Redox State Of Cytochrome Research Articles

The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells

BackgroundMethotrexate is a chemotherapeutic agent used to treat a variety of cancers. However, the occurrence of resistance limits its effectiveness. Cytochrome c in its reduced state is less capable of triggering the apoptotic cascade. Thus, we set up to study the relationship among redox state of cytochrome c, apoptosis and the development of resistance to methotrexate in MCF7 human breast cancer cells.ResultsCell incubation with cytochrome c-reducing agents, such as tetramethylphenylenediamine, ascorbate or reduced glutathione, decreased the mortality and apoptosis triggered by methotrexate. Conversely, depletion of glutathione increased the apoptotic action of methotrexate, showing an involvement of cytochrome c redox state in methotrexate-induced apoptosis. Methotrexate-resistant MCF7 cells showed increased levels of endogenous reduced glutathione and a higher capability to reduce exogenous cytochrome c. Using functional genomics we detected the overexpression of GSTM1 and GSTM4 in methotrexate-resistant MCF7 breast cancer cells, and determined that methotrexate was susceptible of glutathionylation by GSTs. The inhibition of these GSTM isoforms caused an increase in methotrexate cytotoxicity in sensitive and resistant cells.ConclusionsWe conclude that overexpression of specific GSTMs, GSTM1 and GSTM4, together with increased endogenous reduced glutathione levels help to maintain a more reduced state of cytochrome c which, in turn, would decrease apoptosis, thus contributing to methotrexate resistance in human MCF7 breast cancer cells.

Effect of Calcium on the Oxidative Phosphorylation Cascade in Skeletal Muscle Mitochondria

Calcium is believed to regulate mitochondrial oxidative phosphorylation, thereby contributing to the maintenance of cellular energy homeostasis. Skeletal muscle, with an energy conversion dynamic range of up to 100-fold, is an extreme case for evaluating the cellular balance of ATP production and consumption. This study examined the role of Ca(2+) in the entire oxidative phosphorylation reaction network in isolated skeletal muscle mitochondria and attempted to extrapolate these results back to the muscle, in vivo. Kinetic analysis was conducted to evaluate the dose-response effect of Ca(2+) on the maximal velocity of oxidative phosphorylation (V(maxO)) and the ADP affinity. Force-flow analysis evaluated the interplay between energetic driving forces and flux to determine the conductance, or effective activity, of individual steps within oxidative phosphorylation. Measured driving forces [extramitochondrial phosphorylation potential (ΔG(ATP)), membrane potential, and redox states of NADH and cytochromes b(H), b(L), c(1), c, and a,a(3)] were compared with flux (oxygen consumption) at 37 °C; 840 nM Ca(2+) generated an ~2-fold increase in V(maxO) with no change in ADP affinity (~43 μM). Force-flow analysis revealed that Ca(2+) activation of V(maxO) was distributed throughout the oxidative phosphorylation reaction sequence. Specifically, Ca(2+) increased the conductance of Complex IV (2.3-fold), Complexes I and III (2.2-fold), ATP production/transport (2.4-fold), and fuel transport/dehydrogenases (1.7-fold). These data support the notion that Ca(2+) activates the entire muscle oxidative phosphorylation cascade, while extrapolation of these data to the exercising muscle predicts a significant role of Ca(2+) in maintaining cellular energy homeostasis.

Casting New Physicochemical Light on the Fundamental Biological Processes in Single Living Cells by Using Raman Microspectroscopy

This Personal Account highlights the capabilities of spontaneous Raman microspectroscopy for studying fundamental biological processes in a single living cell. Raman microspectroscopy provides time- and space-resolved vibrational Raman spectra that contain detailed information on the structure and dynamics of biomolecules in a cell. By using yeast as a model system, we have made great progress in the development of this methodology. The results that we have obtained so far are described herein with an emphasis placed on how three cellular processes, that is, cell-division, respiration, and cell-death, are traced and elucidated with the use of time- and space-resolved structural information that is extracted from the Raman spectra. For cell-division, compositional- and structural changes of various biomolecules are observed during the course of the whole cell cycle. For respiration, the redox state of mitochondrial cytochromes, which is inferred from the resonance Raman bands of cytochromes, is used to evaluate the respiration activity of a single cell, as well as that of isolated mitochondrial particles. Special reference is made to the "Raman spectroscopic signature of life", which is a characteristic Raman band at 1602 cm(-1) that is found in yeast cells. This signature reflects the cellular metabolic activity and may serve as a measure of mitochondrial dysfunction. For cell-death, "cross-talk" between the mitochondria and the vacuole in a dying cell is suggested.

Systematic investigation of changes in oxidized cerebral cytochrome c oxidase concentration during frontal lobe activation in healthy adults

Using transcranial near-infrared spectroscopy (NIRS) to measure changes in the redox state of cerebral cytochrome c oxidase (Δ[oxCCO]) during functional activation in healthy adults is hampered by instrumentation and algorithm issues. This study reports the Δ[oxCCO] response measured in such a setting and investigates possible confounders of this measurement. Continuous frontal lobe NIRS measurements were collected from 11 healthy volunteers during a 6-minute anagram-solving task, using a hybrid optical spectrometer (pHOS) that combines multi-distance frequency and broadband components. Only data sets showing a hemodynamic response consistent with functional activation were interrogated for a Δ[oxCCO] response. Simultaneous systemic monitoring data were also available. Possible influences on the Δ[oxCCO] response were systematically investigated and there was no effect of: 1) wavelength range chosen for fitting the measured attenuation spectra; 2) constant or measured, with the pHOS in real-time, differential pathlength factor; 3) systemic hemodynamic changes during functional activation; 4) changes in optical scattering during functional activation. The Δ[oxCCO] response measured in the presence of functional activation was heterogeneous, with the majority of subjects showing significant increase in oxidation, but others having a decrease. We conclude that the heterogeneity in the Δ[oxCCO] response is physiological and not induced by confounding factors in the measurements.

Mapping of Redox State of Mitochondrial Cytochromes in Live Cardiomyocytes Using Raman Microspectroscopy

This paper presents a nonivasive approach to study redox state of reduced cytochromes , and of complexes II and III in mitochondria of live cardiomyocytes by means of Raman microspectroscopy. For the first time with the proposed approach we perform studies of rod- and round-shaped cardiomyocytes, representing different morphological and functional states. Raman mapping and cluster analysis reveal that these cardiomyocytes differ in the amounts of reduced cytochromes , and . The rod-shaped cardiomyocytes possess uneven distribution of reduced cytochromes , and in cell center and periphery. Moreover, by means of Raman spectroscopy we demonstrated the decrease in the relative amounts of reduced cytochromes , and in the rod-shaped cardiomyocytes caused by H2O2-induced oxidative stress before any visible changes. Results of Raman mapping and time-dependent study of reduced cytochromes of complexes II and III and cytochrome in cardiomyocytes are in a good agreement with our fluorescence indicator studies and other published data.

Mitochondrial involvement in regulation of apoptosis via redox state of external cytochrome c

Mitochondrial involvement in regulation of apoptosis via redox state of external cytochrome c

Long‐Distance Electron Transfer by G. sulfurreducens Biofilms Results in Accumulation of Reduced c‐Type Cytochromes

So close, but yet so far: G. sulfurreducens c-type cytochromes become reduced as biofilms grow on electrodes beyond a few cell thicknesses, even if the electrode is poised well above the potential required to oxidize all cytochromes. Cytochrome redox state also lags behind rapid potential changes during voltammetry, but only when the films are multiple cell layers thick, as would be expected if diffusional or exchange-based kinetics controls electron transfer between cytochromes.

Quantitative analysis of the redox states of cytochromes in a living L929 (NCTC) cell by resonance Raman microspectroscopy

Raman spectra and images of a living L929 (NCTC) cell have been measured with 532 nm excitation. Both reduced and oxidized forms of cytochromes b and c (cyt b and cyt c) have been observed in situ without any pretreatment. The redox states of cyts b and c have been assessed quantitatively with a spectral analysis. It has been found that reduced cyt c is more abundant than oxidized cyt c, while oxidized cyt b is slightly more abundant than reduced cyt b in a living cell.

Label-free Raman observation of cytochrome c dynamics during apoptosis

We performed label-free observation of molecular dynamics in apoptotic cells by Raman microscopy. Dynamic changes in cytochrome c distribution at the Raman band of 750cm(-1) were observed after adding an apoptosis inducer to the cells. The comparison of mitochondria fluorescence images and Raman images of cytochrome c confirmed that changes in cytochrome c distribution can be distinguished as release of cytochrome c from mitochondria. Our observation also revealed that the redox state of cytochrome c was maintained during the release from the mitochondria. Monitoring mitochondrial membrane potential with JC-1 dye confirmed that the observed cytochrome c release was associated with apoptosis.

Resonance Raman quantification of the redox state of cytochromes b and cin‐vivo and in‐vitro

We observe the redox state changes with respiration of cytochromes b and c in mitochondria in a living Saccharomyces cerevisiae cell as well as in isolated mitochondria with the very use of Raman microspectroscopy. The possibility of monitoring the respiration activity of mitochondria in vivo and in vitro by Raman microspectroscopic quantification of the cytochrome redox states is suggested. It will lead to a new means to assess mitochondrial respiration activity in vivo and in vitro without using any labelling or genetic manipulation.

Tert-Butylthiacalix[4]arene monolayers as a biomimetic model for the oxidation of antioxidants with cytochrome c

The possibility of designing a model of sensor to antioxidants based on thiacalix[4]arene monolayers with immobilized cytochrome c was shown. The molecular surface area S0 of thiacalix[4]arene in the monolayers and the surface pressure coefficient −dπ/dS (elasticity) reflect changes in the redox state of cytochrome c in the presence of dihydroquercetin and ascorbic acid in the aqueous subphase. The absorption spectra in the visible and UV ranges of solutions of the subphase and transferred thiacalix[4]arene monolayers with immobilized cytochrome c confirm the oxidation of the antioxidants to quinones and formation of the reduced form of cytochrome c.

Electrochemical insight into the mechanism of electron transport in biofilms of Geobacter sulfurreducens

Electroactive bacterial biofilms can be produced on a polarized electrode by forcing its use as the final electron acceptor for bacterial respiration. This strategy offers the researcher the unique possibility to control the respiration process with extreme precision. The production of current, the accumulation of charge and the conducting properties of electroactive biofilms has been interrogated in this work through very basic electrochemical techniques including chronopotentiometry, chronoamperometry and cyclic voltammetry. Presented results indicate that charge can be accumulated in the biofilm conductive network, that network conductivity does not represent a limit for current production and that both the steady state current and the amount of accumulated charge depend on the redox state of cytochromes wiring the cells to the electrode. A model of biofilm conduction is presented as well.

Linking Spectral and Electrochemical Analysis to Monitor c‐type Cytochrome Redox Status in Living Geobacter sulfurreducens Biofilms

Biofilm synesthesia: A simultaneous spectral and electrochemical analysis reveals the redox status of cytochromes within living electroactive biofilms of Geobacter. The results confirm that the primary electron reservoirs within these biofilms are c-type cytochromes and suggest which cytochromes act as kinetic bottlenecks to electron transfer.

NOBILETIN TRANSIENTLY INCREASES THE PRODUCTION OF NITRIC OXIDE AND CHANGES THE ACTIVITY OF SUCCINATE DEHYDROGENASE IN HUMAN BLOOD LYMPHOCYTES

The citrus flavonoids exhibit abilities to modulate the inflammatory response and carcinogenesis via several different mechanisms. We have studied the effects of two major citrus flavonoids – nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone), and sinensetin (5,6,7,3′,4′-pentamethoxyflavone) on the spontaneous synthesis of nitric oxide (NO), succinate dehydrogenase (SDH) activity and the copper redox state of cytochrome c oxidase in human peripheral blood lymphocytes. Our results showed that in contrast to sinensetin, nobiletin causes a transient enhancement in the production of NO, which leads to a subsequent compensatory elevation of cytochrome c oxidase and SDH. High positive correlation between NO production and level of oxidation of copper in cytochrome oxidase and inverse correlation with the activity of SDH were found. We propose that temporal alterations of mitochondrial enzymes could mediate immunomodulatory and anticarcinogenic effects of nobiletin. PRACTICAL APPLICATIONS Positive correlation between nitric oxide production and level of oxidation of copper in cytochrome oxidase and inverse correlation with the activity of succinate dehydrogenase (SDH) were found in peripheral blood lymphocytes after treatment of cells by nobiletin. These results demonstrate that the beneficial immunomodulatory and anticarcinogenic effects of nobiletin possibly involve the transient inhibition of mitochondrial respiratory chain and subsequent compensatory elevation of SDH activity. Our findings indicate new steps in the molecular mechanism of nobiletin effects, suggesting new potential pharmacological actions of nutritional citrus flavonoids.

Spectral Characterization of Mitochondrial Cytochrome Redox States and Identification of Crossover Points within the Electron Transport Chain

As we begin to appreciate the complexity of respiratory control within the mitochondrion, we find it apparent that simultaneous monitoring of all respiratory complexes is required to effectively evaluate the system. Towards this goal, we developed a spectrophotometric technique utilizing a center-mounted sample in an integrating sphere to minimize the effects of scattering in turbid mitochondrial suspensions. With adequate signal to noise to collect spectra every 100 milliseconds spanning 400 to 700 nm, this system allows us to acquire absorbance data of all cytochromes in porcine heart mitochondria (1 nmol cyt a/mL) simultaneously with redundant measurements in both the alpha and soret regions. Spectra were analyzed using a least squares fitting routine with model optical spectra of electron-transfer components (flavin adenine dinucleotide and cyt bH,bL, c1, c, and a). The redox state of cyt a was found to correlate linearly with oxygen consumption rate. Surprisingly, a redox crossover event between cyt b (more oxidized) and cyt c (more reduced) was observed during the state 4 to 3 transition rather than the more commonly-observed crossover between cyt c and a. Furthermore, this crossover between cyt b and c was dependent on matrix Ca2+. In Ca2+-depleted mitochondria, the overall respiratory rate was lower and the crossover occurred between cyt c and a. Upon replenishment of matrix Ca2+, the crossover shifted back to cyt b and c, concomitant with a two- to three-fold enhancement of state 3 respiratory rate. These data demonstrate the feasibility of monitoring the entire redox chain in intact mitochondria and provide the first evidence that Ca2+, in addition to its known effects on matrix dehydrogenases and Complex V, may alter the relationship between redox state and electron flux within Complex III.

Feedback stabilization involving redox states of c-type cytochromes in living bacteria

The formal potential of c-type cytochromes expressed in the bacterial outer membrane was dependent on their redox state functioning in mediating extracellular electron transfer. Its negative (positive) shifting accompanying with the oxidization (reduction) of c-type cytochromes can serve to stabilize the redox states in a feedback manner.

Mitochondrial cytochrome redox states and respiration in acute pulmonary oxygen sensing

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism to optimise lung gas exchange. We aimed to decipher the proposed oxygen sensing mechanism of mitochondria in HPV. Cytochrome redox state was assessed by remission spectrophotometry in intact lungs and isolated pulmonary artery smooth muscle cells (PASMC). Mitochondrial respiration was quantified by high-resolution respirometry. Alterations were compared with HPV and hypoxia-induced functional and molecular readouts on the cellular level. Aortic and renal arterial smooth muscle cells (ASMC and RASMC, respectively) served as controls. The hypoxia-induced decrease of mitochondrial respiration paralleled HPV in isolated lungs. In PASMC, reduction of respiration and mitochondrial cytochrome c and aa3 (complex IV), but not of cytochrome b (complex III) matched an increase in matrix superoxide levels as well as mitochondrial membrane hyperpolarisation with subsequent cytosolic calcium increase. In contrast to PASMC, RASMC displayed a lower decrease in respiration and no rise in superoxide, membrane potential or intracellular calcium. Pharmacological inhibition of mitochondria revealed analogous kinetics of cytochrome redox state and strength of HPV. Our data suggest inhibition of complex IV as an essential step in mitochondrial oxygen sensing of HPV. Concomitantly, increased superoxide release from complex III and mitochondrial membrane hyperpolarisation may initiate the cytosolic calcium increase underlying HPV.

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides. Methods The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry. Results We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c. Conclusions Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione. General significance Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.

Mitochondria as decision‐makers in cell death

Mitochondria play an essential role in both cell health and death. Increasing experimental evidence suggests that mitochondria are involved in active control of cell death processes at several levels including (1) mitochondrial membrane permeabilization and release of proapoptotic proteins, (2) post-cytochrome c regulation of caspase activation, and (3) supply of energy for execution of death program. The purpose of this review is to discuss the main mechanisms by which alterations in mitochondrial outer membrane permit the translocation of proapoptotic proteins into cytosol, how mitochondria "make decisions" on the mode of cell death, and how they regulate caspase activation by changing the redox state of cytosolic cytochrome c. The interventions into these processes may constitute an important strategy for the pharmacological prevention of unwanted cell death in various pathologies or, conversely, for facilitation of anticancer therapy.

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O 2) conditions. The system measures the concentrations of O 2 and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O 2 concentration and electron turnover of the enzyme. At a high O 2 concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O 2. At low O 2 (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O 2 consumption. At both high and low O 2 concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.