The rate of nitrite reduction in leaves as indicated by O₂ and CO₂ exchange during photosynthesis

Light response (at 300 ppm CO(2) and 10-50 ppm O(2) in N(2)) and CO(2) response curves [at absorbed photon fluence rate (PAD) of 550 μmol m(-2) s(-1)] of O(2) evolution and CO(2) uptake were measured in tobacco (Nicotiana tabacum L.) leaves grown on either NO(3)(-) or NH(4)(+) as N source and in potato (Solanum tuberosum L.), sorghum (Sorghum bicolor L. Moench), and amaranth (Amaranthus cruentus L.) leaves grown on NH(4)NO(3). Photosynthetic O(2) evolution in excess of CO(2) uptake was measured with a stabilized zirconia O(2) electrode and an infrared CO(2) analyser, respectively, and the difference assumed to represent the rate of electron flow to acceptors alternative to CO(2), mainly NO(2)(-), SO(4)(2-), and oxaloacetate. In NO(3)(-)-grown tobacco, as well as in sorghum, amaranth, and young potato, the photosynthetic O(2)-CO(2) flux difference rapidly increased to about 1 μmol m(-2) s(-1) at very low PADs and the process was saturated at 50 μmol quanta m(-2) s(-1). At higher PADs the O(2)-CO(2) flux difference continued to increase proportionally with the photosynthetic rate to a maximum of about 2 μmol m(-2) s(-1). In NH(4)(+)-grown tobacco, as well as in potato during tuber filling, the low-PAD component of surplus O(2) evolution was virtually absent. The low-PAD phase was ascribed to photoreduction of NO(2)(-) which successfully competes with CO(2) reduction and saturates at a rate of about 1 μmol O(2) m(-2) s(-1) (9% of the maximum O(2) evolution rate). The high-PAD component of about 1 μmol O(2) m(-2) s(-1), superimposed on NO(2)(-) reduction, may represent oxaloacetate reduction. The roles of NO(2)(-), oxaloacetate, and O(2) reduction in the regulation of ATP/NADPH balance are discussed.     

Is C4 photosynthesis less phenotypically plastic than C3 photosynthesis?

C4 photosynthesis is a complex specialization that enhances carbon gain in hot, often arid habitats where photorespiration rates can be high. Certain features unique to C4 photosynthesis may reduce the potential for phenotypic plasticity and photosynthetic acclimation to environmental change relative to what is possible with C3 photosynthesis. During acclimation, the structural and physiological integrity of the mesophyll-bundle sheath (M-BS) complex has to be maintained if C4 photosynthesis is to function efficiently in the new environment. Disruption of the M-BS structure could interfere with metabolic co-ordination between the C3 and C4 cycles, decrease metabolite flow rate between the tissues, increase CO2 leakage from the bundle sheath, and slow enzyme activity. C4 plants have substantial acclimation potential, but in most cases lag behind the acclimation responses in C3 plants. For example, some C4 species are unable to maintain high quantum yields when grown in low-light conditions. Others fail to reduce carboxylase content in shade, leaving substantial over-capacity of Rubisco and PEP carboxylase in place. Shade-tolerant C4 grasses lack the capacity for maintaining a high state of photosynthetic induction following sunflecks, and thus may be poorly suited to exploit subsequent sunflecks compared with C3 species. In total, the evidence indicates that C4 photosynthesis is less phenotypically plastic than C3 photosynthesis, and this may contribute to the more restricted ecological and geographical distribution of C4 plants across the Earth.     

Adenosine A₂a receptors and O₂ sensing in development

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O? sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(?A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(?A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(?A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(?A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(?A) receptors play virtually no role in O? sensing by the carotid bodies, but brain A(?A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(?A) receptors have been implicated in O? sensing by carotid glomus cells, while central A(?A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(?A) receptors are crucially involved in the transduction mechanisms of O? sensing in fetal carotid bodies and brains. Postnatally, central A(?A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O? sensing in carotid chemoreceptors, particularly in developing lambs.     

When did oxygenic photosynthesis evolve?

The atmosphere has apparently been oxygenated since the 'Great Oxidation Event' ca 2.4 Ga ago, but when the photosynthetic oxygen production began is debatable. However, geological and geochemical evidence from older sedimentary rocks indicates that oxygenic photosynthesis evolved well before this oxygenation event. Fluid-inclusion oils in ca 2.45 Ga sandstones contain hydrocarbon biomarkers evidently sourced from similarly ancient kerogen, preserved without subsequent contamination, and derived from organisms producing and requiring molecular oxygen. Mo and Re abundances and sulphur isotope systematics of slightly older (2.5 Ga) kerogenous shales record a transient pulse of atmospheric oxygen. As early as ca 2.7 Ga, stromatolites and biomarkers from evaporative lake sediments deficient in exogenous reducing power strongly imply that oxygen-producing cyanobacteria had already evolved. Even at ca 3.2 Ga, thick and widespread kerogenous shales are consistent with aerobic photoautrophic marine plankton, and U-Pb data from ca 3.8 Ga metasediments suggest that this metabolism could have arisen by the start of the geological record. Hence, the hypothesis that oxygenic photosynthesis evolved well before the atmosphere became permanently oxygenated seems well supported.     

Leaf photosynthesis and carbohydrates of CO₂-enriched maize and grain sorghum exposed to a short period of soil water deficit during vegetative development

Among C₄ species, sorghum is known to be more drought tolerant than maize. The objective was to evaluate differences in leaf gas exchanges, carbohydrates, and two enzyme activities of these nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) C₄ subtype monocots in response to water deficit and CO₂ concentration ([CO₂]). Maize and sorghum were grown in pots in sunlit environmental-controlled chambers. Treatments included well watered (WW) and water stressed (WS) (water withheld at 26 days) and daytime [CO₂] of 360 (ambient) and 720 (elevated) μmol mol⁻¹. Midday gas exchange rates, concentrations of nonstructural carbohydrates, and activities of sucrose-phosphate synthase (SPS) and adenosine 5′-diphosphoglucose pyrophosphorylase (ADGP) were determined for fully expanded leaf sections. There was no difference in leaf CO₂ exchange rates (CER) between ambient and elevated [CO₂] control plants for both maize and sorghum. After withholding water, leaf CER declined to zero after 8 days in maize and 10 days for sorghum. Sorghum had lower stomatal conductance and transpiration rates than maize, which resulted in a longer period of CER under drought. Nonstructural carbohydrates of both control maize and sorghum were hardly affected by elevated [CO₂]. Under drought, however, increases in soluble sugars and decreases in starch were generally observed for maize and sorghum at both [CO₂] levels. For stressed maize and sorghum, decreases in starch occurred earlier and were greater at ambient [CO₂] than at elevated [CO₂]. For maize, drought did not meaningfully affect SPS activity. However, a decline in SPS activity was observed for drought-stressed sorghum under both [CO₂] treatments. There was an increase in ADGP activity in maize under drought for both [CO₂] treatments. Such a response in ADGP to drought, however, did not occur for sorghum. The generally more rapid response of maize than sorghum to drought might be related to the more rapid growth of leaf area of maize.     

How does protein phosphorylation regulate photosynthesis?

Phosphorylation of light-harvesting antenna proteins redirects absorbed light energy between reaction centres of photosynthetic membranes. A generally accepted explanation for this is that electrostatic forces drive the more negatively charged, phosphorylated antenna proteins between membrane domains that differ in surface charge. However, structural studies on soluble phosphoproteins indicate that phosphorylated amino acid side chains have specific effects on molecular recognition, by ligand blocking or by intramolecular interactions which alter protein structure. These studies suggest alternative mechanisms for phosphorylation in control of pairwise protein-protein interactions in biological membranes. Thus, in photosynthesis, the surface charge model is only one possible interpretation.     

The evolution of photosynthesis…again?

‘Replaying the tape’ is an intriguing ‘would it happen again?’ exercise. With respect to broad evolutionary innovations, such as photosynthesis, the answers are central to our search for life elsewhere. Photosynthesis permits a large planetary biomass on Earth. Specifically, oxygenic photosynthesis has allowed an oxygenated atmosphere and the evolution of large metabolically demanding creatures, including ourselves. There are at least six prerequisites for the evolution of biological carbon fixation: a carbon-based life form; the presence of inorganic carbon; the availability of reductants; the presence of light; a light-harvesting mechanism to convert the light energy into chemical energy; and carboxylating enzymes. All were present on the early Earth. To provide the evolutionary pressure, organic carbon must be a scarce resource in contrast to inorganic carbon. The probability of evolving a carboxylase is approached by creating an inventory of carbon-fixation enzymes and comparing them, leading to the conclusion that carbon fixation in general is basic to life and has arisen multiple times. Certainly, the evolutionary pressure to evolve new pathways for carbon fixation would have been present early in evolution. From knowledge about planetary systems and extraterrestrial chemistry, if organic carbon-based life occurs elsewhere, photosynthesis—although perhaps not oxygenic photosynthesis—would also have evolved.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

Synthesis of functional SiO₂-coated graphene oxide nanosheets decorated with Ag nanoparticles for H₂O₂ and glucose detection

In this paper, we report on the first preparation of well-defined SiO(2)-coated graphene oxide (GO) nanosheets (SiO(2)/GO) without prior GO functionalization by combining sonication with sol-gel technique. The functional SiO(2)/GO nanocomposites (F-SiO(2)/GO) obtained by surface functionalization with NH(2) group were subsequently employed as a support for loading Ag nanoparticles (AgNPs) to synthesize AgNP-decorated F-SiO(2)/GO nanosheets (AgNP/F-SiO(2)/GO) by two different routes: (1) direct adsorption of preformed, negatively charged AgNPs; (2) in situ chemical reduction of silver salts. The morphologies of these nanocomposites were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is found that the resultant AgNP/F-SiO(2)/GO exhibits remarkable catalytic performance for H(2)O(2) reduction. This H(2)O(2) sensor has a fast amperometric response time of less than 2s. The linear range is estimated to be from 1×10(-4) M to 0.26 M (r=0.998) and the detection limit is estimated to be 4 × 10(-6) M at a signal-to-noise ratio of 3, respectively. We also fabricated a glucose biosensor by immobilizing glucose oxidase (GOD) into AgNP/F-SiO(2)/GO nanocomposite-modified glassy carbon electrode (GCE) for glucose detection. Our study demonstrates that the resultant glucose biosensor can be used for the glucose detection in human blood serum.     

Do mesocosms influence photosynthesis and soil respiration?

Mesocosms, enclosed outdoor experimental systems, are commonly used in terrestrial ecology. They are frequently used to study the effects of elevated CO₂ and temperature on terrestrial ecosystem processes. Despite their advantages and frequent use it is important to verify, through explicit measures, that mesocosms reliably model the larger system. In this study, fully-coupled, soil–litter–plant mesocosms were constructed in Corvallis using native soil and litter, and planted with Douglas-fir (Pseudotsuga menziesii Mirb. Franco) seedlings. Needle photosynthesis and soil respiration were measured repeatedly over a 21-month period in mesocosms and compared to measurements made at two field sites (Toad Creek and Falls Creek) planted at the same density as the mesocosms. Under the temperature and soil moisture conditions, photosynthetic and soil respiration rates in the mesocosms were not significantly different than the rates at Toad Creek, where the soil and litter in the mesocosms were collected. In contrast, the soil at Falls Creek was different than the soil in the mesocosms and at Toad Creek and photosynthetic and soil respiration rates at Falls Creek were significantly different than at the other two sites. The lack of significant differences between rates measured in the mesocosms in Corvallis and at the Toad Creek field site indicate that the mesocosms did not cause significant artifacts in the data and that the results for these rates in the mesocosms can be extrapolated to field settings with comparable edaphic conditions.     

Can improvement in photosynthesis increase crop yields?

The yield potential (Y_p) of a grain crop is the seed mass per unit ground area obtained under optimum growing conditions without weeds, pests and diseases. It is determined by the product of the available light energy and by the genetically determined properties: efficiency of light capture (?_i), the efficiency of conversion of the intercepted light into biomass (?_c) and the proportion of biomass partitioned into grain (η). Plant breeding brings η and ?_i close to their theoretical maxima, leaving ?_c, primarily determined by photosynthesis, as the only remaining major prospect for improving Y_p. Leaf photosynthetic rate, however, is poorly correlated with yield when different genotypes of a crop species are compared. This led to the viewpoint that improvement of leaf photosynthesis has little value for improving Y_p. By contrast, the many recent experiments that compare the growth of a genotype in current and future projected elevated [CO₂] environments show that increase in leaf photosynthesis is closely associated with similar increases in yield. Are there opportunities to achieve similar increases by genetic manipulation? Six potential routes of increasing ?_c by improving photosynthetic efficiency were explored, ranging from altered canopy architecture to improved regeneration of the acceptor molecule for CO₂. Collectively, these changes could improve ?_c and, therefore, Y_p by c. 50%. Because some changes could be achieved by transgenic technology, the time of the development of commercial cultivars could be considerably less than by conventional breeding and potentially, within 10–15 years.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

CO₂ utilizing microbes--a comprehensive review

CO₂ fixing microbes are the species primarily engaged in complexing the inorganic carbon dioxide to organic carbon compounds. There are many microorganisms from archaeal and bacterial domain that can fix carbon dioxide through six known CO₂ fixing pathways. These organisms are ubiquitous and can survive in wide range of aerobic and anaerobic habitats. This review focuses on the prior research, that has been conducted in this field and presents a summarized overview of all the mechanisms (along with their genes and enzymes) used by these microbes for CO₂ incorporation. In addition, this review provides a better understanding of diversity and taxonomy of CO₂ fixing microorganisms. The information presented here will motivate researchers to further explore the diversity of CO₂ fixing microorganisms as well as to decipher the underlying mechanisms of CO₂ utilization.     

VO₂ requirements of boxing exercises

The purpose of this study was to quantify the physiological requirements of various boxing exercises such as sparring, pad work, and punching bag. Because it was not possible to measure the oxygen uptake (VO?) of "true" sparring with a collecting gas valve in the face, we developed and validated a method to measure VO? of "true" sparring based on "postexercise" measurements. Nine experienced male amateur boxers (Mean ± SD: age = 22.0 ± 3.5 years, height = 176.0 ± 8.0 cm, weight = 71.4 ± 10.9 kg, number of fights = 13.0 ± 9.5) of regional and provincial level volunteered to participate in 3 testing sessions: (a) maximal treadmill test in the LAB, (b) standardized boxing training in the GYM, and (c) standardized boxing exercises in the LAB. Measures of VO?, heart rate (HR), blood lactate concentration [LA], rated perceived exertion level, and punching frequencies were collected. VO? values of 43.4 ± 5.9, 41.1 ± 5.1, 24.7 ± 6.1, 30.4 ± 5.8, and 38.3 ± 6.5 ml·kg?1·min?1 were obtained, which represent 69.7 ± 8.0, 66.1 ± 8.0, 39.8 ± 10.4, 48.8 ± 8.5, and 61.7 ± 10.3%VO?peak for sparring, pad work, and punching bag at 60, 120, and 180 b·min?1, respectively. Except for lower VO? values for punching the bag at 60 and 120 b·min?1 (p < 0.05), there was no VO? difference between exercises. Similar pattern was obtained for %HRmax with respective values of 85.5 ± 5.9, 83.6 ± 6.3, 67.5 ± 3.5, 74.8 ± 5.9, and 83.0 ± 6.0. Finally, sparring %HRmax and [LA] were slightly higher in the GYM (91.7 ± 4.3 and 9.4 ± 2.2 mmol·L?1) vs. LAB (85.5 ± 5.9 and 6.1 ± 2.3 mmol·L?1). Thus, in this study simulated LAB sparring and pad work required similar VO? (43-41 ml·kg?1·min?1, respectively), which corresponds to ~70%VO?peak. These results underline the importance of a minimum of aerobic fitness for boxers and draw some guidelines for the intensity of training.     

Effects of olive leaf polyphenols against H₂O₂ toxicity in insulin secreting β-cells

In pancreatic β-cells, although H?O? is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H?O? (0.035 mM) for 45 min. H?O? alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H?O?-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H?O?-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.     

Plant respiration: Controlled by photosynthesis or biomass?

Two simplifying hypotheses have been proposed for whole‐plant respiration. One links respiration to photosynthesis; the other to biomass. Using a first‐principles carbon balance model with a prescribed live woody biomass turnover, applied at a forest research site where multidecadal measurements are available for comparison, we show that if turnover is fast the accumulation of respiring biomass is low and respiration depends primarily on photosynthesis; while if turnover is slow the accumulation of respiring biomass is high and respiration depends primarily on biomass. But the first scenario is inconsistent with evidence for substantial carry‐over of fixed carbon between years, while the second implies far too great an increase in respiration during stand development—leading to depleted carbohydrate reserves and an unrealistically high mortality risk. These two mutually incompatible hypotheses are thus both incorrect. Respiration is not linearly related either to photosynthesis or to biomass, but it is more strongly controlled by recent photosynthates (and reserve availability) than by total biomass.     

Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H₂O₂

Oxidative stress is a threat to mammalian cells. To better understand the molecular response and mechanism underlying oxidative stress, we applied two‐dimensional polyacrylamide gel electrophoresis and matrix‐assisted laser desorption ionization time‐of‐fight mass spectrometry analysis to identify differential nuclear protein profiling of mouse fibroblast NIH‐3T3 cells exposed to mild‐H₂O₂. Thirteen differentially expressed proteins were identified by MS and two of them were further validated by Western blot. The results revealed that exposure to mild‐H₂O₂ for 12 h cause up‐regulated expression of DJ‐1, glutathione S‐transferase P 1, DNA ligase I, dynamin 2, nucleophosmin, and down‐regulated expression of nucleoside diphosphate kinase A, enolase‐α, barrier‐to‐autointegration factor 1, metastasis associated protein 1, glycosytransferase‐like domain containing protein 1, synaptonemal complex protein 1, alpha‐centractin, bromodomain, and PHD finger containing 1 (BRPF1). Most of the identified proteins are supported as nuclear proteins localized by previous research. The findings may provide some clues to elucidate cell responses to H₂O₂ and the potential mechanism underlying protection against oxidative stress in fibroblast cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Protective effects of cynaroside against H₂O₂-induced apoptosis in H9c2 cardiomyoblasts

Flavonoids with potent anti-oxidative effects are the major effective components in traditional herbal medicine used in treating cardiovascular diseases. Cynaroside is a flavonoid compound that exhibits anti-oxidative capabilities. However, little is known about its effect on oxidative injury to cardiac myocytes and the underlying mechanisms. This study was designed to investigate the protective effects of cynaroside against H(2) O(2) -induced apoptosis in H9c2 cardiomyoblasts. H9c2 cells were pretreated with cynaroside for 4 h before exposure to 150 μM H(2) O(2) for 6 h. H(2) O(2) treatment caused severe injury to the H9c2 cells, which was accompanied by apoptosis, as revealed by analysis of cell nuclear morphology, through Annexin V FITC/PI staining and caspase proteases activation. Cynaroside pretreatment significantly reduced the apoptotic rate by enhancing the endogenous anti-oxidative activity of superoxide dismutase, glutathione peroxidase, and catalase, thereby inhibiting intracellular reactive oxygen species (ROS) generation. Moreover, cynaroside moderated H(2) O(2) -induced disruption of mitochondrial membrane potential, increased the expression of anti-apoptotic protein Bcl-2 while decreased the expression of pro-apoptotic protein Bax, and thereby inhibited the release of apoptogenic factors (cytochrome c and smac/Diablo) from mitochondria in H9c2 cells. Our data also demonstrated that cynaroside pretreatment showed an inhibitory effect on the H(2) O(2) -induced increase in c-Jun N-terminal kinase (JNK) and P53 protein expression. These results suggest that cynaroside prevents H(2) O(2) -induced apoptosis in H9c2 cell by reducing the endogenous production of ROS, maintaining mitochondrial function, and modulating the JNK and P53 pathways.     

Removal of H₂O₂ and generation of superoxide radical: role of cytochrome c and NADH

In cells, mitochondria, endoplasmic reticulum, and peroxisomes are the major sources of reactive oxygen species (ROS) under physiological and pathophysiological conditions. Cytochrome c (cyt c) is known to participate in mitochondrial electron transport and has antioxidant and peroxidase activities. Under oxidative or nitrative stress, the peroxidase activity of Fe³⁺cyt c is increased. The level of NADH is also increased under pathophysiological conditions such as ischemia and diabetes and a concurrent increase in hydrogen peroxide (H₂O₂) production occurs. Studies were performed to understand the related mechanisms of radical generation and NADH oxidation by Fe³⁺cyt c in the presence of H₂O₂. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with NADH, Fe³⁺cyt c, and H₂O₂ in the presence of methyl-β-cyclodextrin. An EPR spectrum corresponding to the superoxide radical adduct of DMPO encapsulated in methyl-β-cyclodextrin was obtained. This EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase (SOD1). The amount of superoxide radical adduct formed from the oxidation of NADH by the peroxidase activity of Fe³⁺cyt c increased with NADH and H₂O₂ concentration. From these results, we propose a mechanism in which the peroxidase activity of Fe³⁺cyt c oxidizes NADH to NAD^•, which in turn donates an electron to O₂, resulting in superoxide radical formation. A UV-visible spectroscopic study shows that Fe³⁺cyt c is reduced in the presence of both NADH and H₂O₂. Our results suggest that Fe³⁺cyt c could have a novel role in the deleterious effects of ischemia/reperfusion and diabetes due to increased production of superoxide radical. In addition, Fe³⁺cyt c may play a key role in the mitochondrial “ROS-induced ROS-release” signaling and in mitochondrial and cellular injury/death. The increased oxidation of NADH and generation of superoxide radical by this mechanism may have implications for the regulation of apoptotic cell death, endothelial dysfunction, and neurological diseases. We also propose an alternative electron transfer pathway, which may protect mitochondria and mitochondrial proteins from oxidative damage.