Oxidation Of Cytochrome Research Articles

The influence of carbon dioxide on cerebral metabolism and oxygen consumption: combining multimodal monitoring with dynamic systems modelling.

Hypercapnia increases cerebral blood flow. The effects on cerebral metabolism remain incompletely understood although studies show an oxidation of cytochrome c oxidase, Complex IV of the mitochondrial respiratory chain. Systems modelling was combined with previously published non-invasive measurements of cerebral tissue oxygenation, cerebral blood flow, and cytochrome c oxidase redox state to evaluate any metabolic effects of hypercapnia. Cerebral tissue oxygen saturation and cytochrome oxidase redox state were measured with broadband near infrared spectroscopy and cerebral blood flow velocity with transcranial Doppler ultrasound. Data collected during 5-min hypercapnia in awake human volunteers were analysed using a Fick model to determine changes in brain oxygen consumption and a mathematical model of cerebral hemodynamics and metabolism (BrainSignals) to inform on mechanisms. Either a decrease in metabolic substrate supply or an increase in metabolic demand modelled the cytochrome oxidation in hypercapnia. However, only the decrease in substrate supply explained both the enzyme redox state changes and the Fick-calculated drop in brain oxygen consumption. These modelled outputs are consistent with previous reports of CO2 inhibition of mitochondrial succinate dehydrogenase and isocitrate dehydrogenase. Hypercapnia may have physiologically significant effects suppressing oxidative metabolism in humans and perturbing mitochondrial signalling pathways in health and disease.

Synthesis of Cerium Oxide Nanoparticles in a Bacterial Nanocellulose Matrix and the Study of Their Oxidizing and Reducing Properties

A soft synthesis of nanoceria with non-stoichiometric composition (33% Ce3+/67% Ce4+) named CeO2 NPs in bacterial cellulose (BC) matrix in the form of aerogel and hydrogel with controlled CeO2 NPs content was proposed. The advantage of CeO2 NPs synthesis in BC is the use of systemic antacid API-trisamine as a precursor, which did not destruct cellulose at room temperature and enabled a reduction in the duration of synthesis and the number of washes. Moreover, this method resulted in the subsequent uniform distribution of CeO2 NPs in the BC matrix due to cerium (III) nitrate sorption in the BC matrix. CeO2 NPs (0.1-50.0%) in the BC matrix had a fluorite structure with a size of 3-5 nm; the specific surface area of the composites was 233.728 m2/g. CeO2 NPs in the BC-CeO2 NPs composite demonstrated SOD-like activity in the processes of oxidation and reduction of cytochrome c (cyt c3+/cyt c2+), as well as epinephrine to inhibit its auto-oxidation in aqueous solutions by 33-63% relative to the control. In vitro experiments on rat blood showed a decrease in the MDA level and an increase in the activity of antioxidant defense enzymes-SOD by 24% and G6PDH by 2.0-2.5 times. Therefore, BC-CeO2 NPs can be proposed for wound healing as antioxidant material.

Capacity and kinetics of light-induced cytochrome oxidation in intact cells of photosynthetic bacteria

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivaxgelatinosus and Rhodobactersphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria.

Isolation and Characterization of Tunicates (Ascidian: Phallusia sp.) associative marine bacteria in agar media with Pb heavy metal addition

Tunicates or Ascidian (sea squirt) is a very diverse and cosmopolitan organism, also could live in contaminated environment. Tunicates could harbor various associative bacteria that could produce potential secondary metabolites and adapt to heavy metals. This research aimed to isolate and characterize associative bacteria of Phallusia sp. (Ascidian) using Sea Water Complete Agar (SWCA) media with Pb heavy metals addition. Associative bacteria were isolated from different parts of Phallusia sp. body tissue (basal, middle, and apical part), using several concentrations of Pb added to the media. Three different concentration was applied, K-as control (non-added Pb), K+ (0.008 mgL-1 Pb), and P (0.05 mgL-1 Pb). Result showed that total count of associative bacteria from different parts of body tissue was not significantly different, in neither of Pb concentrations (ANOVA, P value of 0.16). There were 24 isolates with different colonies successively found from all media. Most of the isolate’s colony has white pigmentation, dominated by Gram-negative bacteria. Motility assay showed that all bacteria isolate was motile, with 16 isolates could hydrolyse starch. Lipase activity was found in 20 isolates, and all isolates have catalase, and glucose and mannose fermentative activity. There were 21 isolates with cytochrome oxidation activity. Morphological and physiological characterization suggest that associative bacteria belonged to five groups of bacteria, including four different genera (Alteromonas, Bacillus, Deleya, and Vibrio). Kruskal-Wallis test result showed that the genus composition between each Pb concentration was insignificantly different, with a P value of 0.878.

Homogeneous Cytochrome 579 Is an Octamer That Reacts Too Slowly With Soluble Iron to Be the Initial Iron Oxidase in the Respiratory Chain of Leptospirillum ferriphilum.

The exact role that cytochrome 579 plays in the aerobic iron respiratory chain of Leptospirillum ferriphilum is unclear. This paper presents genomic, structural, and kinetic data on the cytochrome 579 purified from cell-free extracts of L. ferriphilum cultured on soluble iron. Electrospray mass spectrometry of electrophoretically homogeneous cytochrome 579 yielded two principal peaks at 16,015 and 16,141 Daltons. N-terminal amino acid sequencing of the purified protein yielded data that were used to determine the following: there are seven homologs of cytochrome 579; each homolog possesses the CXXCH heme-binding motif found in c-type cytochromes; each of the seven sequenced strains of L. ferriphilum expresses only two of the seven homologs of the cytochrome; and each homolog contains an N-terminal signal peptide that directs the mature protein to an extra-cytoplasmic location. Static light scattering and macroion mobility measurements on native cytochrome 579 yielded masses of 125 and 135 kDaltons, respectively. The reduced alkaline pyridine hemochromogen spectrum of the purified cytochrome had an alpha absorbance maximum at 567 nm, a property not exhibited by any known heme group. The iron-dependent reduction and oxidation of the octameric cytochrome exhibited positively cooperative kinetic behavior with apparent Hill coefficients of 5.0 and 3.7, respectively, when the purified protein was mixed with mM concentrations of soluble iron. Consequently, the extrapolated rates of reduction at sub-mM iron concentrations were far too slow for cytochrome 579 to be the initial iron oxidase in the aerobic respiratory chain of L. ferriphilum. Rather, these observations support the hypothesis that the acid-stable cytochrome 579 is a periplasmic conduit of electrons from initial iron oxidation in the outer membrane of this Gram-negative bacterium to a terminal oxidase in the plasma membrane.

Bionic design of cytochrome c oxidase-like single-atom nanozymes for oxygen reduction reaction in enzymatic biofuel cells

Designing an artificial enzyme, from the perspective of bionics, to mimic the catalytic activity of natural enzymes is highly desirable but remains challenges. In response to the simulation of biological structure, we developed cytochrome c oxidase (CcO)-like single-atom nanozymes with FeN 5 active centers (FeN 5 SAs) in this work. Similar to the spatial structure of heme a 3 in natural CcO, the active center of FeN 5 SAs is axial N-coordinated heme-like structure and can be served as oxygen-binding site to complete oxygen reduction reaction (ORR) in respiratory electron transport chain by catalyzing the oxidation of cytochrome c (Cyt c). Depending on this bionic structure, furthermore, FeN 5 SAs exhibited competitive electrocatalytic performance towards ORR with a half-wave potential of 0.67 V (vs. RHE) at neutral condition. Coupling a glucose dehydrogenase (GDH) bioanode, the FeN 5 SAs-based glucose/O 2 enzymatic biofuel cell (EBFC) obtained a maximum power density of 149.2 ± 4.0 μW cm −2 with an open circuit potential of 0.40 ± 0.01 V. In this study, inspired by the native structure of enzymes, we develop CcO-like FeN 5 SAs and expand its application in EBFCs, which may provide an rational research approach to advance the development of nanozymes. Inspired by the native structure of enzymes, FeN 5 SAs, with remarkably high cytochrome c oxidases-like activity (K M , 4.2 × 10 −5 M) and competitive electrocatalytic performance towards ORR ( E 1/2 , 0.61 V, neutral condition), were developed in this work. Coupling a glucose dehydrogenase bioanode, the FeN 5 SAs-based glucose/O 2 EBFC obtained a maximum power density of 149.2 ± 4.0 μW cm −2 with an open circuit potential of 0.40 ± 0.01 V. • Inspired by the native structure of enzymes, CcO-like FeN 5 single-atom nanozymes were developed in this work. • FeN 5 single-atom nanozymes display remarkably high CcO-like activity and competitive electrocatalytic performance towards ORR. • The study of FeN 5 single-atom nanozymes boosts the cross-merging between single-atom nanomaterials and artificial enzymes.

Total Internal Reflection Tip-Enhanced Raman Spectroscopy of Cytochrome c.

Surface and tip-enhanced Raman spectroscopies in total internal reflection (TIR-SERS and TIR-TERS) are used to characterize the oxidation, spin, and ligation state of cytochrome c (Cc) molecules electrostatically bound on a hydrophilic thiol self-assembled monolayer. TIR-SERS spectra of this model hemoprotein show marker bands typical of the 6cLS ferric state of Cc. The performances of the TIR-TERS technique as a function of the incidence angle are described, showing in particular a significant electromagnetic enhancement of the Raman signal under p-polarized light excitation. TIR-TERS spectra of Cc confirm the 6cLS ferric state assignment deduced from TIR-SERS spectra, thus demonstrating the possibility of probing with nanoscale spatial resolution the 6cLS oxidized form of Cc that is potentially implicated in cell apoptotic processes. The minimal far-field contribution of the sample in TIR-TERS also offers promising perspectives for future nanoscale chemical characterizations of photosensitive biomolecules in complex biological media.

Bioelectrochemistry of Cytochrome c in a closed bipolar electrochemical cell

The reversible oxidation and reduction of Cytochrome c (Cyt c) is demonstrated with a closed bipolar electrochemical cell (CBPEC). Herein, a 4-electrode configuration was studied with the opposite poles of the bipolar electrode resting in separate aqueous and organic electrolyte solutions, respectively. Using biocompatible indium tin oxide (ITO) slides as the bipolar electrode poles, we investigated the influence of the redox potential of the reductant (decamethyferrocene or dimethylferrocene) in an α,α,α-trifluorotoluene organic phase on the observed voltammetry. Reversible electron transfer was only observed between Cyt c and decamethylferrocene. Use of the weaker dimethylferrocene as the reductant required a larger external bias of the driving electrodes to initiate the electron transfer reaction between the two poles of the bipolar electrode. Consequently, the surface of the ITO slide at the aqueous pole experienced a significant negative cathodic potential and underwent irreversible reduction. The biphasic setup using the 4-electrode CBPEC provides insights into electron transfer processes at an interface between two immiscible electrolyte solutions (ITIES), highlighting the strong probability of observing interfacial electron transfer between decamethylferrocene (but not dimethylferrocene) and Cyt c within the short ~1 V polarisable potential window available at an ITIES.

Ergodicity-Breaking in Thermal Biological Electron Transfer? Cytochrome C Revisited.

It was recently suggested that certain redox proteins operate in an ergodicity-breaking regime to facilitate biological electron transfer (ET). A signature for this is a large variance reorganization free energy (several electronvolts) but a significantly smaller Stokes reorganization free energy due to incomplete protein relaxation on the time scale of the ET event. Here we investigate whether this picture holds for oxidation of cytochrome c in aqueous solution, at various levels of theory including classical molecular dynamics with two additive and one electronically polarizable force field, and QM/MM calculations with the QM region treated by full electrostatic DFT embedding and by the perturbed matrix method. Sampling the protein and energy gap dynamics over more than 250 ns, we find no evidence for ergodicity-breaking effects. In particular, the inclusion of electronic polarizability of the heme group at QM/MM levels did not induce nonergodic effects, contrary to previous reports by Matyushov et al. The well-known problem of overestimation of reorganization free energies with additive force fields is cured when the protein and solvent are treated as electronically polarizable. Ergodicity-breaking effects may occur in other redox proteins, and our results suggest that long simulations, ideally on the ET time scale, with electronically polarizable force fields are required to obtain strong numerical evidence for them.

Discrete Ligand Binding and Electron Transfer Properties of ba3-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments.

Cytochrome c oxidase (C cO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven C cO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other C cOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase.

Surfactant protein D regulates caspase-8-mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation

Surfactant-associated protein D (SP-D) is a soluble innate immune collectin present on many mucosal surfaces. We recently showed that SP-D suppresses the extrinsic pathway of apoptosis by downregulating caspase-8 activation. However, the effects of SP-D on the intrinsic pathway of apoptosis are not clearly understood. In the intrinsic pathway, cytochrome c is released by mitochondria into the cytoplasm. Oxidation of cytochrome c by cytochrome c oxidase activates the apoptosome and caspase-9 cascade. Both caspase-8- and caspase-9-mediated branches are activated in the intrinsic pathway of apoptosis; however, little is known about the relevance of the caspase-8 pathway in this context. Here we studied the effects of SP-D on different branches of the intrinsic pathway of apoptosis using UV-irradiated Jurkat T-cells. We found that SP-D does not inhibit the caspase-9 branch of apoptosis and the relevance of the caspase-8-related branch became apparent when the caspase-9 pathway was inhibited by blocking cytochrome c oxidase. Under these conditions, SP-D reduces the activation of caspase-8, executioner caspase-3 and exposure of phosphatidylserine (PS) on the membranes of dying cells. By contrast, SP-D increases the formation of nuclear and membrane blebs. Inhibition of caspase-8 confirms the effect of SP-D is unique to the caspase-8 pathway. Overall, SP-D suppresses certain aspects of the intrinsic pathway of apoptosis via reduction of caspase-8 activation and PS flipping while at the same time increasing membrane and nuclear bleb formation. This novel regulatory aspect of SP-D could help to regulate intrinsic pathway of apoptosis to promote effective blebbing and breakdown of dying cells.

Mimicking a Natural Enzyme System: Cytochrome c Oxidase-Like Activity of Cu2O Nanoparticles by Receiving Electrons from Cytochrome c.

Inorganic nanomaterials-based artificial enzymes (nanozymes) have received considerable attention over the past years. However, the substrates studied for nanozymes have so far been limited to small organic molecules. The catalytic oxidation of biomacromolecules, such as proteins, by nanozymes has not yet been reported to date. In this study, we report that cuprous oxide nanoparticles (Cu2O NPs) possess cytochrome c oxidase (CcO)-like activity and catalyze the oxidation of cytochrome c (Cyt c), converting it from the ferrous state to the ferric state under atmospheric oxygen conditions. Furthermore, the CcO-like activity of Cu2O NPs is pH- and size-dependent. The lower the solution pH and the smaller the particle size, the higher the CcO-like activity. The artificial Cyt c-Cu2O NPs system closely mimics the native Cyt c-CcO enzyme system, which opens new vistas in enzyme construction and potential applications.

Abstract 169: Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma

Abstract Tumor hypoxia is prevalent in head and neck squamous cell carcinoma (HNSCC), where it limits radiotherapy outcomes. Hypoxia-activated prodrugs (HAPs) have been developed to target hypoxic regions of tumors. These agents undergo oxygen-sensitive reductive activation, thereby delivering cytotoxic species within hypoxic cells. This study investigated the efficacy and sensitivity determinants of the clinical-stage HAP evofosfamide (TH-302) using molecularly-characterized models of HNSCC. We deployed a collection of 27 HPV-negative HNSCC cell lines derived from lesions of varying TNM stages and primary, nodal or recurrent sites. The collection was characterized for gene expression by RNA-seq, from which somatic variants were also called. Their transcriptomic features were investigated in the context of pan-cancer TCGA data by hierarchical clustering. The potency and hypoxic selectivity of 3 HAPs - evofosfamide, PR-104A and SN30000 - were assessed by antiproliferative assay in 22 lines and compared to bromo-isophosphoramide mustard (Br-IPM), cisplatin and 5-FU. The antitumor activity of evofosfamide (50 mg/kg qdx5 for 2-3 cycles with or without a single 10 Gy dose of radiation on day 5 of cycle 1) was evaluated in HNSCC xenografts in addition to a PDX isolated from an SCC of the glottic larynx. The hypoxic fraction at baseline and after 5 days of treatment was quantified by pimonidazole staining. Genetic modifiers of sensitivity to evofosfamide and its cytotoxic metabolite Br-IPM were explored through whole-genome CRISPR-Cas9 screens using the GeCKO v2 library. High-throughput screens with a custom shRNA pool were performed in one HNSCC and two pancreatic ductal adenocarcinoma cell lines to identify reductases responsible for the activation of evofosfamide in hypoxic cells. Evofosfamide was more potent and more selective for hypoxic HNSCC cells in vitro than PR-104A or SN30000. Cell line sensitivity to evofosfamide was correlated with Br-IPM and cisplatin but not with PR-104A, SN30000 or 5-FU, indicating distinct sensitivity determinants. Evidence of antitumor activity with evofosfamide was observed in vivo. CRISPR screens identified potential evofosfamide sensitivity genes that were reproducibly enriched following drug exposure. Reductase-focused RNA interference screens defined a cluster of sensitivity genes that mapped to mitochondrial electron transport, whereas shRNA’s targeted against presumed activating enzymes such as POR were not enriched. Concentration-dependent oxidation of cytochrome a and decreased respiration was observed in cells exposed to evofosfamide, suggesting reduction by mitochondrial complexes. This study provides a rationale for the clinical evaluation of evofosfamide with radiotherapy in genetically defined subsets of HNSCC patients. Citation Format: Francis W. Hunter, Avik Shome, Dan Li, Way W. Wong, Peter Tsai, Nooriyah Poonawala, Purvi M. Kakadiya, Troy M. Ketelä, Maria K. Kondratyev, Courtney R. Lynch, Tet-Woo Lee, Khanh B. Tran, Jules B. Devaux, Rachel Zussman, Cho R. Hong, Dennis Kee, Andrew M. Macann, Anthony J. Hickey, Stefan K. Bohlander, Cristin G. Print, William R. Wilson, Bradly G. Wouters, Stephen M. Jamieson. Preclinical efficacy and sensitivity determinants of evofosfamide in molecularly defined models of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 169. doi:10.1158/1538-7445.AM2017-169

Broadband near-infrared spectroscopy can detect cyanide-induced cytochrome aa3 inhibition in rats: a proof of concept study.

Clinically available near-infrared spectroscopy (NIRS) devices use two to five wavelengths of light to measure the relative amounts of oxyhemoglobin and deoxyhemoglobin in tissue to determine tissue hemoglobin oxygen saturation (StO2). In addition to StO2, broadband NIRS devices (using hundreds of wavelengths of light) may be able to measure the oxidation state of mitochondrial cytochrome aa3 (Cytox) which reflects the subcellular energetic state. We hypothesize that broadband NIRS devices can measure Cytox independent of changes in hemoglobin saturation. In this prospective non-randomized study, 20 male Sprague-Dawley rats (300 g) were anesthetized with isoflurane, tracheally intubated, and ventilated with 100% O2 containing 2% isoflurane. They were subsequently instrumented with a broadband NIRS device that used a halogen light source coupled to an emitting fibreoptic cable. Three receiving fibreoptic cables were utilized; one analyzed the light source and the other two were directed at the base of the skull. Each receiving fibre was connected to a spectrometer to measure light intensity. Sodium cyanide (NaCN) 5 mg·kg-1 iv was injected in order to produce cytochrome aa3 reduction. Two to three minutes after injection, oxygen was eliminated and 100% nitrogen (i.e., anoxia) was used for ventilation in order to induce a reduction in both cytochrome aa3 and hemoglobin desaturation. Changes in the cytochrome oxidation state and hemoglobin oxygenation were calculated using a broadband algorithm and compared before and after both the NaCN and anoxia interventions. The NaCN injection resulted in a decrease in median [interquartile range (IQR)] deoxyhemoglobin (-0.014 [-0.29 to -0.005] arbitrary units [AU]; P < 0.001), an increase in oxyhemoglobin (0.013 [-0.011 to 0.031] AU; P < 0.001), and a reduction in cytochrome aa3 (-0.015 [-0.020 to -0.011] AU; P < 0.001). Anoxia resulted in an increase in median [IQR] deoxyhemoglobin (0.13 [0.11 to 0.18] AU; P < 0.001), a decrease in oxyhemoglobin (-0.17 [-0.22 to -0.15] AU; P < 0.001), and a reduction in cytochrome aa3 (-0.04 [-0.06 to -0.03] AU; P < 0.001). Broadband NIRS can effectively measure the directionality of changes in both Cytox and StO2 by uncoupling the cytochrome and hemoglobin signals through inhibition of the electron transport chain and anoxia.

Optical monitoring of retinal respiration in real time: 670 nm light increases the redox state of mitochondria

Mitochondria play a key role in ageing and disease. Their membrane potentials and ATP production decline with age and this is associated with progressive inflammation, cell loss and death. Here we use broadband Near-Infrared Spectroscopy (NIRS) to non-invasively measure in-vivo changes in aged retinal mitochondrial respiration following exposure to 670 nm, which improves mitochondrial performance and reduces inflammation. Low power NIR light was shone into the eye via a fibre optic and the reflection monitored to measure signature changes in the oxidation of cytochrome c oxidase (COX) in complex IV of the electron transport chain. Changes in retinal haemodynamics and oxygenation were also recorded simultaneously with COX by measuring changes in oxygenated and deoxygenated haemoglobin (Δ[HbO2] and Δ[HHb]). Retinae of aged rats exposed to 670 nm for 5 mins showed consistent progressive increases in oxidation of COX 5 mins post exposure. This remained significantly greater than baseline for up to 2 h. This was not seen when retinae were exposed to 420 nm light of the same power or when no light was applied. 670 nm exposure significantly increased total haemoglobin concentration (Δ[HbT] = Δ[HbO2] +Δ[HHb]) but not haemoglobin difference (Δ[HbDiff] = Δ[HbO2] -Δ[HHb]). There were no changes in blood metrics in association with 420 nm light or when no light exposure was given. Hence, brief 670 nm exposure that is associated with reduced inflammation has a significant positive impact on the redox state of COX in aged retinae. The relative redox state of retinal COX may provide a valuable biomarker in ageing and macular degeneration where declining mitochondrial function is implicated.

Chiral Selectivity in Inter-reactant Recognition and Electron Transfer of the Oxidation of Horse Heart Cytochrome c by Trioxalatocobaltate(III).

Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and horse heart cytochrome c (cyt c). This choice is prompted by strong ion-pair formation that enables separation into inter-reactant interaction (chiral "recognition") and ET within the ion pair ("stereoselectivity"). Chiral selectivity was first addressed experimentally. Λ-[Co(Ox)3](3-) was found to be both the more strongly bound and faster reacting enantiomer expressed respectively by the ion-pair formation constant KX and ET rate constant kET(X) (X = Λ and Δ), with KΛ/KΔ and kET(Λ)/kET(Δ) both ≈1.1-1.2. rac-[Co(Ox)3](3-) behavior is intermediate between those of Λ- and Δ-[Co(Ox)3](3-). Chirality was next analyzed by quantum-mechanical ET theory combined with density functional theory and statistical mechanical computations. We also modeled the ion pair K(+)·[Co(Ox)3](3-) in order to address the influence of the solution ionic strength. The complex structure of cyt c meant that this reactant was represented solely by the heme group including the chiral axial ligands L-His and L-Met. Both singlet and triplet hemes as well as hemes with partially deprotonated propionic acid side groups were addressed. The computations showed that the most favorable inter-reactant configuration involved a narrow distance and orientation space very close to the contact distance, substantiating the notion of a reaction complex and the equivalence of the binding constant to a bimolecular reaction volume. The reaction is significantly diabatic even at these short inter-reactant distances, with electronic transmission coefficients κel(X) = 10(-3)-10(-2). The computations demonstrated chirality in both KX and κel(X) but no chirality in the reorganization and reaction free energy (driving force). As a result of subtle features in both KX and κel(X) chirality, the "operational" chirality κET(Λ)KΛ/κET(Δ)KΔ emerges larger than unity (1.1-1.2) from the molecular modeling as in the experimental data.

Molecular cloning and expression analysis of cytochrome c oxidase subunit II from Sitophilus zeamais

Cytochrome c oxidase subunit II (COX II) containing a dual core CuA active site is one of the core subunits of mitochondrial Cytochrome c oxidase (Cco), which plays a significant role in the physiological process. In this report, the full-length cDNA of COXII gene was cloned from Sitophilus zeamais, which had an open reading frame (ORF) of 684 bp encoding 227 amino acids residues. The predicted COXII protein had a molecular mass of 26.2 kDa with pI value of 6.37. multiple sequence alignment and phylogenetic analysis indicated that Sitophilus zeamais COXII had high sequence identity with the COXII of other insect species. The gene was subcloned into the expression vector pET-32a, and induced by isopropyl β-d-thiogalactopyranoside (IPTG) in E. coli Transetta (DE3) expression system. Finally the recombinant COXII with 6-His tag was purified using affinity chromatography with Ni2+-NTA agarose. Western Blotting (WB) showed the recombinant protein was about 44 kD, and the concentration of fusion protein was 50 μg/mL. UV-spectrophotometer and infrared spectrometer analysis showed that recombinant COXII could catalyze the oxidation of substrate Cytochrome C (Cyt c), and influenced by allyl isothiocyanate (AITC). By using molecular docking method, It was found that a sulfur atom of AITC structure could form a length of 2.9 Å hydrogen bond with Leu-31. These results suggested that tag-free COXII was functional and one of the action sites of AITC, which will be helpful to carry out a point mutation in binding sites for the future research.

Paraquat inhibits progesterone synthesis in human placental mitochondria

IntroductionHuman placenta mitochondria produces huge amounts of progesterone necessary for maintaining the pregnancy. Lipid peroxidation in human placental mitochondria inhibits progesterone synthesis and that inhibition can be reversed by superoxide dismutase and other antioxidants. Paraquat (PQ) a highly toxic herbicide generates superoxide radical inside cells and induces lipid peroxidation. Hence, it is supposed to stimulate lipid peroxidation in human placental mitochondria and in consequence to inhibit a placental mitochondrial steroidogenesis. MethodsPlacentas were obtained from normal pregnancies. All experiments were done using isolated human placental mitochondria. Mitochondrial lipid peroxidation was determined as tiobarbituric acid reactive substances (TBARS). A conversion of cholesterol to pregnenolone or pregnenolone to progesterone was measured using radiolabeled steroids and thin layer chromatography. ResultsPQ enhanced the iron-dependent lipid peroxidation as also PQ heightened the inhibitory action of this process on progesterone synthesis in isolated human placental mitochondria. Paradoxically, a superoxide dismutase (SOD) reversed the inhibition of progesterone synthesis only minimally although it strongly inhibited PQ stimulated iron-dependent lipid peroxidation. When iron was absent, PQ stimulated only negligible lipid peroxidation but strongly inhibited progesterone synthesis. SOD had no effect on inhibition of progesterone synthesis by PQ. PQ strongly inhibited of the conversion of cholesterol to pregnenolone but had not got any influence on the enzymatic activity of mitochondrial 3β-hydroxysteroid dehydrogenase. PQ strongly decreased the efficiency of NADPH-dependent cytochrome P450 reduction as well as it promoted the rapid oxidation of the pre-reduced mitochondrial cytochrome P450. However PQ has not inhibited combined activity of adrenodoxin reductase and adrenodoxin. DiscussionWe conclude that the most important reason of the inhibition of progesterone synthesis by PQ is the escape of electrons from cytochrome P450scc to that compound what leads to cytochrome oxidation and, in consequence the inhibition of the reaction catalyzed by it. The action of PQ described here should be considered as potentially harmful for pregnancy and fetal development.

Effect of Red-to-Near Infrared Light on the Reaction of Isolated Cytochrome c Oxidase with Cytochrome c.

Our primary hypothesis was that red-to-near infrared (R-NIR) irradiation would have an effect on the kinetics parameters of the reaction of cytochrome c with isolated cytochrome c oxidase (CCO), and that the magnitude and direction of these changes could be interpreted in the context of the reaction schemes proposed by other authors. New values for the milimolar extinction coefficients of cytochrome c were also determined. Definitive answers to the fundamental processes involved in red-to-near infrared photobiomodulation (R-NIR-PBM) have not been obtained. The consensus is that the electron transport chain enzyme CCO is the target for R-NIR-PBM. This work was undertaken to explore the effect of R-NIR on the activity of isolated CCO. Scans for cytochrome c were obtained in both reduced and oxidized states, and values for the extinction coefficients were calculated. Activity assays were performed by following the oxidation state of cytochrome c at 550 or 415 nm. R-NIR effects on CCO activity were evaluated by pre-irradiating the enzyme at 670 or 830 nm, or by irradiating the reaction mixture with 660 nm light. Milimolar extinction coefficients (L-1 cm-1) were: ɛ550red = 29.1 ± 0.4, ɛ550ox = 8.60 ± 0.15, ɛ415red = 140 ± 2, and ɛ415ox = 89.0 ± 1.1. Reduced-oxidized extinction coefficients were: δɛ550red-ox = 20.5 ± 0.2, and δɛ415red-ox = 51.0 ± 2.0. The second order rate constants k' for irradiated CCO did not show a statistically significant difference from controls. The oxidation of cytochrome c by isolated CCO has not been shown to be affected by R-NIR irradiation, whether applied prior to or concurrently with the enzymatic assays. This lack of effect by R-NIR calls into question the CCO activity model of R-NIR photobiomodulation.

Assessment of the oxidative potential of nanoparticles by the cytochrome c assay: assay improvement and development of a high-throughput method to predict the toxicity of nanoparticles.

Oxidative stress has increasingly been demonstrated as playing a key role in the biological response induced by nanoparticles (NPs). The acellular cytochrome c oxidation assay has been proposed to determine the intrinsic oxidant-generating capacity of NPs. Yet, there is a need to improve this method to allow a rapid screening to classify NPs in terms of toxicity. We adapted the cytochrome c assay to take into account NP interference, to improve its sensitivity and to develop a high-throughput method. The intrinsic oxidative ability of a panel of NPs (carbon black, Mn2O3, Cu, Ag, BaSO4, CeO2, TiO2 and ZnO) was measured with this enhanced test and compared to other acellular redox assays. To assess whether their oxidative potential correlates with cellular responses, we studied the effect of insoluble NPs on the human bronchial epithelial cell line NCI-H292 by measuring the cytotoxicity (WST-1 assay), pro-inflammatory response (IL-8 cytokine production and expression) and antioxidant defense induction (SOD2 and HO-1 expression). The adapted cytochrome c assay had a greatly increased sensitivity allowing the ranking of NPs in terms of their oxidative potential by using the developed high-throughput technique. Besides, a high oxidative potential revealed to be predictive for toxic effects as Mn2O3 NPs induced a strong oxidation of cytochrome c and a dose-dependent cytotoxicity, pro-inflammatory response and antioxidant enzyme expression. BaSO4, which presented no intrinsic oxidative potential, had no cellular effects. Nevertheless, CeO2 and TiO2 NPs demonstrated no acellular oxidant-generating capacity but induced moderate cellular responses. In conclusion, the novel cytochrome c oxidation assay could be used for high-throughput screening of the intrinsic oxidative potential of NPs. However, acellular redox assays may not be sufficient to discriminate among low-toxicity NPs, and additional tests are thus needed.