How does cytochrome c oxidase inhibition cause cell death?

How does cytochrome c oxidase inhibition cause cell death?

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I realise the inhibition of cytochrome c oxidase prevents the release of H+ ions into the intermembrane space, and that the ion gradient is required for ATP synthase action. However, I'm not sure how this causes cell death.

Does this:

a) Prevent oxidation of Cytochrome C, preventing electron movement the cytochrome bc1 complex, which in turn prevents electron movement from earlier parts in the chain, shutting down the electron transport chain altogether? Thus, no ATP is produced and the cell dies due to lack of ATP.

b) Prevent oxidation of Cytochrome C, but not preventing oxidation in the other protein complexes*, merely reduce the number of H+ ions available for ATP synthase action, lowering the amount of ATP produced, and the cell dies due to low ATP levels.

c) other.

I realise in either case, the ATP count is merely lowered, as ATP is produced by other methods, so my statement 'lack of ATP' is not totally explicit.

*This being the case, how are the released electrons dealt with by the cell? Are there electron acceptors to remove this?

Inhibition is rarely binary - it's almost always subjected to stoichiometric effects. So the answer could be either option, depending on the amount of the inhibitor present in the system. If there is a huge amount of the inhibitor in the cell, it may more or less completely ablate ATP production by the ETC.

On the other hand, if there isn't enough of the inhibitor to shut down all cytochrome C oxidase, you'd only see a reduction in the amount of ATP produced by the ETC (which could still be sufficient to kill the cell).

It also depends on the type of inhibition taking place. Is it reversible or does it bind permanently? Does it degrade quickly? These aspects are also important to consider.

A peptide inhibitor of cytochrome c/inositol 1,4,5-trisphosphate receptor binding blocks intrinsic and extrinsic cell death pathways

Departments of * Neuroscience, † Pharmacology and Molecular Sciences, and ‡ Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Damian B. van Rossum

Departments of * Neuroscience, † Pharmacology and Molecular Sciences, and ‡ Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Randen L. Patterson

Departments of * Neuroscience, † Pharmacology and Molecular Sciences, and ‡ Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Solomon H. Snyder

Departments of * Neuroscience, † Pharmacology and Molecular Sciences, and ‡ Psychiatry and Behavioral Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205

Cytochrome c oxidase deficiency

There are currently 4 known forms of COX deficiency. The range and severity of signs and symptoms can vary widely from case to case.

In one form, referred to as the benign infantile mitochondrial myopathy type, symptoms may be limited to the skeletal muscles. Episodes of lactic acidosis may occur and can cause life-threatening complications if left untreated. However, with appropriate treatment, individuals with this form of the condition may spontaneously recover within the first few years of life. [1]

In the second form of the disorder, referred to as the infantile mitochondrial myopathy type, the skeletal muscles as well as several other tissues (such as the heart, kidney, liver, brain, and/or connective tissue) are affected. Symptoms associated with this form typically begin within the first few weeks of life and may include muscle weakness heart problems kidney dysfunction failure to thrive difficulties sucking, swallowing, and/or breathing and/or hypotonia . Affected infants may also have episodes of lactic acidosis. [1]

The third form of COX deficiency is thought to be a systemic form of the condition and is referred to as Leigh's disease. This form is characterized by progressive degeneration of the brain as well as dysfunction of several other organs including the heart, kidneys, muscles, and/or liver. Symptoms of this form, which predominantly involve the central nervous system , may begin between three months and two years of age and may include loss of previously acquired motor skills and/or head control poor sucking ability loss of appetite vomiting irritability and possible seizures . Intellectual disability may also occur. [1]

In the fourth form of COX deficiency, the French-Canadian type, the brain (as in Leigh's disease) and liver are particularly affected in addition to the skeletal muscles and connective tissues. However, in this form, the kidneys and heart appear to have near-normal enzyme activity. Individuals with this form may have developmental delay hypotonia slight facial abnormalities Leigh's disease strabismus ataxia liver degeneration and/or episodes of lactic acidosis. [1]

Although some mildly affected individuals survive into adolescence or adulthood, this condition is often fatal in childhood. [2]

This table lists symptoms that people with this disease may have. For most diseases, symptoms will vary from person to person. People with the same disease may not have all the symptoms listed. This information comes from a database called the Human Phenotype Ontology (HPO) . The HPO collects information on symptoms that have been described in medical resources. The HPO is updated regularly. Use the HPO ID to access more in-depth information about a symptom.


Cytochrome c oxidase deficiency can have different inheritance patterns depending on the gene involved.

When this condition is caused by mutations in genes within nuclear DNA, it is inherited in an autosomal recessive pattern , which means both copies of the gene in each cell have mutations. The parents of an individual with an autosomal recessive condition each carry one copy of the mutated gene, but they typically do not show signs and symptoms of the condition.

When this condition is caused by mutations in genes within mtDNA, it is inherited in a mitochondrial pattern , which is also known as maternal inheritance. This pattern of inheritance applies to genes contained in mtDNA. Because egg cells, but not sperm cells, contribute mitochondria to the developing embryo, children can only inherit disorders resulting from mtDNA mutations from their mother. These disorders can appear in every generation of a family and can affect both males and females, but fathers do not pass traits associated with changes in mtDNA to their children.


Wild-Type S. cerevisiae Stationary Cells Undergo a PCD Process Induced by Acetic Acid

The study was focused on the role of mitochondria in the previously reported acetic acid-induced PCD process in S. cerevisiae W303-1A (Ludovico et al., 2001a). Stationary cells rather than exponential cells were used for that purpose. Cells from stationary growth phase are more advantageous because they possess fully active mitochondria and display a higher mitochondrial mass. When exposed to different acetic acid concentrations at pH 3.0, and in the presence of glucose, S. cerevisiae stationary cells also committed to a PCD process. This conclusion is based on the detection of cell death accompanied by DNA strand breaks evaluated by the TUNEL assay (our unpublished data), an evident apoptotic marker, and by the fact that cycloheximide inhibited cell death (Figure1). Consistent with the recognized higher resistance of stationary cells to different stress agents, the effect was only observed for higher acetic acid concentrations. Actually, although acetic acid concentrations >120 mM are necrotic for exponential cells, they induce a PCD process in stationary cells. In fact, 140 mM acetic acid concentration induces, after 200 min, ∼50% of loss of cell viability evaluated by cfu and ∼30% of TUNEL-positive cells. This concentration (140 mM) was selected to perform mitochondrial function analysis. Incubation with acetic acid concentrations >200 mM resulted in no detectable TUNEL staining (our unpublished data).

Fig. 1. Programmed cell death of S. cerevisiae W303-1A stationary cells induced by acetic acid is partially inhibited by cycloheximide and is independent of oxidative phosphorylation. Relative survival (percentage of cfu on YEPD agar plates 100% corresponds to the number of cfu at time 0) of cells incubated for 200 min with 140 mM acetic acid in the absence (▪) or presence of cycloheximide (□) or oligomycin (▴).

To evaluate whether the acetic acid-induced PCD in S. cerevisiae is affected by the inhibition of oxidative phosphorylation, the treatment with acetic acid was carried out in the presence of oligomycin. Figure 1 shows that cell death was not affected by the drug.

Cytochrome c Is Translocated from Mitochondria to Cytosol during Acetic Acid-induced PCD Process

To check whether acetic acid-induced PCD process was accompanied by a release of CytC from mitochondria to cytosol, the levels of CytC in mitochondria and in the PMS (containing soluble cytosolic proteins) from S. cerevisiae W303-1A cells undergoing a acid-induced PCD were detected by Western blot analysis. The amount of CytC present in mitochondria of cells treated with 140 mM acetic acid was decreased by two- to threefold compared with the mitochondria from untreated cells (Figure 2). This portion of “lost” CytC in mitochondria from acetic acid treated cells was detected in its PMS, whereas no CytC was detected in the PMS of untreated control cells (Figure 2). Levels of other mitochondrial proteins, such as COX subunits II and V were not detected in PMS (our unpublished data), although COX II was found to be reduced in mitochondria (described below). Decrease of the CytC amount in mitochondria was confirmed by cytochrome spectra analysis of mitochondria from treated and untreated cells. As shown in Figure3A, there was some decrease in the amount of cytochromes c+c1 extracted from the mitochondrial membranes.

Fig. 2. In S. cerevisiae W303-1A cytochromec is released from mitochondria to the cytosol during PCD induced by acetic acid. CytC was detected by immunodetection in mitochondria (5, 10, and 20 μg of protein) and postmitochondrial supernatants obtained from S. cerevisiae stationary untreated cells (A) or cells treated with 140 mM acetic acid (B).

Fig. 3. Release of CytC parallels a reduction of cytochrome c oxidase in acid-treated cells. (A) Cytochrome spectra. Cytochrome spectra of S. cerevisiaemitochondria isolated from untreated cells (full line) or from cells treated with 140 mM acetic acid (dashed line). The α absorption bands corresponding to cytochromes a+a3 have maxima at 603 nm. The corresponding maximum for cytochromeb is 560 nm and for cytochromec+c1, 550 nm. (B) Steady-state levels of COX II subunit are decreased in mitochondria from acid-treated cells. Immunodetection of COX II and V and ATPase 6 subunits in mitochondrial membranes of S. cerevisiae untreated cells (A) or cells treated with 140 mM acetic acid (B).

The levels of CytC were also evaluated in mitochondria and in the PMS from S. cerevisiae ATP10 mutant cells, which do not undergo a PCD process induced by acetic acid as shown hereafter. CytC was not detected in PMS and it was found to be at a normal level in mitochondria, compared with untreated cells (our unpublished data). These results indicate that CytC is specifically translocated from mitochondria to the cytosol during the acid-induced PCD.

ROS Are Produced in Mitochondria during Acetic Acid-induced PCD Process

S. cerevisiae W303-1A stationary cells stained with MitoTracker Red CM-H2Xros and treated with 140 mM acetic acid were analyzed by flow cytometry. An increase in the red fluorescence indicative of cells with an increased mitochondrial ROS production could only be detected after 100 min of treatment (our unpublished data). The results obtained after 200 min of treatment showed a heterogeneous population and a second subpopulation (∼45%) with a higher mean fluorescence intensity (Figure4). The epifluorescence analysis showed that this subpopulation display a bright red fluorescence localized in mitochondria (our unpublished data). Moreover, killed cells displayed a highest red fluorescence corresponding to an unspecific cell staining (our unpublished data).

Fig. 4. ROS are produced in mitochondria ofS. cerevisiae W303-1A cells treated with acetic acid. Overlay of red fluorescence histograms obtained for cells stained with MitoTracker Red CM-H2XRos. Untreated cells (thin gray line), or treated cells with 140 mM acetic acid (thick black line).

Acetic Acid-induced PCD Is Accompanied by Mitochondrial Alterations

The respiratory capacity of mitochondria isolated from control and acetic acid-treated cells was assayed polarographically by measuring oxygen uptake with NADH as substrate. The effect on respiration of exogenously added CytC was evaluated in a set of experiments. Results presented in Table 1 show that mitochondria isolated from cells treated with 140 mM acetic acid have a dramatically reduced oxygen consumption, with a decrease of nearly 75% in NADH oxidase activity. Although an increase of ∼2.8-fold of the oxygen consumption was observed after addition of CytC, the inability to fully restore the respiration rate with this addition suggested that an enzymatic portion of the respiratory chain could be intrinsically affected.

Table 1. Respiratory activities of mitochondria from control and acetic acid treated cells Mitochondria were assayed polarographically for NADH oxidase. The specific activities reported (expressed as nanomoles of O2 per minute per milligram of protein) were corrected for KCN-insensitive respiration. Cytochrome oxidase activity (expressed as micromoles of cytochrome coxidized per minute per milligram of mitochondrial protein) was assayed spectrophotometrically in mitochondria permeabilized with potassium deoxycholate by measuring oxidation of ferrocytochrome c at 550 nm. NADH cytochrome c reductase (expressed as micromoles of cytochrome c reduced per minute per milligram of mitochondrial protein) was also measured spectrophotometrically as described under MATERIALS AND METHODS. The rates measured in at least two independent assays did not differ by >10%. The values reported are the average of the two assays.

To establish the biochemical basis for the decrease in the respiratory activity of mitochondria, the NADH-CytC reductase and COX activities of isolated mitochondria from untreated and acid-treated cells, were measured. As shown in Table 1, the treatment with 140 mM acetic acid resulted in a decrease of 50% of COX activity in mitochondrial membranes, whereas NADH-CytC reductase activity was essentially identical to that obtained with mitochondria from untreated cells. These results confirm that COX complex was affected, whereas complexbc1 was unaffected by the treatment. Cytochromes spectra were recorded in isolated mitochondria (Figure 3A) to clarify the observed respiratory chain alterations (Table 1). In agreement with the observed lower COX activity, a decrease in the amount of cytochromes a+a3 in mitochondrial membranes of cells treated with acetic acid was observed, whereas the levels of cytochrome b were not affected (Figure3A).

When the COX subunits II and V were analyzed by immunodetection of mitochondrial proteins obtained from cells treated with 140 mM acetic acid, the amount of COX II protein but not COX V was found to be lower than in the control (Figure 3B). Additionally, the amount of cytochromeb from bc1 complex and of subunit 6 of ATPase remained identical to control (Figure 3B).

Some studies on mammalian apoptosis reported an increase in ΔΨm after a lethal stimulus, with ΔΨm decreasing later in the death process (Vander Heiden et al., 1997, 1999). Consistently with such observations, in our study, acetic acid treatment induced a transient slight hyperpolarization followed by a depolarization (our unpublished data). However, although with a lower ΔΨm, cells maintained the specific mitochondria staining indicating that mitochondria membrane integrity is still preserved (our unpublished data).

Mitochondrial Respiration Is Essential for S. cerevisiae to Undergo a PCD Process Induced by Acetic Acid

The requirement of mitochondria function in the PCD process induced by acetic acid was analyzed by the study of three S. cerevisiae W303-1A mutant strains, namely, the ρ 0 , lacking mitochondrial DNA the nullATP10 mutant, deleted in an assembly factor of mitochondrial ATPase and the null CYC3 mutant, deleted in the gene encoding a heme lyase, essential for the covalent binding of the heme group to isoform 1 and 2 of apocytochrome c (Pearce and Sherman, 1995). It was observed that these mutant strains were more resistant to death induced by acetic acid, comparatively to the wild-type strain. In addition, cycloheximide had no effect on survival (Figure 5) and no TUNEL-positive cells were found, for any of the acetic acid concentrations tested, even for concentrations inducing a percentage of dead cells identical to that obtained for the wild type (our unpublished data).

Fig. 5. Respiratory-deficient mutant cells of S. cerevisiae W303-1A are more resistant to acetic acid and acid-induced death is not inhibited by cycloheximide. Relative survival (percentage of cfu on YEPD agar plates 100% corresponds to the number of cfu at time 0) of wild-type, ρ 0 (lacking mitochondrial DNA), ΔATP10 (depleted of ATPase), and ΔCYC3 (depleted of CytC) cells of S. cerevisiae incubated for 200 min with acetic acid in the absence (black bars) or presence (white bars) of cycloheximide.

Materials and Methods


Sodium ascorbate, L-arginine, sodium dithionite, S-ethylisothiourea (S-EITU), myxothiazol, sodium nitrite, soybean protease inhibitor, tetracycline and N,N,N′,N′ tetramethyl-p-phenylenediamine (TMPD) were purchased from Aldrich-Sigma. Cell culture media, hygromycin and trypsin-EDTA were from Invitrogen. Blasticidin was from Calbiochem.

Plasmid preparation and transfection

The tetracycline-inducible cell line Tet-iNOS 293 that stably expresses NOS from human chondrocytes was obtained as described previously (Mateo et al., 2003). Briefly, the cDNA encoding the complete coding region of the NOS gene (GenBank accession no. X73029) was cloned into the inducible expression vector pcDNA5/FRT/TO (Invitrogen) using PCR primers designed to contain restriction sites for HindIII and XhoI at the 5′ and 3′ ends, respectively, giving as a result the pcDNA5/FRT/TO-iNOS DNA construct. For transfection, 2×10 6 Flp-In™ T-REx™-293 cells (Invitrogen), which stably express the tetracycline repressor, were co-transfected with 0.3 μg of pcDNA5/FRT/TO-iNOS and 3 μg of Flp recombinase expression plasmid (pOG44) using 7.5 μl of lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Forty-eight hours after transfection, the cells were selected in growth medium supplemented with 200 μg ml -1 hygromycin B and 15 μg ml -1 blasticidin.

Cell culture and induction of human NOS in Tet-iNOS293 cells

Tet-iNOS 293 cells were cultured in T-175 flasks with Phenol-Red-free Dulbecco's modified Eagle's medium (DMEM) containing 25 mM D-glucose, 4 mM glutamine, 15 μg ml -1 blasticidin, 200 μg ml -1 hygromycin B and 10% (v/v) of heat-inactivated foetal bovine serum (HI-FBS), as previously described (Mateo et al., 2003). Maximal expression of human NOS in Tet-iNOS293 cells was achieved by 15 hours incubation with induction medium (DMEM containing 25 mM D-glucose, 4 mM glutamine, 10% HI-FBS, 1.5 μg ml -1 tetracycline and 500 μM S-EITU the latter is added to avoid generation of NO from L-arginine in the medium that is necessary for NOS dimerisation and consequent enzyme activity). Under these conditions we achieved a sensitive and reproducible NO generation from NOS in response to small concentrations of exogenous L-arginine. A basal production of NO was evident in these cells (see Results) even in the absence of exogenous L-arginine, most probably due to the conversion of endogenously-generated L-arginine by NOS once the inhibitor (S-EITU) was removed.

Cells were harvested by trypsinisation, then centrifuged at 115 g for 10 minutes and re-suspended at a concentration of 5×10 6 cells ml -1 in Hanks-VLS solution (20 mM HEPES, 5.5 mM D-glucose, 5.37 mM KCl, 1.26 mM CaCl2, 0.5 mM MgCl2, 0.4 mM MgSO4, 137 mM NaCl, 4.2 mM NaHCO3, 0.34 mM Na2HPO4 and 1% dialysed HI-FBS). Cell viability was always over 95%, as measured by the Trypan Blue exclusion method. Cell suspensions were placed in a water bath at 37°C with constant agitation (80 rpm) to ensure that they remained well-oxygenated and at a constant temperature throughout the experiments. After 1 hour incubation to remove the NOS inhibitor, cells were centrifuged again and re-suspended at a cell concentration of approximately 2×10 7 cells ml -1 in Hanks-VLS solution. Cells were then incubated for another hour at 37°C in the water bath before placing them in the VLS chamber in aliquots of 1 ml final volume. At the end of each experiment aliquots of 50 μl were diluted in triplicate, in 10 ml of isotonic buffer, and cell-counting was immediately carried out using a Coulter Counter (Z Series, Beckman Coulter FL). L-arginine was always added when the concentration of O2 in the chamber reached (approximately) 70 μM. This [O2] was selected because both parameters of our study, respiration and the redox state of cytochromes aa3 and cc1, remained independent of [O2] above ∼50 μM (in the absence of exogenous L-arginine, significant changes from the baselines occurred in cytochromes aa3, and respiration at 34.4±1.1 and 9.8±1.1 μM O2, respectively).

Simultaneous measurement of cytochrome redox states, respiration and NO release from exogenous L-arginine

The visible-light spectroscopy (VLS) system is essentially the same as described before (Hollis et al., 2003) with some improvements in the optical system and the detection of NO. Briefly, the diffraction grating has been replaced with one blazed at 400 nm (Horiba-Jobin Yvon, Stanmore, UK), for greater sensitivity of light detection in the visible region, and the optical fibres with those of a larger core diameter and numerical aperture (Thorlabs, Ely, UK), for enhanced delivery and collection of light. These improvements to the system allow the sampling rate to be increased from 50 to 100 Hz, although averaging is maintained such that a data point or spectrum is recorded every 500 mseconds. The NO electrode has been replaced by a nanosensor (amiNO-700 Innovative Instruments, FL), calibrated using standard concentrations of NaNO2 in the reaction with 10 mM KI and 100 mM H2SO4 at 37°C. The sensitivity of the electrode to the generation of NO was on average over 250 pA nM -1 . Calibration of the Clarke-type O2 electrode (Rank Bros., Cambridge, UK) and corrections for its time response and background O2 consumption were carried out as previously described (Hollis et al., 2003). The rate of mitochondrial respiration (VO2) was determined as the time derivative of the [O2].

Measured changes in optical attenuation are converted into changes in the redox states of the mitochondrial cytochromes by two multi-wavelength linear least-squares fits of their specific absorption coefficients (Hollis et al., 2003). To account for non-absorption changes in the attenuation spectra, a first-order background has been included in the least-squares fitting algorithm, described by (x·λ)+y, where λ is the wavelength and x and y are the free (variable) coefficients of the first-order background. Simulations have been carried out to demonstrate the ability of the fitting to recover redox-dependent changes in cytochrome concentrations within the range of the redox changes observed here (data not shown).

The measurement of pathlength, required for the absolute determination of redox-dependent changes in cytochrome concentrations, was carried out as previously described (Hollis et al., 2003). Within the range of cell concentrations used here, a significant (P<0.001) negative correlation between cell concentration and pathlength was observed. Thus, the pathlength (β) was calculated from the cell concentration using the equation β=p· [cell]+q, where [cell] is the cell concentration and the coefficients p and q were -0.23±0.01 cm 10 -7 cells ml -1 and 2.56±0.03 cm, respectively (n=16).

Quantification of changes in respiration rate and reduction of cytochromes aa3 and cc1

Changes in the redox states of cytochromes aa3 and cc1 are expressed as percentage changes varying between 0% at the [O2]-independent baseline (prior to addition of L-arginine at ∼70 μM O2) and 100% when fully reduced at anoxia. It should be noted, however, that the redox states of cytochromes aa3 and cc1 at the [O2]-independent baseline are not 0%, i.e. fully oxidised. This assignation was designed to provide a better comparison with VO2, for which the maximal value during the steady-state was normalised to 100%. Using the method described below to determine total cytochrome concentrations, the percentage of cytochromes aa3 and cc1 in their reduced forms at the [O2]-independent baseline was estimated to be 11.8±1.4 and 11.4±1.6%, respectively (n=6). For the case of cytochromes aa3 this predominantly (80-90%) reflects the redox state of cytochrome a and is not used to draw conclusions about the redox species populating the catalytic centre (a3·CuB). The measurement of cytochromes cc1 comprise a combined signal from cytochrome c1 of complex bc1 and cytochrome c in the intermembrane space, although the ratio of c:c1 in Tet-iNOS293 cells is undetermined.

Estimation of total concentration of CcO and turnover number (TN) of the enzyme in vivo

The total CcO concentration ([CcO]total) was estimated in respiring cells by measuring from the baseline the maximal reduction of cytochrome aa3 at anoxia (Δ[aa3]red) and, in an independent experiment, the maximal oxidation obtained after the addition of 1 μM of myxothiazol (Δ[aa3]oxi), i.e. [CcO]total= (Δ[aa3]red+Δ[aa3]oxi)/β, where β is the pathlength determined from cell number as described above. Electron turnover (eTN) of the enzyme (in electrons per second) was then estimated from the total concentration of the enzyme and the maximal VO2 during the [O2]-independent phase (VO2max) using the equation eTN=VO2max·4/[CcO]total, the factor 4 accounting for the number of electrons in one turnover cycle of CcO, i.e. the consumption of one molecule of O2.

Statistical analysis

The mean ± standard deviation was determined for the quantitative analysis of the results. The (two-tailed) Z-test was used to determine statistically significant changes in VO2 and cytochromes aa3 and cc1 from their [O2]-independent baseline values, and the (one-tailed) t-test was used to determine statistically significant correlations between dependent and independent variables (pathlength vs cell concentration and eTN vs decrease in respiration).

Physical Properties

Molecular mass: 3
12,384 Da (equine)
12,327 Da (bovine)
12,384 Da (pigeon)
12,588 Da (Saccharomyces cerevisiae)
12,233 Da (human)

Isoelectric point (pI): 4 range of 10.0 – 10.5 (equine)
Spectral properties: 5 (equine)
λmax = 550 nm (reduced form)
E mM = 29.5 (reduced form, 0.1 M phosphate buffer, pH 6.8)
E mM = 8.4 (oxidized form, 0.1 M phosphate buffer, pH 6.8)

Recommended storage time for aqueous solutions
Storage at –20 °C (freezer): 6 months
Storage at 2-8 °C (refrigerator): 2 weeks
Storage at 20-25 °C (ambient temperature): 3 days

Mitochondrial Uncoupling and CO

Mitochondrial oxidative metabolism is accompanied by ROS generation due to the incomplete reduction of oxygen into anion superoxide. Under proper control, ROS generation functions as ubiquitous signaling factors. However, under pathological conditions, reversion of electron flow might result in persistent and damaging generation of ROS, thus mild mitochondrial uncoupling is an inherent cellular mechanism to limit oxidative stress. Uncoupling consists of energy dissipation by the leakage of proton through the inner membrane, causing a compensatory increase on oxygen consumption, which is not coupled with ATP production. Iacono and colleagues have demonstrated that CORM-3 protects mitochondria against oxidative stress by inducing mild uncoupling state (Iacono et al., 2011). Low micromolar concentrations of CORM-3 increase oxygen consumption under state 2 of respiration (in an ADP independent manner), indicating an uncoupling effect between oxygen reduction and ATP production. Moreover, an inhibitor of succinate dehydrogenase (complex II), malonate significantly reverses the CORM-3-induced uncoupling effect. Likewise, inhibitors of uncoupling protein (UCP) and ATP/ADP translocator (ANT), which are proteins involved in mitochondrial uncoupling process, also prevented mitochondrial uncoupling due to low concentrations of CORM-3. Whenever respiration is initiated at complex II by using succinate as substrate, there is a reversion of electron transfer to complex I with higher levels of ROS production. Under these conditions, CORM-3 prevented excessive ROS generation, limiting oxidative stress (Iacono et al., 2011). In contrast, when electron transfer is physiologically initiated by addition of pyruvate/malate, CORM-3 promotes ROS generation at complex III due to complex IV inhibition, under a concentration-dependent manner. Since Iacono and colleagues generated these data using an in vitro approach (isolated mitochondria), one can speculate whether this CO-promoted mild uncoupling effect is physiologically relevant under pathological conditions.

In a recent and more detailed approach, same authors have found that CO targets phosphate carrier. In fact, by increasing phosphate carrier activity, CO promotes the transport of protons and phosphate inside mitochondria, inducing a mild uncoupling effect (Long et al., 2014).

Inhibition of Oxidative Phosphorylation Induces a Rapid Death of GA-Pretreated Aleurone Cells, But Not of ABA-Pretreated Aleurone Cells

Reactive oxygen species (ROS) mediate programmed cell death in aleurone cells, which is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Plant mitochondria contain two distinct respiratory pathways: respiration through cytochrome c oxidase increases ROS production, whereas respiration through the alternative oxidase pathway lowers it. While studying the effects of GA and ABA on partitioning of respiration between those two pathways during the germinating process, we discovered that oxidative phosphorylation inhibitors like sodium azide and 2, 4-dinitrophenol induce rapid death of GA-pretreated aleurone cells but not of ABA-pretreated cells. Functional aerobic respiration was required for GA signaling, and 6 to 12 hours of GA signaling altered the cellular state of aleurone cells to be extremely susceptible to inhibition of oxidative phosphorylation. Anaerobic conditions were also able to mimic the effects of respiratory inhibitors in specifically inducing cell death in GA-treated cells, but cell death was provoked much more slowly. Cotreatment with various antioxidants did not prevent this process at all, suggesting that no ROS are responsible for this respiratory inhibitor-induced cell death. Our observation implicates that GA may partition all the electrons produced during mitochondrial respiration only to the cytochrome oxidase pathway, which would at least partly contribute to cellular accumulation of ROS.

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Cytochrome c oxidase-modulatory near-infrared light penetration into the human brain: Implications for the noninvasive treatment of ischemia/reperfusion injury

Maik Hüttemann, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University, Detroit, Michigan, USA

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Department of Health Care Sciences, Eugene Applebaum College of Pharmacy & Health Sciences, Wayne State University, Detroit, Michigan, USA

College of Medicine, Dankook University, Cheonan-si, Republic of Korea

Department of Emergency Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA

Department of Biochemistry, Microbiology and Immunology, Wayne State University, Detroit, Michigan, USA

Maik Hüttemann, Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI 48201.

Funding information: Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program, Grant/Award Number: W81XWH-16-1-0175 U.S. National Institutes of Health, Grant/Award Number: R42NS105238


Near-infrared light (IRL) has been evaluated as a therapeutic for a variety of pathological conditions, including ischemia/reperfusion injury of the brain, which can be caused by an ischemic stroke or cardiac arrest. Strategies have focused on modulating the activity of mitochondrial electron transport chain (ETC) enzyme cytochrome c oxidase (COX), which has copper centers that broadly absorb IRL between 700 and 1,000 nm. We have recently identified specific COX-inhibitory IRL wavelengths that are profoundly neuroprotective in rodent models of brain ischemia/reperfusion through the following mechanism: COX inhibition by IRL limits mitochondrial membrane potential hyperpolarization during reperfusion, which otherwise causes reactive oxygen species (ROS) production and cell death. Prior to clinical application of IRL on humans, IRL penetration must be tested, which may be wavelength dependent. In the present study, four fresh (unfixed) cadavers and isolated cadaver tissues were used to examine the transmission of infrared light through human biological tissues. We conclude that the transmission of 750 and 940 nm IRL through 4 cm of cadaver head supports the viability of IRL to treat human brain ischemia/reperfusion injury and is similar for skin with different skin pigmentation. We discuss experimental difficulties of working with fresh cadavers and strategies to overcome them as a guide for future studies.

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