Complexities of complex II: Sulfide metabolism in vivo

High levels of H₂S produced by gut microbiota can block oxygen utilization by inhibiting mitochondrial complex IV. Kumar et al. have shown how cells respond to this inhibition by using the mitochondrial sulfide oxidation pathway and reverse electron transport. The reverse activity of mitochondrial complex II (succinate-quinone oxidoreductase, i.e., fumarate reduction) generates oxidized coenzyme Q, which is then reduced by the mitochondrial sulfide quinone oxidoreductase to oxidize H₂S. This newly identified redox circuitry points to the importance of complex II reversal in mitochondria during periods of hypoxia and cellular stress.     

Structural perspectives on H2S homeostasis

The enzymes involved in H₂S homeostasis regulate its production from sulfur-containing amino acids and its oxidation to thiosulfate and sulfate. Two gatekeepers in this homeostatic circuit are cystathionine beta-synthase, which commits homocysteine to cysteine, and sulfide quinone oxidoreductase, which commits H₂S to oxidation via a mitochondrial pathway. Inborn errors at either locus affect sulfur metabolism, increasing homocysteine-derived H₂S synthesis in the case of CBS deficiency and reducing complex IV activity in the case of SQOR deficiency. In this review, we focus on structural perspectives on the reaction mechanisms and regulation of these two enzymes, which are key to understanding H₂S homeostasis in health and its dysregulation and potential targeting in disease.     

Photorespiration is More Effective than the Mehler Reaction in Protecting the Photosynthetic Apparatus against Photoinhibition

I. Isolated intact chloroplasts: Photosystem II, but not photosystem I, of the electron transport chain is rapidly photoinactivated even by very low intensities of red light when no large proton gradient can be formed and the electron transport chain becomes over-reduced in the absence of oxygen and other reducable substrates. Electron acceptors including oxygen provide protection against photoinactivation. Nevertheless, photosystem II is rapidly, and photosystem I more slowly, photoinactivated by high intensities of red light when oxygen is the only electron acceptor available. Increased damage is observed at increased oxygen concentrations although catalase is added to destroy H₂O₂ formed during oxygen reduction in the Mehler reaction. Photoinactivation can be decreased, but not prevented by ascorbate which reduces hydrogen peroxide inside the chloroplasts and increases coupled electron flow. II. Leaves: Simple measurements of chlorophyll fluorescence permit assessment of damage to photosystem II after exposure of leaves to high intensity illumination. In contrast to isolated chloroplasts, chloroplasts suffer more damage in situ at reduced than at elevated oxygen concentrations. The difference in the responses is due to photorespiration which is active in leaves, but not in isolated chloroplasts. After photosynthesis and photorespiration are inhibited by feeding glyceraldehyde to leaves, photoinactivation is markedly increased, although oxygen reduction in the Mehler reaction is not affected by glyceraldehyde. In the presence of reduced CO₂ levels, photorespiratory reactions, but not the Mehler reaction, can prevent the overreduction of the electron transport chain. Over-reduction indicates ineffective control of photosystem II activity. Effective control is needed for protection of the electron transport chain against photoinactivation. It is suggested to be made possible by coupled cyclic electron flow around photosystem I which is facilitated by the redox poising resulting from the interplay between photorespiratory carbohydrate oxidation and the refixation of evolved CO₂.     

Luminal sulfide and large intestine mucosa: friend or foe?

Hydrogen sulfide (H₂S) is present in the lumen of the human large intestine at millimolar concentrations. However, the concentration of free (unbound) sulfide is in the micromolar range due to a large capacity of fecal components to bind the sulfide. H₂S can be produced by the intestinal microbiota from alimentary and endogenous sulfur-containing compounds including amino acids. At excessive concentration, H₂S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen (O₂) consumption. However, the concept that sulfide is simply a metabolic troublemaker toward colonic epithelial cells has been challenged by the discovery that micromolar concentration of H₂S is able to increase the cell respiration and to energize mitochondria allowing these cells to detoxify and to recover energy from luminal sulfide. The main product of H₂S metabolism by the colonic mucosa is thiosulfate. The enzymatic activities involved in sulfide oxidation by the colonic epithelial cells appear to be sulfide quinone oxidoreductase considered as the first and rate-limiting step followed presumably by the action of sulfur dioxygenase and rhodanese. From clinical studies with human volunteers and experimental works with rodents, it appears that H₂S can exert mostly pro- but also anti-inflammatory effects on the colonic mucosa. From the available data, it is tempting to propose that imbalance between the luminal concentration of free sulfide and the capacity of colonic epithelial cells to metabolize this compound will result in an impairment of the colonic epithelial cell O₂ consumption with consequences on the process of mucosal inflammation. In addition, endogenously produced sulfide is emerging as a prosecretory neuromodulator and as a relaxant agent toward the intestinal contractibility. Lastly, sulfide has been recently described as an agent involved in nociception in the large intestine although, depending on the experimental design, both pro- and anti-nociceptive effects have been reported.     

Coupled cyclic electron transport in intact chloroplasts and leaves of C3 plants: Does it exist? if so,what is its function?

Transthylakoid proton transport based on Photosystem I-dependent cyclic electron transport has been demonstrated in isolated intact spinach chloroplasts already at very low photon flux densities when the acceptor side of Photosystem I (PS I) was largely closed. It was under strict redox control. In spinach leaves, high intensity flashes given every 50 s on top of far-red, but not on top of red background light decreased the activity of Photosystem II (PS II) in the absence of appreciable linear electron transport even when excitation of PS II by the background light was extremely weak. Downregulation of PS II was a consequence of cyclic electron transport as shown by differences in the redox state of P700 in the absence and the presence of CO₂ which drained electrons from the cyclic pathway eliminating control of PS II. In the presence of CO₂, cyclic electron transport comes into play only at higher photon flux densities. At H⁺/e=3 in linear electron transport, it does not appear to contribute much ATP for carbon reduction in C3 plants. Rather, its function is to control the activity of PS II. Control is necessary to prevent excessive reduction of the electron transport chain. This helps to protect the photosynthetic apparatus of leaves against photoinactivation under light stress.     

Modulation of redox regulatory molecules and electron transport chain activity in muscle of air breathing fish Heteropneustes fossilis under air exposure stress

Responses of redox regulatory system to long-term survival (>18 h) of the catfish Heteropneustes fossilis in air are not yet understood. Lipid and protein oxidation level, oxidant (H₂O₂) generation, antioxidative status (levels of superoxide dismutase, catalase, glutathione peroxidase and reductase, ascorbic acid and non-protein sulfhydryl) and activities of respiratory complexes (I, II, III and IV) in mitochondria were investigated in muscle of H. fossilis under air exposure condition (0, 3, 6, 12 and 18 h at 25 °C). The increased levels of both H₂O₂ and tissue oxidation were observed due to the decreased activities of antioxidant enzymes in muscle under water deprivation condition. However, ascorbic acid and non-protein thiol groups were the highest at 18 h air exposure time. A linear increase in complex II activity with air exposure time and an increase up to 12 h followed by a decrease in activity of complex I at 18 h were observed. Negative correlation was observed for complex III and V activity with exposure time. Critical time to modulate the above parameters was found to be 3 h air exposure. Dehydration induced oxidative stress due to modulation of electron transport chain and redox metabolizing enzymes in muscle of H. fossilis was clearly observed. Possible contribution of redox regulatory system in muscle tissue of the fish for long-term survival in air is elucidated. Results of the present study may be useful to understand the redox metabolism in muscle of fishes those are exposed to air in general and air breathing fishes in particular.     

Purification, molecular weight, amino acid, and subunit composition of arylsulfatase A from human liver.

In isolated hepatic mitochondria, the oxidation of NAD⁺-dependent substrates was decreased after chronic consumption of ethanol or by the addition of acetaldehyde in vitro. Reversed electron transport from succinate to NAD^?, which requires transfer of electrons through the NADH dehydrogenase complex and energy transduction through coupling site 1, was depressed by ethanol feeding and by acetaldehyde in vitro, whereas NADH formation from glutamate, which is mediated directly by substrate oxidation and is not energy-dependent, was slightly increased. By contrast, reactions involving the terminal portion of the phosphorylation chain, e.g., ATP-³²P exchange or dinitrophenolstimulated ATPase activity, were not affected. Adenine nucleotide translocase activity was not altered by chronic consumption of ethanol or the addition of acetaldehyde in vitro. These data suggest that the NADH-ubiquinone oxidoreductase complex of the respiratory chain, a segment which contains several iron-sulfur centers which participate in electron transport and energy transduction, may be impaired by chronic consumption of ethanol and is especially sensitive to inhibition by acetaldehyde in vitro. Neither energy coupling sites II or III, nor the terminal reactions of oxidative phosphorylation share this sensitivity. CO₂ production from various labeled intermediates of the citric acid cycle was depressed after chronic consumption of ethanol and after the addition of acetaldehyde. Acetate had no effect on these reactions, indicating that the inhibition by acetaldehyde is not mediated via acetate. Impairment of the activities of the respiratory chain and the citric acid cycle, or both, may explain the decreases in oxygen uptake and CO₂ production from citric acid cycle intermediates and fatty acids, as well as the increase in ketone body production, found in mitochondria from ethanolfed rats.     

Sulfide Oxidation by a Noncanonical Pathway in Red Blood Cells Generates Thiosulfate and Polysulfides

A cardioprotectant at low concentrations, H₂S is a toxin at high concentrations and inhibits cytochrome c oxidase. A conundrum in H₂S homeostasis is its fate in red blood cells (RBCs), which produce H₂S but lack the canonical mitochondrial sulfide oxidation pathway for its clearance. The sheer abundance of RBCs in circulation enhances the metabolic significance of their clearance strategy for H₂S, necessary to avoid systemic toxicity. In this study, we demonstrate that H₂S generation by RBCs is catalyzed by mercaptopyruvate sulfurtransferase. Furthermore, we have discovered the locus of sulfide oxidation in RBCs and describe a new role for an old protein, hemoglobin, which in the ferric or methemoglobin state binds H₂S and oxidizes it to a mixture of thiosulfate and hydropolysulfides. Our study reveals a previously undescribed route for the biogenesis of hydropolysulfides, which are increasingly considered important for H₂S-based signaling, but their origin in mammalian cells is unknown. An NADPH/flavoprotein oxidoreductase system restores polysulfide-carrying hemoglobin derivatives to ferrous hemoglobin, thus completing the methemoglobin-dependent sulfide oxidation cycle. Methemoglobin-dependent sulfide oxidation in mammals is complex and has similarities to chemistry reported for the dissolution of iron oxides in sulfidic waters and during bioleaching of metal sulfides. The catalytic oxidation of H₂S by hemoglobin explains how RBCs maintain low steady-state H₂S levels in circulation, and suggests that additional hemeproteins might be involved in sulfide homeostasis in other tissues.     

Hydrogen sulfide,a signaling molecule in plant stress responses

Gaseous molecules, such as hydrogen sulfide(H_2S)and nitric oxide(NO), are crucial players in cellular and(patho)physiological processes in biological systems. The biological functions of these gaseous molecules, which were first discovered and identified as gasotransmitters in animals, have received unprecedented attention from plant scientists in recent decades. Researchers have arrived at the consensus that H_2S is synthesized endogenously and serves as a signaling molecule throughout the plant life cycle.However, the mechanisms of H_2S action in redox biology is still largely unexplored. This review highlights what we currently know about the characteristics and biosynthesis of H_2S in plants. Additionally,we summarize the role of H_2S in plant resistance to abiotic stress. Moreover, we propose and discuss possible redox-dependent mechanisms by which H_2S regulates plant physiology.     

Glutaredoxin 2 prevents H2O2-induced cell apoptosis by protecting complex I activity in the mitochondria

Glutaredoxin 2 (Grx2) belongs to the oxidoreductase family and is an isozyme of glutaredoxin 1 (Grx1) present in the mitochondria, however its function is not well understood. The purpose of this study is to evaluate the potential anti-apoptotic function of Grx2 by examining its ability to protect complex I in the mitochondrial electron transport system using human lens epithelial cells as a model. We found that cells treated with 200 μM hydrogen peroxide (H₂O₂) for 24 h exhibited decreased viability and became apoptotic with corresponding Bax up-regulation, Bcl-2 down-regulation, caspase 3 activation and mitochondrial cytochrome c leakage. Grx2 over-expression (OE) could protect cells against H₂O₂-induced damage while Grx2 knockdown (KD) showed the opposite effect. Under the same conditions, H₂O₂ treatment caused 50% inactivation of complex I activity in control cells (vector only), 75% in Grx2 KD cells but only 20% in Grx2 OE cells. Furthermore, the inactivated complex I in the H₂O₂-treated cells could be protected mostly by importing the purified nascent Grx2 protein, but not the Grx2 protein mutated at the active site with C70S, or C73S, or with C70S plus C73S. Immunoprecipitation study also revealed that Grx2 co-precipitated with complex I, but not complex II, in the mitochondrial lysate. Thus, the mechanism of Grx2 protection against H₂O₂-induced apoptosis is likely associated with its ability to preserve complex I.     

The amylin peptide implicated in type 2 diabetes stimulates copper-mediated carbonyl group and ascorbate radical formation

Human amylin (hA), which is toxic to islet β-cells, can self-generate H₂O₂, and this process is greatly enhanced in the presence of Cu(II) ions. Here we show that carbonyl groups, a marker of oxidative modification, were formed in hA incubated in the presence of Cu(II) ions or Cu(II) ions plus H₂O₂, but not in the presence of H₂O₂ alone. Furthermore, under similar conditions (i.e., in the presence of both Cu(II) ions and H₂O₂), hA also stimulated ascorbate radical formation. The same observations concerning carbonyl group formation were made when the histidine residue (at position 18) in hA was replaced by alanine, indicating that this residue does not play a key role. In complete contrast to hA, rodent amylin, which is nontoxic, does not generate H₂O₂, and binds Cu(II) ions only weakly, showed none of these properties. We conclude that the hA-Cu(II)/Cu(I) complex is redox active, with electron donation from the peptide reducing the oxidation state of the copper ions. The complex is capable of forming H₂O₂ from O₂ and can also generate ^•OH via Fenton chemistry. These redox properties of hA can explain its ability to stimulate copper-mediated carbonyl group and ascorbate radical formation. The formation of reactive oxygen species from hA in this way could hold the key to a better understanding of the damaging consequences of amyloid formation within the pancreatic islets of patients with type 2 diabetes mellitus.     

Spectroscopic characterization of a Ni-organic radical intermediate in the aerobic oxidation of methanol catalyzed by a Ni(II)(polyoximate) complex

In the aerobic oxidation of methanol catalyzed by a Ni(II)(TRISOX) complex [H₃TRISOX = tris(1-propan-2-onyl oxime)amine], an intermediate is observed spectroscopically. The intensities of both the UV-Vis absorption and electron paramagnetic resonance (EPR) spectra associated with this intermediate maximize during the time period of maximum formaldehyde production, and decrease as the methanol oxidation activity decreases. The UV-Vis spectrum has prominent features at 350, 420, and 535 nm. The EPR spectrum is centered at g = 2.00 and shows splittings of 28 ± 5 G. Both of these spectra are consistent with characterization of the intermediate as including one or more iminoxyl radicals derived from the oximate groups of the TRISOX ligand. Spectroscopic features very similar to those in the air-oxidized intermediate are observed in electrochemically oxidized samples, suggesting that the electrochemically generated complex will be a useful model for the intermediate observed during catalytic turnover. The crystal structure of a Ni(II) complex with an intermediate protonation state of the ligand, [Ni(II)₂(H₂TRISOX)₂(μ₂:η¹-ONO₂)](NO₃) · (CH₃CN) · 5(H₂O), 4, has been structurally characterized. Comparison to the previously reported [Ni(II)(H₂TRISOX)(CH₃CN)]₂(ClO₄)₂, 3, shows that bis(μ-oximate) dimers can form either with or without an additional bridging ligand. Addition of the nitrato bridge decreases the Ni-Ni distance from 3.5752(13) Å in 3 to 3.2014(4) Å in 4. It is intriguing to note that the reactions catalyzed by the Ni(II)(TRISOX) complex, the net transfer of two hydrogen atoms from an alcohol or amine substrate to O₂, are the same reactions catalyzed by several different metalloenzymes that also incorporate both a redox active metal and a redox active organic component in their active sites.     

Redox properties of heme peroxidases

Peroxidases are heme enzymes found in bacteria, fungi, plants and animals, which exploit the reduction of hydrogen peroxide to catalyze a number of oxidative reactions, involving a wide variety of organic and inorganic substrates. The catalytic cycle of heme peroxidases is based on three consecutive redox steps, involving two high-valent intermediates (Compound I and Compound II), which perform the oxidation of the substrates. Therefore, the thermodynamics and the kinetics of the catalytic cycle are influenced by the reduction potentials of three redox couples, namely Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺. In particular, the oxidative power of heme peroxidases is controlled by the (high) reduction potential of the latter two couples. Moreover, the rapid H₂O₂-mediated two-electron oxidation of peroxidases to Compound I requires a stable ferric state in physiological conditions, which depends on the reduction potential of the Fe³⁺/Fe²⁺ couple. The understanding of the molecular determinants of the reduction potentials of the above redox couples is crucial for the comprehension of the molecular determinants of the catalytic properties of heme peroxidases.This review provides an overview of the data available on the redox properties of Fe³⁺/Fe²⁺, Compound I/Fe³⁺, Compound I/Compound II and Compound II/Fe³⁺ couples in native and mutated heme peroxidases. The influence of the electron donor properties of the axial histidine and of the polarity of the heme environment is analyzed and the correlation between the redox properties of the heme group with the catalytic activity of this important class of metallo-enzymes is discussed.     

A Refined Analysis of Superoxide Production by Mitochondrial sn-Glycerol 3-Phosphate Dehydrogenase

The oxidation of sn-glycerol 3-phosphate by mitochondrial sn-glycerol 3-phosphate dehydrogenase (mGPDH) is a major pathway for transfer of cytosolic reducing equivalents to the mitochondrial electron transport chain. It is known to generate H₂O₂ at a range of rates and from multiple sites within the chain. The rates and sites depend upon tissue source, concentrations of glycerol 3-phosphate and calcium, and the presence of different electron transport chain inhibitors. We report a detailed examination of H₂O₂ production during glycerol 3-phosphate oxidation by skeletal muscle, brown fat, brain, and heart mitochondria with an emphasis on conditions under which mGPDH itself is the source of superoxide and H₂O₂. Importantly, we demonstrate that a substantial portion of H₂O₂ production commonly attributed to mGPDH originates instead from electron flow through the ubiquinone pool into complex II. When complex II is inhibited and mGPDH is the sole superoxide producer, the rate of superoxide production depends on the concentrations of glycerol 3-phosphate and calcium and correlates positively with the predicted reduction state of the ubiquinone pool. mGPDH-specific superoxide production plateaus at a rate comparable with the other major sites of superoxide production in mitochondria, the superoxide-producing center shows no sign of being overreducible, and the maximum superoxide production rate correlates with mGPDH activity in four different tissues. mGPDH produces superoxide approximately equally toward each side of the mitochondrial inner membrane, suggesting that the Q-binding pocket of mGPDH is the major site of superoxide generation. These results clarify the maximum rate and mechanism of superoxide production by mGPDH.     

Polyphenol Oxidation by Vicia faba Chloroplast Membranes: STUDIES ON THE LATENT MEMBRANE-BOUND POLYPHENOL OXIDASE AND ON THE MECHANISM OF PHOTOCHEMICAL POLYPHENOL OXIDATION

The mechanism whereby light effects polyphenol oxidation was examined with Vicia faba chloroplast membranes known to contain a bound latent polyphenol oxidase. Results obtained with the inhibitors 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-idopropyl-p-benzoquinone (DBMIB) indicated an involvement of the non-cyclic electron transport pathway in the light-dependent oxidation of polyphenols, such as dihydroxyphenylalanine (DOPA). Further evidence was provided by experiments in which (a) DOPA replaced H₂O as electron donor for the photoreduction of NADP, (b) NADP replaced O₂ as electron acceptor in the photochemical oxidation of DOPA, and (c) the variable fluorescence associated with photosystem II was increased by DOPA. The photochemical oxidation of DOPA by V. faba chloroplast membranes was insensitive to KCN and to antibodies against purified latent polyphenol oxidase. The results are consistent with the conclusion that the light-dependent oxidation of polyphenols by V. faba chloroplast membranes is achieved independently of the latent membrane-bound polyphenol oxidase. Electrons derived from polyphenols seem to enter the noncyclic electron transport chain on the oxidizing side of photosystem II and to react with O₂ at an unidentified site on the photosystem I side of the DCMU/DBMIB blocks.     

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H₂O₂. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H₂O₂ directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H₂O₂ production at a fairly high rate of about 300 pmol H₂O₂·min⁻¹·mg protein⁻¹ when oxidation of ubiquinol is prevented and complex II is uninhibited. This H₂O₂ production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site II_F of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H₂O₂ production (rotenone to inhibit sites in complex I (site I_Q and, indirectly, site I_F), myxothiazol to inhibit site III_Qo in complex III, and malonate plus atpenin A5 to inhibit site II_F in complex II), dihydroorotate dehydrogenase generates superoxide/H₂O₂, at a small but significant rate (23 pmol H₂O₂·min⁻¹·mg protein⁻¹), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H₂O₂ directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.     

Coordination Chemistry and Redox Processes in Siderophore-Mediated Iron Transport

In this mini-review we present an environmental iron mobility/transport scheme consisting of inter-related controls, whereby the first coordination shell of iron modulates the iron redox potential (E_1/2), and the oxidation state of iron controls the chemistry of the first coordination sphere and therefore the immediate chemical environment of the iron. Siderophores (microbially generated iron specific chelators) may be viewed as iron redox mediators. Siderophore chelation of environmental iron in a reduced (Fe(II)) oxidation state results in facile air oxidation of iron due to the negative redox potentials observed for Fe-siderophore complexes. This solubilizes the iron and locks it into a specific coordination environment, thereby preventing hydrolysis and precipitation. The high-spin Fe³⁺ → Fe⁺ electron transfer process may be viewed as a switch that controls the thermodynamic stability and kinetic lability of the first coordination shell. Reduction of iron(III)-siderophore complexes to iron(II)-siderophore complexes decreases thermodynamic stability, increases the rate of siderophore ligand exchange, and increases the ease of siderophore donor atom protonation, thus facilitating a rapid turnover of the first coordination shell. Results are presented for iron-siderophore pH and oxidation state dependent speciation studies that are relevant to environmental and microbial iron mobility and transport.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Analysis of Dichlorodihydrofluorescein and Dihydrocalcein as Probes for the Detection of Intracellular Reactive Oxygen Species

Dihydrocalcein (H₂-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H₂-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H₂-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5?min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H₂-calcein but increased the oxidation of H₂-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H₂-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H₂-DCF. In contrast, DPI inhibited H₂-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H₂-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H₂-calcein, but not H₂-DCF inhibited complex I activity.

These observations indicate that H₂-DCF is an indicator for intracellular ROS, whereas the oxidation of H₂-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.