Redox State Of Cytochrome Research Articles

Effect of glycerol on photobleaching of cytochrome Raman lines in frozen yeast cells.

We applied a Raman spectroscopy approach to investigate the effect of a cryoprotectant on the redox state of cytochromes on freezing yeast cells. The redox activity of cytochromes was studied using time-resolved photobleaching of the resonance Raman lines. It is found that ice formation causes a drastic change in the redox state of cytochromes in cells frozen without cryoprotectant, whereas in the presence of glycerol the effects of ice formation are more gradual. The photobleaching rate of cells frozen in glycerol solution shows a gradual slowing with temperature decrease and an abrupt slowdown below - 48°C. This abrupt decrease was interpreted as originating from changes in protein conformational dynamics. Our findings provide important new insights into the transition from active to inactive cytochrome states as cells undergo freezing in the presence and absence of cryoprotectant.

Surface Recognition and Electron Transfer in Electroactive Bacterial Biofilms: Principal Component Analysis

Bacterial electroactive biofilms attract researchers’ attention due to their ability for generation of electric current when electrons travel for very long, up to several tens microns, distance. It was demonstrated that this electron transfer (ET) ability correlates with the accumulation in the BF huge amounts of a c-type cytochrome (Cyt), but molecular nature of this Cyt, its spatial localization, interaction with the environment, regulation and the controlling factors are not completely understood. Bacterial cell polarity is an internal cell asymmetry that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Biochemical analysis and molecular imaging demonstrate that sensing of energy and nutrients by the cell correlates with asymmetric protein, lipid and other molecule distributions within the cell. In the present work, we develop a new experimental approach for analyzing redox potential spatial distribution within individual bacterial cells and use this approach for identification how attachment to inorganic mineral, mica affects redox state inside individual G. sulfurreducens cells. The approach is based on confocal Raman microscopy allowing for precise estimation of redox state and localization of subcellular cytochromes. Applying Gaussian deconvolution and principal component analysis we are able to identify spectral signatures of cytochromes in different redox states and intracellular locations. Our results give a new tool for quick identification of internal and surface exposed cytochromes and their localization at bacterial cell sides facing the substrate and the solution. They open up the possibility for analyzing electrochemical potentials inside individual bacterial cells and indicate that Cyt redox state might be a driving force switching cell metabolic activity that leads to irreversible cell attachment to electrode, cell-to-cell association, and biofilm formation.

Real‐Time Monitoring of the Binding/Dissociation and Redox States of a Single Transition Metal Ions

Fluorescence resonance energy transfer (FRET) between organic fluorophores is conventionally used to monitor binding/dissociation and conformational change of macromolecules. Here we use FRET between a cyanine dye and single transition metal ions (tmFRET) to monitor the binding/dissociation of transition metal ions to a synthetic polypeptide, and show that different transition metal ion species can be clearly distinguished based on their binding lifetimes and FRET efficiencies. Furthermore, we demonstrate that the redox state of cytochrome c can be successfully monitored at the single‐ion level using the tmFRET.

Cardiac performance is limited by oxygen delivery to the mitochondria in the crystalloid-perfused working heart.

The left ventricular working, crystalloid-perfused heart is used extensively to evaluate basic cardiac function, pathophysiology, and pharmacology. Crystalloid-perfused hearts may be limited by oxygen delivery, as adding oxygen carriers increases myoglobin oxygenation and improves myocardial function. However, whether decreased myoglobin oxygen saturation impacts oxidative phosphorylation (OxPhos) is unresolved, since myoglobin has a much lower affinity for oxygen than cytochrome c oxidase (COX). In the present study, a laboratory-based synthesis of an affordable perfluorocarbon (PFC) emulsion was developed to increase perfusate oxygen carrying capacity without impeding optical absorbance assessments. In left ventricular working hearts, along with conventional measurements of cardiac function and metabolic rate, myoglobin oxygenation and cytochrome redox state were monitored using a novel transmural illumination approach. Hearts were perfused with Krebs-Henseleit (KH) or KH supplemented with PFC, increasing perfusate oxygen carrying capacity by 3.6-fold. In KH-perfused hearts, myoglobin was deoxygenated, consistent with cytoplasmic hypoxia, and the mitochondrial cytochromes, including COX, exhibited a high reduction state, consistent with OxPhos hypoxia. PFC perfusate increased aortic output from 76 ± 6 to 142 ± 4 ml/min and increased oxygen consumption while also increasing myoglobin oxygenation and oxidizing the mitochondrial cytochromes. These results are consistent with limited delivery of oxygen to OxPhos resulting in an adapted lower cardiac performance with KH. Consistent with this, PFCs increased myocardial oxygenation, and cardiac work was higher over a wider range of perfusate Po2. In summary, heart mitochondria are limited by oxygen delivery with KH; supplementation of KH with PFC reverses mitochondrial hypoxia and improves cardiac performance, creating a more physiological tissue oxygen delivery. NEW & NOTEWORTHY Optical absorbance spectroscopy of intrinsic chromophores reveals that the commonly used crystalloid-perfused working heart is oxygen limited for oxidative phosphorylation and associated cardiac work. Oxygen-carrying perfluorocarbons increase myocardial oxygen delivery and improve cardiac function, providing a more physiological mitochondrial redox state and emphasizing cardiac work is modulated by myocardial oxygen delivery.

Effect of Surface Binding on Individual Bacterial Cells: Raman and AFM Approaches

Bacterial cell polarity is an internal cell asymmetry that correlates with the cell ability to sense energy and metabolite sources, chemical signals, quorum signals, toxins, and movement in the desired directions. This ability also plays central role in cell attachment to various surfaces and biofilm formation. Biochemical analysis and molecular imaging demonstrate that sensing of energy and nutrients by the cell correlates with asymmetric protein, lipid and other molecule distributions within the cell. In the present work, we develop an experimental approach and analyze how attachment to inorganic mineral, mica affects Cyt redox state inside individual G. sulfurreducens cells. The approach is based on confocal Raman microscopy allowing for simultaneous detection of Raman spectra of individual bacterial cell and the substrate and allowing for precise estimation of the border between them. Applying Gaussian deconvolution and principal component analysis we are able to identify spectral signatures of cytochrome redox states and their intracellular location. Our results demonstrate that attachment of G. sulfurreducens cells to a muscovite (mica) selectively changes Cyt redox state at the cell side facing the mineral. This leads to the formation of a gradient of redox potential needed for directed electron transfer within the cell. Our results open the possibility for analyzing electrochemical potentials inside individual bacterial cells. They indicate that Cyt redox state might be a driving force switching cell metabolic activity that leads to irreversible cell attachment to electrode, cell-to-cell association, and biofilm formation.

Raman probing of lipids, proteins, and mitochondria in skeletal myocytes: a case study on obesity

We propose a novel approach to assess simultaneously lipid composition in lipid droplets, the redox state of cytochromes, and the relative amount of [Fe–S] clusters in the electron transport chain in the mitochondria of skeletal myocytes by means of near‐infrared Raman spectroscopy. Mitochondria dysfunction, disruption of oxidative phosphorylation, and change in lipid metabolism may be a consequence of the obesity‐related insulin resistance and diabetes mellitus. Lipid metabolism affects the state of the mitochondrial electron transport chain although these relations are not well understood. We demonstrate the applicability of the proposed approach in a case study of myocytes of an obese patient before and after the gastric bypass surgery in comparison with a healthy lean donor. Ratios from chosen Raman peaks were calculated and compared between the different subjects. We show that the suggested technique allows to estimate qualitatively the relative amount of cholesterol and unsaturated lipids, ordering of lipid phase in lipid droplets, changes in the redox state of c‐type and b‐type cytochromes, and the relative amount of [Fe–S] clusters in the mitochondria of intact myocytes. The proposed approach opens perspectives to uncover relations between the functional state of mitochondrial cytochromes and lipid composition and to develop a monitoring tool once it is applied to a wide cohort of patients. Copyright © 2017 John Wiley & Sons, Ltd.

Image reconstruction of oxidized cerebral cytochrome C oxidase changes from broadband near-infrared spectroscopy data.

.In diffuse optical tomography (DOT), overlapping and multidistance measurements are required to reconstruct depth-resolved images of oxy- () and deoxy- (HHb) hemoglobin concentration changes occurring in the brain. These can be considered an indirect measure of brain activity, under the assumption of intact neurovascular coupling. Broadband systems also allow changes in the redox state of cytochrome c oxidase (oxCCO) to be measured, which can be an important biomarker when neurovascular coupling is impaired. We used DOT to reconstruct images of , , and from data acquired with a broadband system. Four healthy volunteers were measured while performing a visual stimulation task (4-Hz inverting checkerboard). The broadband system was configured to allow multidistance and overlapping measurements of the participants’ visual cortex with 32 channels. A multispectral approach was employed to reconstruct changes in concentration of the three chromophores during the visual stimulation. A clear and focused activation was reconstructed in the left occipital cortex of all participants. The difference between the residuals of the three-chromophore model and of the two-chromophore model (recovering only and ) exhibits a spectrum similar to that of oxCCO. These results form a basis for further studies aimed to further optimize image reconstruction of .

Interaction between in vivo bioluminescence and extracellular electron transfer in Shewanella woodyi via charge and discharge.

Extracellular electron transfer (EET) and bioluminescence are both important for microbial growth and metabolism, but the mechanism of interaction between EET and bioluminescence is poorly understood. Herein, we demonstrate an exclusively respiratory luminous bacterium, Shewanella woodyi, which possesses EET ability and electron communication at the interface of S. woodyi and solid substrates via charge and discharge methods. Using an electro-chemiluminescence apparatus, our results confirmed that the FMN/FMNH2 content and the redox status of cytochrome c conjointly regulated the bioluminescence intensity when the potential of an indium-tin oxide electrode was changed. More importantly, this work revealed that there is an interaction between the redox reaction of single cells and bioluminescence of group communication via the EET pathway.

Molecular Basis and Consequences of the Cytochrome c-tRNA Interaction

The intrinsic apoptosis pathway occurs through the release of mitochondrial cytochrome c to the cytosol, where it promotes activation of the caspase family of proteases. The observation that tRNA binds to cytochrome c revealed a previously unexpected mode of apoptotic regulation. However, the molecular characteristics of this interaction, and its impact on each interaction partner, are not well understood. Using a novel fluorescence assay, we show here that cytochrome c binds to tRNA with an affinity comparable with other tRNA-protein binding interactions and with a molecular ratio of ∼3:1. Cytochrome c recognizes the tertiary structural features of tRNA, particularly in the core region. This binding is independent of the charging state of tRNA but is regulated by the redox state of cytochrome c. Compared with reduced cytochrome c, oxidized cytochrome c binds to tRNA with a weaker affinity, which correlates with its stronger pro-apoptotic activity. tRNA binding both facilitates cytochrome c reduction and inhibits the peroxidase activity of cytochrome c, which is involved in its release from mitochondria. Together, these findings provide new insights into the cytochrome c-tRNA interaction and apoptotic regulation.

Redox State of Cytochromes in Frozen Yeast Cells Probed by Resonance Raman Spectroscopy

Cryopreservation is a well-established technique used for the long-term storage of biological materials whose biological activity is effectively stopped under low temperatures (suspended animation). Since most biological methods do not work in a low-temperature frozen environment, the mechanism and details of the depression of cellular activity in the frozen state remain largely uncharacterized. In this work, we propose, to our knowledge, a new approach to study the downregulation of the redox activity of cytochromes b and c in freezing yeast cells in a contactless, label-free manner. Our approach is based on cytochrome photobleaching effects observed in the resonance Raman spectra of live cells. Photoinduced and native redox reactions that contributed to the photobleaching rate were studied over a wide temperature range (from −173 to +25°C). We found that ice formation influences both the rate of cytochrome redox reactions and the balance between the reduced and oxidized cytochromes. We demonstrate that the temperature dependence of native redox reaction rates can be well described by the thermal activation law with an apparent energy of 32.5 kJ/mol, showing that the redox reaction rate is ∼1015 times slower at liquid nitrogen temperature than at room temperature.

Probing cytochrome c in living mitochondria with surface-enhanced Raman spectroscopy.

Selective study of the electron transport chain components in living mitochondria is essential for fundamental biophysical research and for the development of new medical diagnostic methods. However, many important details of inter- and intramembrane mitochondrial processes have remained in shadow due to the lack of non-invasive techniques. Here we suggest a novel label-free approach based on the surface-enhanced Raman spectroscopy (SERS) to monitor the redox state and conformation of cytochrome c in the electron transport chain in living mitochondria. We demonstrate that SERS spectra of living mitochondria placed on hierarchically structured silver-ring substrates provide exclusive information about cytochrome c behavior under modulation of inner mitochondrial membrane potential, proton gradient and the activity of ATP-synthetase. Mathematical simulation explains the observed enhancement of Raman scattering due to high concentration of electric near-field and large contact area between mitochondria and nanostructured surfaces.

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.

Study of the influence of genes related to muscle oxidative processes on beef color

The biochemical bases of meat color are determined by the concentration and redox state of myoglobin, hemoglobin, cytochromes, and other pigments. Post-mortem depletion of cellular oxygen results in oxidative stresses that consume NADH and affects reducing activity, while enzymatic detoxification influences the cellular oxidative processes, both affecting meat color. The aim of this work was to study the influence of several genes related to cellular oxidative processes that could affect CIELAB meat color parameters. The study was performed in steers that received a grass-based diet combined with grain, hays and silages. Results suggest a possible link between colorimetric parameters (a*, b* and chroma) and SNPs in the GSTP1 gene (P<0.05). Although the influence of the enzymes, encoded by GSTP1 gene, on meat color has been proposed previously at biochemical level and protein expression level, further association studies in different populations and functional studies of proteins are needed to confirm the genetic determination of that gene on meat color.

Intrahepatic triglyceride measurement and estimation of viability in rat fatty livers by near-infrared spectroscopy.

This study investigated the features of fatty livers using near-infrared (NIR) spectroscopy and validated the usefulness of NIR spectroscopy for the measurement of intrahepatic triglyceride (TG) contents and evaluation of viability in fatty livers. In vitro, we examined specific spectra for each purified TG fraction by NIR. In vivo, the differences between the spectra obtained from normal and fatty livers before warm ischemia and the differences between the spectra obtained from each rat liver before and after warm ischemia were subjected to multicomponent analysis. In vitro experiments revealed a specific peak at 925 nm in major TG fractions, and NIR spectroscopy precisely detected changes in TG volume. In vivo experiments revealed that NIR spectroscopy detected TG content changes in rat fatty livers induced by a choline-deficient diet following the addition of purified TG spectrum for NIR spectroscopic analysis in least square curve fitting. The TG level in the fatty livers measured by NIR spectroscopy significantly correlated with the morphometric measurement of lipid content in the livers. NIR spectroscopy also revealed decreased levels of total hemoglobin (Hb) and oxidized Hb and maintenance of homeostasis in cytochrome redox states in fatty livers under normal condition. However, NIR spectroscopy showed irreversible deterioration of hepatic microcirculation, Hb oxygenation and homeostasis of the cytochrome redox states in fatty livers after 60-min warm ischemia reperfusion. These studies demonstrated that NIR spectroscopy can quantitatively measure the intrahepatic TG content in addition to simultaneously evaluating microcirculation and Hb oxygenation.

The relationship between cytochrome redox state and oxygen consumption in isolated mouse and beef heart mitochondria during hypoxia

The relationship between cytochrome redox state and oxygen consumption in isolated mouse and beef heart mitochondria during hypoxia

Mdivi-1 Prevents Apoptosis Induced by Ischemia–Reperfusion Injury in Primary Hippocampal Cells via Inhibition of Reactive Oxygen Species–Activated Mitochondrial Pathway

Apoptosis is one of the major mechanisms of neuronal injury during ischemic-reperfusion (I/R). Mitochondrial division inhibitor (mdivi-1) is a selective inhibitor of mitochondrial fission protein Drp1. The previous experiments support that mdivi-1 reduce I/R injury in the heart model of rat, but the neuroprotective effect of the mdivi-1 is not yet clearly defined at the cellular levels in brain. In our present study, we estimated a brain model of I/R injury invitro by subjecting oxygen and glucose deprivation (OGD) followed by reoxygenation to the cultured rat primary hippocampal cells, which aimed to find the neuroprotective mechanism of mdivi-1. The cell was pretreated with mdivi-1 for 40minutes and then ischemia for 6 hours followed by reperfusion for 20hours. The redox state, cell apoptosis, and expression of Drp1, Bcl-2, Bax, and cytochrome C proteins were measured. The data showed that administration of mdivi-1 at the doses of 50 μM significantly reduced oxidative stress, attenuated cell apoptosis, upregulated Bcl-2 expression, and downregulated Drp1, Bax, and cytochrome C expression. The results suggested that mdivi-1 protected brain from OGD reperfusion injury, which through suppressing the ROS initiated mitochondrial pathway.

Nanobiomolecular multiprotein clusters on electrodes for the formation of a switchable cascadic reaction scheme.

A supramolecular multicomponent protein architecture on electrodes is developed that allows the establishment of bidirectional electron transfer cascades based on interprotein electron exchange. The architecture is formed by embedding two different enzymes (laccase and cellobiose dehydrogenase) and a redox protein (cytochrome c) by means of carboxy-modified silica nanoparticles in a multiple layer format. The construct is designed as a switchable dual analyte detection device allowing the measurement of lactose and oxygen, respectively. As the switching force we apply the electrode potential, which ensures control of the redox state of cytochrome c. The two signal chains are operating in a non-separated matrix and are not disturbed by the other biocatalyst.

Cellular and Molecular Mechanisms of Photobiomodulation (Low-Power Laser Therapy)

Monochromatic (laser) and quasimonochromatic (LED) radiation in the red-to-near infrared optical region both in CW and pulsed modes is used to treat in a nondestructive fashion various soft-tissue and neurologic conditions. Cytochrome c oxidase, the terminal enzyme of mitochondrial respiratory chain, is considered as the photoacceptor. The mechanisms of photobiomodulation on cellular level are based on the electronic excitation of CuA and CuB chromophores in cytochrome c oxidase molecule. As the result, the redox status of cytochrome c oxidase molecule as well as its functional activity is modulated. The primary reactions in mitochondria switch on a set of secondary biochemical reactions (light-sensitive retrograde mitochondrial signaling) with endpoint as gene expression in the nucleus. Also, two specific topics are discussed and reviewed: patch-clamp studies in connection with optical stimulation of nerves and differences in CW and pulsed light action mechanisms when biological objects are irradiated.

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.

Cytochrome c oxidase response to changes in cerebral oxygen delivery in the adult brain shows higher brain-specificity than haemoglobin

The redox state of cerebral mitochondrial cytochrome c oxidase monitored with near-infrared spectroscopy (Δ[oxCCO]) is a signal with strong potential as a non-invasive, bedside biomarker of cerebral metabolic status. We hypothesised that the higher mitochondrial density of brain compared to skin and skull would lead to evidence of brain-specificity of the Δ[oxCCO] signal when measured with a multi-distance near-infrared spectroscopy (NIRS) system. Measurements of Δ[oxCCO] as well as of concentration changes in oxygenated (Δ[HbO2]) and deoxygenated haemoglobin (Δ[HHb]) were taken at multiple source-detector distances during systemic hypoxia and hypocapnia (decrease in cerebral oxygen delivery), and hyperoxia and hypercapnia (increase in cerebral oxygen delivery) from 15 adult healthy volunteers. Increasing source-detector spacing is associated with increasing light penetration depth and thus higher sensitivity to cerebral changes. An increase in Δ[oxCCO] was observed during the challenges that increased cerebral oxygen delivery and the opposite was observed when cerebral oxygen delivery decreased. A consistent pattern of statistically significant increasing amplitude of the Δ[oxCCO] response with increasing light penetration depth was observed in all four challenges, a behaviour that was distinctly different from that of the haemoglobin chromophores, which did not show this statistically significant depth gradient. This depth-dependence of the Δ[oxCCO] signal corroborates the notion of higher concentrations of CCO being present in cerebral tissue compared to extracranial components and highlights the value of NIRS-derived Δ[oxCCO] as a brain-specific signal of cerebral metabolism, superior in this aspect to haemoglobin.