resolution imaging provides the opportunity to identify sites of photooxidative stress response by ROS accumulation. This technique was applied to grapevine during the first 7 days after transfer from in vitro to ex vitro under an irradiance 4-fold higher than in vitro. ROS accumulation was detected in the first days of analysis, which gradually decreased to levels comparable to greenhouse leaves. O2 was uniformly distributed while H2O2 accumulated preferentially in veins, wounds and stomatal guard and surrounding cells. To evaluate the role of H2O2 in stomatal functioning and its crosstalk with abscisic acid (ABA) we focused on the percentage of coloured structures, stomatal aperture and ABA concentration. We propose that the high H2O2 level triggered by increased light is responsible for the activation of a signalling pathway over stomatal cells, in a process apparently irrespective of ABA regulation prior to root protrusion. This could explain the gain of function of a low yet consistent percentage of stomatal cells, essential for plant survival during the ontogenic period in analysis.  相似文献   

14.
Using open top chambers, the effects of elevated O3 (80 nmol mol−1) and elevated CO2 (700 μmol mol−1), alone and in combination, were studied on young trees of Quercus mongolica. The results showed that elevated O3 increased malondialdehyde content and decreased photosynthetic rate after 45 days of exposure, and prolonged exposure (105 days) induced significant increase in electrolyte leakage and reduction of chlorophyll content. All these changes were alleviated by elevated CO2, indicating that oxidative stress on cell membrane and photosynthesis was ameliorated. After 45 days of exposure, elevated O3 stimulated activities of superoxide dismutase (SOD, EC 1.15.1.1) and ascorbate peroxidase (APX, EC 1.11.1.11), but the stimulation was dampened under elevated CO2 exposure. Furthermore, ascorbate (AsA) and total phenolics contents were not higher in the combined gas treatment than those in elevated O3 treatment. It indicates that the protective effect of elevated CO2 against O3 stress was achieved hardly by enhancing ROS scavenging ability after 45 days of exposure. After 105 days of exposure, elevated O3 significantly decreased activities of SOD, catalase (CAT, EC 1.11.1.6) and APX and AsA content. Elevated CO2 suppressed the O3-induced decrease, which could ameliorate the oxidative stress in some extent. In addition, elevated CO2 increased total phenolics content in the leaves both under ambient O3 and elevated O3 exposure, which might contribute to the protection against O3-induced oxidative stress as well.  相似文献   

15.
This mini-review briefly summarizes our current knowledge on the reaction pattern of light-driven water splitting and the structure of Photosystem II that acts as a water:plastoquinone oxidoreductase. The overall process comprises three types of reaction sequences: (a) light-induced charge separation leading to formation of the radical ion pair P680+•QA−•; (b) reduction of plastoquinone to plastoquinol at the QB site via a two-step reaction sequence with QA−• as reductant and (c) oxidative water splitting into O2 and four protons at a manganese-containing catalytic site via a four-step sequence driven by P680+• as oxidant and a redox active tyrosine YZ acting as mediator. Based on recent progress in X-ray diffraction crystallographic structure analysis the array of the cofactors within the protein matrix is discussed in relation to the functional pattern. Special emphasis is paid on the structure of the catalytic sites of PQH2 formation (QB-site) and oxidative water splitting (Mn4O x Ca cluster). The energetics and kinetics of the reactions taking place at these sites are presented only in a very concise manner with reference to recent up-to-date reviews. It is illustrated that several questions on the mechanism of oxidative water splitting and the structure of the catalytic sites are far from being satisfactorily answered.  相似文献   

16.
Short-term effects of 3,5-L-diiodothyronine (T2) administration to hypothyroid rats on FoF1-ATP synthase activity were investigated in liver mitochondria. One hour after T2 injection, state 4 and state 3 respiration rates were noticeably stimulated in mitochondria subsequently isolated. FoF1-ATP synthase activity, which was reduced in mitochondria from hypothyroid rats as compared to mitochondria from euthyroid rats, was significantly increased by T2 administration in both the ATP-synthesis and hydrolysis direction. No change in β-subunit mRNA accumulation and protein amount of the α-β subunit of FoF1-ATP synthase was found, ruling out a T2 genomic effect. In T2-treated rats, changes in the composition of mitochondrial phospholipids were observed, cardiolipin (CL) showing the greatest alteration. In mitochondria isolated from hypothyroid rats the decrease in the amount of CL was accompanied by an increase in the level of peroxidised CL. T2 administration to hypothyroid rats enhanced the level of CL and decreased the amount of peroxidised CL in subsequently isolated mitochondria, tending to restore the CL value to the euthyroid level. Minor T2-induced changes in mitochondrial fatty acid composition were detected. Overall, the enhanced FoF1-ATP synthase activity observed following injection of T2 to hypothyroid rats may be ascribed, at least in part, to an increased level of mitochondrial CL associated with decreased peroxidation of CL.  相似文献   

17.
The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc1 complex. The types of ROS produced (O2??, 1O2, and, possibly, H2O2) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ p ?? (possible sources for O2??), the Rieske iron–sulfur cluster (possible source of O2?? and/or 1O2), Chl a (possible source of 1O2), and heme c n (possible source of O2?? and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.  相似文献   

18.
Complex I is the main O2 producer of the mitochondrial respiratory chain. O2 release is low with NAD-linked substrates and increases strongly during succinate oxidation, which increases the QH2/Q ratio and is rotenone sensitive. We show that the succinate dependent O2 production (measured as H2O2 release) is inhibited by propargylamine containing compounds (clorgyline, CGP 3466B, rasagiline and TVP-1012). The inhibition does not affect membrane potential and is unaffected by ΔpH modifications. Mitochondrial respiration is similarly unaffected. The propargylamines inhibition of O2 /H2O2 production is monitored also in the presence of the Parkinson's disease toxin dopaminochrome which stimulates O2 release. Propargylamine-containing compounds are the first pharmacological inhibitors described for O2 release at Complex I.  相似文献   

19.
Differences in maximal yields of chlorophyll variable fluorescence (Fm) induced by single turnover (ST) and multiple turnover (MT) excitation are as great as 40%. Using mutants of Chlamydomonas reinhardtii we investigated potential mechanisms controlling Fm above and beyond the QA redox level. Fm was low when the QB binding site was occupied by PQ and high when the QB binding site was empty or occupied by a PSII herbicide. Furthermore, in mutants with impaired rates of plastoquinol reoxidation, Fm was reached rapidly during MT excitation. In PSII particles with no mobile PQ pool, Fm was virtually identical to that obtained in the presence of PSII herbicides. We have developed a model to account for the variations in maximal fluorescence yields based on the occupancy of the QB binding site. The model predicts that the variations in maximal fluorescence yields are caused by the capacity of secondary electron acceptors to reoxidize QA.  相似文献   

20.
In the present study mitochondrial respiratory function of fibroblasts from a patient affected by early-onset Parkinsonism carrying the homozygous W437X nonsense mutation in the PINK1 gene has been thoroughly characterized. When compared with normal fibroblasts, the patient’s fibroblast mitochondria exhibited a lower respiratory activity and a decreased respiratory control ratio with cellular ATP supply relying mainly on enhanced glycolytic production. The quantity, specific activity and subunit pattern of the oxidative phosphorylation complexes were normal. However, a significant decrease of the cellular cytochrome c content was observed and this correlated with a reduced cytochrome c oxidase in situ-activity. Measurement of ROS revealed in mitochondria of the patient’s fibroblasts enhanced O2•− and H2O2 production abrogated by inhibition of complex I. No change in the glutathione-based redox buffering was, however, observed. Special issue article in honor of Anna Maria Giuffrida-Stella.  相似文献   

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