Oxidation Of Cytochrome Research Articles

Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects

The effect of NO between cytochromes b and c of the mitochondrial respiratory chain were studied using submitochondrial particles (SMP) from bovine heart and GSNO and SPER-NO as NO sources. Succinate-cytochrome c reductase (complex II-III) activity (222±4nmol/min. mg protein) was inhibited by 51% in the presence of 500μM GSNO and by 48% in the presence of 30μM SPER-NO, in both cases at ~1.25μM NO. Neither GSNO nor SPER-NO were able to inhibit succinate-Q reductase activity (complex II; 220±9nmol/min. mg protein), showing that NO affects complex III. Complex II-III activity was decreased (36%) when SMP were incubated with l-arginine and mtNOS cofactors, indicating that this effect is also produced by endogenous NO. GSNO (500μM) reduced cytochrome b562 by 71%, in an [O2] independent manner. Hyperbolic increases in O2•- (up to 1.3±0.1nmol/min. mg protein) and H2O2 (up to 0.64±0.05nmol/min. mg protein) productions were observed with a maximal effect at 500μM GSNO. The O2•-/H2O2 ratio was 1.98 in accordance with the stoichiometry of the O2•- disproportionation. Moreover, H2O2 production was increased by 72–74% when heart coupled mitochondria were exposed to 500μM GSNO or 30μM SPER-NO. SMP incubated in the presence of succinate showed an EPR signal (g=1.99) compatible with a stable semiquinone. This EPR signal was increased not only by antimycin but also by GSNO and SPER-NO. These signals were not modified under N2 atmosphere, indicating that they are not a consequence to the effect of NOx species on complex III area. These results show that NO interacts with ubiquinone-cytochrome b area producing antimycin-like effects. This behaviour comprises the inhibition of electron transfer, the interruption of the oxidation of cytochromes b, and the enhancement of [UQH•]ss which, in turn, leads to an increase in O2•- and H2O2 mitochondrial production rates.

Cytochrome redox states and respiratory control in mouse and beef heart mitochondria at steady-state levels of hypoxia.

Mitochondrial control of cellular redox states is a fundamental component of cell signaling in the coordination of core energy metabolism and homeostasis during normoxia and hypoxia. We investigated the relationship between cytochrome redox states and mitochondrial oxygen consumption at steady-state levels of hypoxia in mitochondria isolated from beef and mouse heart (BHImt, MHImt), comparing two species with different cardiac dynamics and local oxygen demands. A low-noise, rapid spectrophotometric system using visible light for the measurement of cytochrome redox states was combined with high-resolution respirometry. Monophasic hyperbolic relationships were observed between oxygen consumption, JO2, and oxygen partial pressure, Po2, within the range <1.1 kPa (8.3 mmHg; 13 μM). P50j (Po2 at 0.5·Jmax) was 0.015 ± 0.0004 and 0.021 ± 0.003 kPa (0.11 and 0.16 mmHg) for BHImt and MHImt, respectively. Maximum oxygen consumption, Jmax, was measured at saturating ADP levels (OXPHOS capacity) with Complex I-linked substrate supply. Redox states of cytochromes aa3 and c were biphasic hyperbolic functions of Po2. The relationship between cytochrome oxidation state and oxygen consumption revealed a separation of distinct phases from mild to severe and deep hypoxia. When cytochrome c oxidation increased from fully reduced to 45% oxidized at 0.1 Jmax, Po2 was as low as 0.002 kPa (0.02 μM), and trace amounts of oxygen are sufficient to partially oxidize the cytochromes. At higher Po2 under severe hypoxia, respiration increases steeply, whereas redox changes are small. Under mild hypoxia, the steep slope of oxidation of cytochrome c when flux remains more stable represents a cushioning mechanism that helps to maintain respiration high at the onset of hypoxia.

Possible pathogenic mechanism of propofol infusion syndrome involves coenzyme q.

Propofol is a short-acting intravenous anesthetic agent. In rare conditions, a life-threatening complication known as propofol infusion syndrome can occur. The pathophysiologic mechanism is still unknown. Some studies suggested that propofol acts as uncoupling agent, others suggested that it inhibits complex I or complex IV, or causes increased oxidation of cytochrome c and cytochrome aa3, or inhibits mitochondrial fatty acid metabolism. Although the exact site of interaction is not known, most hypotheses point to the direction of the mitochondria. Eight rats were ventilated and sedated with propofol up to 20 h. Sequential biopsy specimens were taken from liver and skeletal muscle and used for determination of respiratory chain activities and propofol concentration. Activities were also measured in skeletal muscle from a patient who died of propofol infusion syndrome. In rats, authors detected a decrease in complex II+III activity starting at low tissue concentration of propofol (20 to 25 µM), further declining at higher concentrations. Before starting anesthesia, the complex II+III/citrate synthase activity ratio in liver was 0.46 (0.25) and in skeletal muscle 0.23 (0.05) (mean [SD]). After 20 h of anesthesia, the ratios declined to 0.17 (0.03) and 0.12 (0.02), respectively. When measured individually, the activities of complexes II and III remained normal. Skeletal muscle from one patient taken in the acute phase of propofol infusion syndrome also shows a selective decrease in complex II+III activity (z-score: -2.96). Propofol impedes the electron flow through the respiratory chain and coenzyme Q is the main site of interaction with propofol.

Specific methionine oxidation of cytochrome c in complexes with zwitterionic lipids by hydrogen peroxide: potential implications for apoptosis.

Cytochrome c (Cyt-c) has been previously shown to participate in cardiolipin (CL) oxidation and, therefore, in mitochondrial membrane permeabilization during the early events of apoptosis. The gain in this function has been ascribed to specific CL/Cyt-c interactions. Here we report that the cationic protein Cyt-c is also able to interact electrostatically with the main lipid components of the mitochondrial membranes, the zwitterionic lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE), through the mediation of phosphate anions that bind specifically to amino groups in the surfaces of protein and model membranes. In these complexes, Cyt-c reacts efficiently with H2O2 at submillimolar levels, which oxidizes the sulfur atom of the axial ligand Met80. The modified protein is stable and presents significantly enhanced peroxidatic activity. Based on these results, we postulate that the rise of H2O2 concentrations to the submillimolar levels registered during initiation of the apoptotic program may represent one signaling event that triggers the gain in peroxidatic function of the Cyt-c molecules bound to the abundant PE and PC membrane components. As the activated protein is a chemically stable species, it can potentially bind and oxidize important targets, such as CL.

Mitochondria dysfunction in lung cancer-induced muscle wasting in C2C12 myotubes.

Aims: Cancer cachexia is a syndrome which results in severe loss of muscle mass and marked fatigue. Conditioned media from cachexia-inducing cancer cells triggers metabolic dysfunction in skeletal muscle, including decreased mitochondrial respiration, which may contribute to fatigue. We hypothesized that Lewis lung carcinoma conditioned medium (LCM) would impair the mitochondrial electron transport chain (ETC) and increase production of reactive oxygen species, ultimately leading to decreased mitochondrial respiration. We incubated C2C12 myotubes with LCM for 30 min, 2, 4, 24 or 48 h. We measured protein content by western blot; oxidant production by 2′,7′-dichlorofluorescin diacetate (DCF), 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate (DAF), and MitoSox; cytochrome c oxidase activity by oxidation of cytochrome c substrate; and oxygen consumption rate (OCR) of intact myotubes by Seahorse XF Analyzer. Results: LCM treatment for 2 or 24 h decreased basal OCR and ATP-related OCR, but did not alter the content of mitochondrial complexes I, III, IV and V. LCM treatment caused a transient rise in reactive oxygen species (ROS). In particular, mitochondrial superoxide (MitoSOX) was elevated at 2 h. 4-Hydroxynonenal, a marker of oxidative stress, was elevated in both cytosolic and mitochondrial fractions of cell lysates after LCM treatment. Conclusion: These data show that lung cancer-conditioned media alters electron flow in the ETC and increases mitochondrial ROS production, both of which may ultimately impair aerobic metabolism and decrease muscle endurance.

External mitochondrial NADH-dependent reductase of redox cyclers: VDAC1 or Cyb5R3?

It was reported that VDAC1 possesses an NADH oxidoreductase activity and plays an important role in the activation of xenobiotics in the outer mitochondrial membrane. In the present work, we evaluated the participation of VDAC1 and Cyb5R3 in the NADH-dependent activation of various redox cyclers in mitochondria. We show that external NADH oxidoreductase caused the redox cycling of menadione ≫ lucigenin>nitrofurantoin. Paraquat was predominantly activated by internal mitochondria oxidoreductases. An increase in the ionic strength stimulated and suppressed the redox cycling of negatively and positively charged acceptors, as was expected for the Cyb5R3-mediated reduction. Antibodies against Cyb5R3 but not VDAC substantially inhibited the NADH-related oxidoreductase activities. The specific VDAC blockers G3139 and erastin, separately or in combination, in concentrations sufficient for the inhibition of substrate transport, exhibited minimal effects on the redox cycler-dependent NADH oxidation, ROS generation, and reduction of exogenous cytochrome c. In contrast, Cyb5R3 inhibitors (6-propyl-2-thiouracil, p-chloromercuriobenzoate, quercetin, mersalyl, and ebselen) showed similar patterns of inhibition of ROS generation and cytochrome c reduction. The analysis of the spectra of the endogenous cytochromes b5 and c in the presence of nitrofurantoin and the inhibitors of VDAC and Cyb5R3 demonstrated that the redox cycler can transfer electrons from Cyb5R3 to endogenous cytochrome c. This caused the oxidation of outer membrane-bound cytochrome b5, which is in redox balance with Cyb5R3. The data obtained argue against VDAC1 and in favor of Cyb5R3 involvement in the activation of redox cyclers in the outer mitochondrial membrane.

Real-Time Measurements of the Redox States of c-Type Cytochromes in Electroactive Biofilms: A Confocal Resonance Raman Microscopy Study

Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208±49 µA cm−2) and mature (57 days, j = 267±73 µA cm−2) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190±37 µA cm−2) with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.

Synthesis, characterization, biological activity of binuclear Co(II), Cu(II) and mononuclear Ni(II) complexes of bulky multi-dentate thiosemicarbazide

The chelation behavior of 9,10-dihydro-9,10-ethanoanthracene-11,12-dicarbonyl) bis (N-ethylhydrazine-1-carbothioamide) (H6ETS)(1) towards Co2+, Ni2+and Cu2+ have been studied. The spectral data revealed that the ligand acts as a bi- and/or mono-negative multi-dentate. The isolated Ni(II) and Cu(II) complexes are square-planar while the Co(II) is tetrahedral. EPR spectrum of Cu(II) complex confirmed simulated an axial spin-Hamiltonian exhibiting a four-line pattern with nitrogen super-hyperfine couplings originating from imine hydrazinic nitrogen atoms and possess a significant amount of tetrahedral distortion leading to a pseudo-square-planar geometry with unpaired electron has dx2-y2 ground state. Also, the thermal behavior and kinetic parameters were determined. Furthermore, the title compounds were investigated for their antibacterial activity using inhibition zone diameter and for DNA degradation, superoxide-scavenging activity as well as hydroxyl radicals that generated by the oxidation of cytochrome c in L-ascorbic acid/CuSO4–cytochrome c system.

A caged, destabilized, free radical intermediate in the q-cycle.

The Rieske/cytochrome b complexes, also known as cytochrome bc complexes, catalyze a unique oxidant-induced reduction reaction at their quinol oxidase (Qo ) sites, in which substrate hydroquinone reduces two distinct electron transfer chains, one through a series of high-potential electron carriers, the second through low-potential cytochrome b. This reaction is a critical step in energy storage by the Q-cycle. The semiquinone intermediate in this reaction can reduce O2 to produce deleterious superoxide. It is yet unknown how the enzyme controls this reaction, though numerous models have been proposed. In previous work, we trapped a Q-cycle semiquinone anion intermediate, termed SQo , in bacterial cytochrome bc1 by rapid freeze-quenching. In this work, we apply pulsed-EPR techniques to determine the location and properties of SQo in the mitochondrial complex. In contrast to semiquinone intermediates in other enzymes, SQo is not thermodynamically stabilized, and can even be destabilized with respect to solution. It is trapped in Qo at a site that is distinct from previously described inhibitor-binding sites, yet sufficiently close to cytochrome bL to allow rapid electron transfer. The binding site and EPR analyses show that SQo is not stabilized by hydrogen bonds to proteins. The formation of SQo involves "stripping" of both substrate -OH protons during the initial oxidation step, as well as conformational changes of the semiquinone and Qo proteins. The resulting charged radical is kinetically trapped, rather than thermodynamically stabilized (as in most enzymatic semiquinone species), conserving redox energy to drive electron transfer to cytochrome bL while minimizing certain Q-cycle bypass reactions, including oxidation of prereduced cytochrome b and reduction of O2 .

P53 and aging: role of p66Shc

p53 and aging: role of p66Shc

Prognostic relevance of cytochrome C oxidase in primary glioblastoma multiforme.

Patients with primary glioblastoma multiforme (GBM) have one of the lowest overall survival rates among cancer patients, and reliable biomarkers are necessary to predict patient outcome. Cytochrome c oxidase (CcO) promotes the switch from glycolytic to OXPHOS metabolism, and increased CcO activity in tumors has been associated with tumor progression after chemotherapy failure. Thus, we investigated the relationship between tumor CcO activity and the survival of patients diagnosed with primary GBM. A total of 84 patients with grade IV glioma were evaluated in this retrospective cohort study. Cumulative survival was calculated by the Kaplan-Meier method and analyzed by the log-rank test, and univariate and multivariate analyses were performed with the Cox regression model. Mitochondrial CcO activity was determined by spectrophotometrically measuring the oxidation of cytochrome c. High CcO activity was detected in a subset of glioma tumors (∼30%), and was an independent prognostic factor for shorter progression-free survival and overall survival [P = 0.0087 by the log-rank test, hazard ratio = 3.57 for progression-free survival; P<0.001 by the log-rank test, hazard ratio = 10.75 for overall survival]. The median survival time for patients with low tumor CcO activity was 14.3 months, compared with 6.3 months for patients with high tumor CcO activity. High CcO activity occurs in a significant subset of high-grade glioma patients and is an independent predictor of poor outcome. Thus, CcO activity may serve as a useful molecular marker for the categorization and targeted therapy of GBMs.

Chronic nicotine exposure and acute kidney injury: new concepts and experimental evidence

Although the pathological role of smoking in the development of cardiovascular diseases, cancer [1] or chronic obstructive pulmonary diseases has been widely accepted, its impact on kidney function has only recently been recognized, as reviewed in [2, 3]. Epidemiological studies concluded that smoking is an important remediable risk factor for development of proteinuria [4, 5], progression of diabetic nephropathy [6], evolution of chronic kidney disease (CKD) to end-stage renal disease [7– 9] and graft failure in renal transplant patients [2, 10]. At this moment in time, there is a lack of prospective studies evaluating the role of nicotine abuse as a renal risk factor in patients with primary hypertension or primary renal disease [3]. In spite of the clinical and experimental data, the mechanisms responsible for the reported effects have not yet been fully identified. Although some attention has been paid up till now to the role of smoking in CKD, chronic nicotine-mediated exacerbation of acute kidney injury (AKI) may be an underestimated entity, probably masked by other frequently occurring risk factors such as hypertension, diabetes or obesity. Due to the fact that the current therapeutic array of AKI only consists of supportive care and treatment of the underlying causes and considering the growing evidence on the importance of AKI as a precursor to CKD, the issue of smoking as a risk factor of AKI may warrant more attention. In an interesting paper published in the present issue of Nephrology Dialysis Transplantation [11], Arany et al. investigated in detail the influence of chronic nicotine exposure on the exacerbation of ischaemia-reperfusion-induced oxidative stress and AKI in an in vivo and in vitro mouse model. Based on their previous work [12–15], the authors illustrated nicely that chronic nicotine exposure increased the ischaemia-reperfusion-induced AKI-dependent serine 36 phosphorylation (S36A) and ischaemia-reperfusion-induced AKIdependent renal expression of p66shc. The adaptor protein p66shc is a newly recognized mediator of mitochondrial dysfunction, controlling cellular responses to oxidative stress. The activation of p66shc by oxidative stress is accomplished by the phosphorylation at its Ser36 residue, which facilitates its translocation to the mitochondrial intermembrane space and a H2O2-dependent binding to cytochrome c. The oxidation of cytochrome c results in an excessive generation of reactive oxygen species (ROSs), depolarization of the mitochondria and apoptotic or necrotic death of renal cells through inhibition of the prosurvival epidermal growth factor receptor/ras/MEK/ERK pathway [13, 14]. This paper thus unveils a novel mechanism by which nicotine abuse may be involved in the development of AKI. It broadens our knowledge of the previously described pathways [12–15] and may help us to elucidate the exact link between nicotine and the occurrence of acute/CKD. In previous studies, several potential mediators of smokinginduced renal damage have been discussed, which can be subdivided into non-haemodynamic and haemodynamic mechanisms [3, 16]. Besides the smoking-induced activation of the renin–angiotensin system which is one of the multiple complex and heterogeneous mechanisms, Arany et al. focused in detail on the oxidative stress-dependent Ser36 phosphorylation of p66shc through generation of ROSs, mitochondrial depolarization and consequent injury in cultured proximal tubule cells [11]. This process can be compared with the effects of indoxyl sulphate (IS), a uraemic retention product believed to be involved in the progression of chronic damage, on the p53 pathway [17]. IS is synthesized in the liver from indole, which is generated from tryptophan in dietary proteins by the intestinal flora. As a uraemic toxin, it accumulates in serum and the renal tubules when renal function deteriorates. IS stimulates the expression and phosphorylation of p53 through ROS production [18]. Activation of nuclear factorkappa B (NF-κB) and ROS production in proximal tubular cells are responsible for a reduced renal expression of Klotho in CKD [19], which in turn results in fastened senescence of tubular cells and renal damage. The involvement of IS in renal IN F O C U S

An Inherited Heteroplasmic Mutation in Mitochondrial Gene COI in a Patient with Prostate Cancer Alters Reactive Oxygen, Reactive Nitrogen and Proliferation

Mitochondrial DNA (mtDNA) mutations have been found in many cancers but the physiological derangements caused by such mutations have remained elusive. Prostate cancer is associated with both inherited and somatic mutations in the cytochrome c oxidase (COI) gene. We present a prostate cancer patient-derived rare heteroplasmic mutation of this gene, part of mitochondrial respiratory complex IV. Functional studies indicate that this mutation leads to the simultaneous decrease in cytochrome oxidation, increase in reactive oxygen, and increased reactive nitrogen. These data suggest that mitochondrial DNA mutations resulting in increased reactive oxygen and reactive nitrogen generation may be involved in prostate cancer biology.

Porous Silicon/Photosynthetic Reaction Center Hybrid Nanostructure

The purified photosynthetic reaction center protein (RC) from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA) chemistry or via a noncovalent peptide cross-linker. The characteristic resonance mode in the microcavity reflectivity spectrum red shifted by several nanometers upon RC binding, indicating the protein infiltration into the porous silicon (PSi) photonic structure. Flash photolysis experiments confirmed the photochemical activity of RC after its binding to the solid substrate. The kinetic components of the intraprotein charge recombination were considerably faster (τ(fast) = 14 (±9) ms, τ(slow) = 230 (±28) ms with the RC bound through the GTA cross-linker and only τ(fast) = 27 (±3) ms through peptide coating) than in solution (τ(fast) = 120 (±3) ms, τ(slow) = 1387 (±2) ms), indicating the effect of the PSi surface on the light-induced electron transfer in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.

Synaptic and non-synaptic mitochondria in hippocampus of adult rats differ in their sensitivity to hypothyroidism.

Hypothyroidism in humans provokes various neuropsychiatric disorders, movement, and cognitive abnormalities that may greatly depend on the mitochondrial energy metabolism. Brain cells contain at least two major populations of mitochondria that include the non-synaptic mitochondria, which originate from neuronal and glial cell bodies (CM), and the synaptic (SM) mitochondria, which primarily originate from the nerve terminals. Several parameters of oxidative stress and other parameters in SM and CM fractions of hippocampus of adult rats were compared among euthyroid (control), hypothyroid (methimazol-treated), and thyroxine (T4)-treated hypothyroid states. nNOS translocation to CM was observed with concomitant increase of mtNOS's activity in hypothyroid rats. In parallel, oxidation of cytochrome c oxidase and production of peroxides with substrates of complex I (glutamate + malate) were enhanced in CM, whereas the activity of aconitase and mitochondrial membrane potential (ΔΨm) were decreased. Furthermore, the elevation of mitochondrial hexokinase activity in CM was also found. No differences in these parameters between control and hypothyroid animals were observed in SM. However, in contrast to CM, hypothyroidism increases the level of pro-apoptotic K-Ras and Bad in SM. Our results suggest that hypothyroidism induces moderate and reversible oxidative/nitrosative stress in hippocampal CM, leading to the compensatory elevation of hexokinase activity and aerobic glycolysis. Such adaptive activation in glycolytic metabolism does not occur in SM, suggesting that synaptic mitochondria differ in their sensitivity to the energetic disturbance in hypothyroid conditions.

Charge accumulation and electron transfer kinetics in Geobacter sulfurreducens biofilms

Electroactive bacteria can use a polarized electrode as final electron acceptor, allowing the use of electrochemical techniques for a very accurate quantification of its respiration rate. Biofilm cell respiration has been recently demonstrated to continue after the interruption of electrode polarization since these bacteria can store electrons in the haem groups of exocytoplasmic cytochromes. Interestingly, it has been shown that when the electrode is connected again, stored electrons can be recovered as a current superimposed to the basal steady state current produced by biofilm respiration. This work presents a model for the biofilm-catalysed electron transfer mechanism that reproduces the current profile obtained upon electrode reconnection. The model allows the estimation of kinetic parameters for internalization of the reduced substrate by the cells and the subsequent reduction of cell internal cytochromes, the electron transfer to mediators in the exterior of the cell, charge transport across the biofilm matrix to the electrode through fixed mediators and, finally, the oxidation of cytochromes at the biofilm/electrode interface. Based on these estimates, the distribution of stored charge within the biofilm can also be calculated. The results indicate that the processes involved in electron transfer from acetate to internal cytochromes represent the main limitation to current production, showing that both electron transport through the matrix of cytochromes and interfacial electron transfer are orders of magnitude faster than this process. Stored charge, on the other hand, is an order of magnitude higher inside the cells compared with that in the conductive matrix, suggesting that internal cytochromes are approximately ten times more abundant inside the cells than in the conductive matrix.

Redox state-dependent aggregation of mitochondria induced by cytochrome c

Cytochrome c is known to play central role in apoptosis. Here, it is shown that ferricytochrome c, but not ferrocytochrome c is able to directly induce the aggregation of rat liver mitochondria, similar to the effect caused by magnesium ions at high concentrations. The aggregation was revealed by a decrease in light dispersion of mitochondrial suspension and it was confirmed by the optical microscopy. In the medium containing NADH and cytochrome c, mitochondrial aggregation was initiated only after exhaustion of NADH leading to oxidation of cytochrome c. The aggregation induced by 30 μM ferricytochrome c, but not by 5 mM MgCl(2), was completely inhibited by 30-100 μM ferricyanide, thus indicating that ferricyanide-cytochrome c specific interaction prevents mitochondrial aggregation. After completion of the aggregation caused by ferricytochrome c, this effect cannot be readily reversed by subsequent reduction of cytochrome c. The aggregation induced by ferricytochrome c and/or magnesium ions explains masking of the external NADH-oxidase activity of mitochondria in vitro reported in the literature. This new cytochrome c redox state-dependent phenomenon might also be involved in more complex mechanisms controlling aggregation (clustering) of mitochondria in vivo under the influence of pro-apoptotic factors and requires further study.

Spectral components of the α-band of cytochrome oxidase

Oxidative redox titrations of the mitochondrial cytochromes were performed in near-anoxic RAW 264.7 cells by inhibiting complex I. Cytochrome oxidation changes were measured with multi-wavelength spectroscopy and the ambient redox potential was calculated from the oxidation state of endogenous cytochrome c. Two spectral components were separated in the α-band range of cytochrome oxidase and they were identified as the difference spectrum of heme a when it has a high ( a H) or low ( a L) midpoint potential ( E m) by comparing their occupancy during redox titrations carried out when the membrane potential (Δ Ψ) was dissipated with a protonophore to that predicted by the neoclassical model of redox cooperativity. The difference spectrum of a L has a maximum at 605 nm whereas the spectrum of a H has a maximum at 602 nm. The Δ Ψ-dependent shift in the E m of a H was too great to be accounted for by electron transfer from cytochrome c to heme a against Δ Ψ but was consistent with a model in which a H is formed after proton uptake against Δ Ψ suggesting that the spectral changes are the result of protonation. A stochastic simulation was implemented to model oxidation states, proton uptake and E m changes during redox titrations. The redox anti-cooperativity between heme a and heme a 3, and proton binding, could be simulated with a model where the pump proton interacted with heme a and the substrate proton interacted with heme a 3 with anti-cooperativity between proton binding sites, but not with a single proton binding site coupled to both hemes.

Identification of a New Charge-Transfer Transition through the Partial Unfolding of Cytochrome C under Mild Acidic Conditions

Cytochrome c, a model protein, has been isolated from a wide variety of prokaryotic and eukaryotic sources. It is commonly believed that proteins behave identically if it is extracted from the same source. This investigation illustrates the unexpected conformational change on the exposure of oxidized equine heart cytochrome c to TCA, as opposed to the preparation from acetic acid, both obtained from Sigma-Aldrich and dissolved at a concentration of 0.5 mM in 1.0 mM monobasic phosphate buffer. Without further purification an earlier unfolding event was obtained with the TCA exposed protein (pH 7.0, Temp. 338K). To unravel this enigma a variety of purification methods were employed utilizing partial unfolding-refolding protocols. The complete oxidation of cytochrome c was achieved by adjusting the pH of the sample to 11.5, neutralizing the positive patches on the protein surface. After complete oxidization, the protein was passed over a Sephadex G10 column. Subsequently, the pH of the solution was adjusted back to 8.0, which was the starting point of a titration which covered the region between 8.0 and 4.0. Surprisingly we observed a weak absorption band at 625nm, which increased with decreasing pH, reflecting the protonation of a titratable group (pK=5.2). This value strongly indicates a H33 as the involved residue. Normally, this band is indicative of the population of a ferric high spin state of the heme iron. However, in our case the 695nm band, which is an indicator of the intactness of the Fe(III)-M80 bond, remained present without any loss of intensity. Our observation suggest the stabilization of an excited charge transfer state, possibly resulting from an A2u->dπ transition by the positive charge on H33 in a heme cavity modified by the initially present TCA.

The Diheme Cytochrome c4 from Vibrio cholerae Is a Natural Electron Donor to the Respiratory cbb3 Oxygen Reductase

The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb(3) oxygen reductase. The cbb(3) oxygen reductase has been previously isolated and characterized; however, the natural mobile electron donor(s) that shuttles electrons between the bc(1) complex and the cbb(3) oxygen reductase is not known. The most likely candidates are the diheme cytochrome c(4) and monoheme cytochrome c(5), which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c(4) and c(5) from V. cholerae have been cloned and expressed heterologously in Escherichia coli. It is shown that reduced cytochrome c(4) is a substrate for the purified cbb(3) oxygen reductase and can support steady state oxygen reductase activity of at least 300 e(-1)/s. In contrast, reduced cytochrome c(5) is not a good substrate for the cbb(3) oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c(4) does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c(4) indicates that the apparent lack of saturation is due to the strong dependence of K(M) and V(max) on the concentration of oxidized cytochrome c(4). Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c(4) are 240 and 340 mV (vs standard hydrogen electrode), similar to the electrochemical properties of other c(4)-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c(4)-type cytochrome and a cbb(3) oxygen reductase within the beta- and gamma-proteobacterial clades, suggesting that cytochrome c(4) is the likely natural electron donor to the cbb(3) oxygen reductases within these organisms. These would include the beta-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb(3) oxygen reductases are the only terminal oxidases in their respiratory chains, and the gamma-proteobacterium Pseudomonas stutzeri.