Reduced Oxygen Species Research Articles

Development of a DNA-damaging ferrocene amino acid

The oxygen-sensitive ferrocenylacetic acid moiety was incorporated in a water-soluble DNA-damaging agent via the synthon 1,1′-ferrocenediacetic acid anhydride. The extent of plasmid nicking was pH-dependent, with complete cleavage at pH 5.5. The nicking was indiscriminate and required O 2, implicating reduced oxygen species as the actual damaging agent.

Cold defence responses the role of oxidative stress

Low temperatures provoke increased production of heat accompanied by increased respiration, oxygen consumption and the production of partially reduced oxygen species called ROS. ROS induce different forms of cellular oxidative damage, disturb the redox state and can change the activity of several metabolic enzymes. Organisms have developed a functionally connected set of anti-oxidant enzymes and low molecular mass compounds (together termed the ADS) that metabolise primary ROS. If ROS production within cells overwhelms the ADS, oxidative damage arises and oxidative stress can occur. Short-term cold exposure in endotherms leads to oxidative stress. As cold exposure persists organisms develop adaptive changes toward reducing ROS production and increasing the ADS. In contrast, heterotherms and ectotherms as a normal part of their over-wintering strategy slow down metabolism, oxygen consumption and subsequently cause ROS production. Increased baseline activity of key anti-oxidant enzymes as well as 'secondary' enzymatic defence and/or glutathione levels in preparation for a putative oxidative stressful situation arising from tissue re-oxygenation seems to be the preferred evolutionary adaptation of such animals exposed to low environmental temperatures.

Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction

Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.

Modification of surface layer of magnesium oxide via partial dissolution and re-growth of crystallites

A procedure to modify surface layer of metal oxide is presented. By way of partial dissolution and re-growth of crystallites, a new MgO surface layer on the “core” of the original MgO particles was formed. XRD analyses indicate that the new surface layer is different from the original MgO particles in crystallinity. Thus a higher reducibility of surface non-lattice oxygen species is generated. As the extent of dissolution and re-growth of crystallites increased, reducible surface non-lattice oxygen species increased, which led to a lowering of surface non-lattice oxygen concentration on the X%-MgO catalysts in the OCM reaction atmosphere. This is considered to be the major reason for decreasing of CO2 formation.

Cytochrome P450 Reductase: A Harbinger of Diffusible Reduced Oxygen Species

The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1∶1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100∶1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically ‘wasteful’ and potentially ‘hazardous’ owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize “uncoupling” on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work.

Mutational disruption of the nitric oxide synthase/calmodulin interaction causes increased formation of reactive oxygen species

The nitric oxide synthases (NOSs) catalyze the formation of nitric oxide (NO) from L‐arginine. The NOSs are bidomain enzymes, consisting of a heme‐containing oxygenase domain and a flavin‐containing reductase domain, bisected by a calmodulin (CaM) binding site. Occupancy of this site is required for NO formation. Recently, Xia et al. (2009, J. Biol. Chem., 284, 30708) published the structure of human iNOS CaM‐bound FMN domain, showing that the only interactions between the FMN domain and CaM involve R536 of iNOS, which makes a salt bridge with E47 of CaM and forms H‐bonds with N42 of CaM and S562, F565 and A564 in the FMN domain. Mutations of R530, the homologous residue in the murine iNOS reductase domain, and R753 in the nNOS reductase domain, yield enzymes active in electron transfer to cytochrome c and potassium ferricyanide. The rate of ferricyanide reduction is equivalent to that of wild‐type, but cytochrome c reduction is diminished by approximately 60%. NADPH oxidation in the absence of acceptor, however, is elevated by about 40% over that of wild‐type, indicating an increase in reduced oxygen species production by these mutants.Supported by NIH GM052419 to BSSM and LJR.

Astragaloside IV Improves Homocysteine-Induced Acute Phase Endothelial Dysfunction via Antioxidation

Astragaloside IV, the major active component extracted from Astragalus membranaceus, exerts multipotent activities under pathophysiological conditions. Hyperhomocysteinemia, an independent risk factor for cardiovascular disease, induces oxidative stress leading to endothelial dysfunction. We investigated the effect of astragaloside IV on acute phase endothelial dysfunction induced by homocysteine. In a concentration-dependent manner, endothelial dysfunction was induced by homocysteine. In organ bath experiment using rat aortic rings, treatment with astragaloside IV resulted in an improvement of the impaired endothelium-dependent vasorelaxation by homocysteine as reflected by the higher maximal vasorelaxation to acetylcholine. However, the presence of N(omega)-nitro-L-arginine methyl ester hydrochloride could abolish the protective effect of astragaloside IV on homocysteine-induced vasomotor dysfunction. In human umbilical vein endothelial cells culture experiment, exposure to astragaloside IV significantly ameliorated the homocysteine-induced inactivation of nitric oxide-nitric oxide synthase signal pathway via reducing oxygen species and increasing the activity of superoxide dismutase. Additionally, pretreatment with superoxide dismutase showed a similar effect to astragaloside IV on attenuation of the homocysteine-induced endothelial dysfunction. These data support the view that astragaloside IV might be advantageous in the treatment of endothelial dysfunction induced by disturbed nitric oxide-nitric oxide synthase pathway due to oxidative stress in hyperhomocysteinemia.

O 2 reduction by a functional heme/nonheme bis-iron NOR model complex

O(2) reactivity of a functional NOR model is investigated by using electrochemistry and spectroscopy. The electrochemical measurements using interdigitated electrodes show very high selectivity for 4e O(2) reduction with minimal production of partially reduced oxygen species (PROS) under both fast and slow electron flux. Intermediates trapped at cryogenic temperatures and characterized by using resonance Raman spectroscopy under single-turnover conditions indicate that an initial bridging peroxide intermediate undergoes homolytic O--O bond cleavage generating a trans heme/nonheme bis-ferryl intermediate. This bis ferryl species can oxygenate 2 equivalents of a reactive substrate.

Evaluation of reactor and catalyst performance in methane partial oxidation over modified nickel catalysts

The process of catalytic partial oxidation (CPO) of methane was investigated at moderate furnace temperatures (500–973 K) and 1 atm over a series of modified Ni catalysts. By exposing the catalysts to different GHSV (15–600 N l CH 4/g h), the conditions ranged from approaching equilibrium at bed exit gas temperatures to oxygen break-through with associated effects on methane conversion and product composition. By modifying Ni catalysts we illustrate that the expected availability of reducible oxygen species influence the combustion activity of the catalyst. This is reasoned from observations of stronger temperature gradients and a higher maximum gas temperature with modifiers such as Co, Mo, Pt, Pd, Rh and to some degree Cr, as compared to a pure Ni catalyst. Modifiers which are generally difficult to reduce, such as oxides of W, Fe and Mn did not increase combustion activity at the bed entrance. At conditions where oxygen break-through was observed, the formation of coupled products, in particular ethane was promoted. It is reasoned that hetero-homogeneous coupling may be involved. The pure Pt and Pt-modified Ni catalysts were found to promote ethane formation even at 100% oxygen conversion. Interestingly, we here report results suggesting a direct route to synthesis gas over Rh, most likely working in parallel to a combustion reforming mechanism at high GHSV. This is argued based on an evaluation of water-gas-shift and the H 2/CO ratio at low bed exit gas temperatures. The conclusion appears reasonable even when accounting for a potentially higher surface temperature than gas temperature.

Role of a distal pocket in the catalytic O 2 reduction by cytochrome c oxidase models immobilized on interdigitated array electrodes

Five iron porphyrins with different superstructures were immobilized on self-assembled-monolayer (SAM)-coated interdigitated-array (IDAs) gold-platinum electrodes. The selectivity of the catalysts i.e., limited formation of partially reduced oxygen species (PROS) in the electrocatalytic reduction of dioxygen, is a function of 2 rates: (i) the rate of electron transfer from the electrode to the catalyst, which is controlled by the length, and conjugation of the linker from the catalyst to the electrode and (ii) the rate of bound oxygen (superoxide) hydrolysis, which correlates with the presence of a water cluster in the gas-binding pocket influencing the rate of oxygen binding; these factors are controlled by the nature of the porphyrin superstructure. The structurally biomimetic Tris-imidazole model is the most selective.

Occurrence, phylogeny, structure, and function of catalases and peroxidases in cyanobacteria

Cyanobacteria have evolved approximately 3x10(9) years ago from ancient phototrophic microorganisms that already lived on our planet Earth. By opening the era of an aerobic, oxygen-containing biosphere, they are the true pacemakers of geological and biological evolution. Cyanobacteria must have been among the first organisms to elaborate mechanisms for the detoxification of partially reduced oxygen species including (hydrogen) peroxide. Since there is still an suprising lack of knowledge on the type, role, and mechanism(s) of peroxide-degrading enzymes in these bacteria, all 44 fully or partially sequenced genomes for haem and non-haem catalases and peroxidases have been critically analysed based on well known structure-function relationships of the corresponding oxidoreductases. It is demonstrated that H(2)O(2)-dismutating enzymes are mainly represented by bifunctional (haem) catalase-peroxidases and (binuclear) manganese catalases, with the latter being almost exclusively found in diazotrophic species. Several strains even lack a gene that encodes an enzyme with catalase activity. Two groups of peroxidases are found. Genes encoding putative (primordial) haem peroxidases (with homology to corresponding mammalian enzymes) and vanadium-containing iodoperoxidases are found only in a few species, whereas genes encoding peroxiredoxins (1-Cys, 2-Cys, type II, and Q-type) are ubiquitous in cyanobacteria. In addition, approximately 70% contain NADPH-dependent glutathione peroxidase-like proteins. The occurrence and phylogeny of these enzymes is discussed, as well as the present knowledge of their physiological role(s).

Using Functional Models to Understand the Four-Electron Reduction of Dioxygen by Cytochrome c Oxidase Under Steady State Conditions

A new functional biomimetic model of cytochrome c oxidase is reported. It is synthesized in 32 steps. Single turnover reaction with oxygen leads to the same intermediates as the ones reported in the enzyme: Fe(III)O2·-/Cu(I)/PhOH (oxygen complex), and Fe(IV)=O/Cu(II)/PhO· (PM). A sister model derivatized with an alkyne was immobilized on azide-functionnalized self-assembled monolayer (SAM) coated gold electrodes by Cu(I)-catalyzed click chemistry. The rate of electron transfer from the electrode to the model was controlled by the length of the linker in the SAM. Steady-state turnover was achieved at biologically relevant electron transfer, where CuB and Tyr244 mimics rapidly deliver their electrons, hence minimizing the release of partially reduced oxygen species (PROS).

Influence of Metals and Reduced Oxygen Species on the Photooxidation of 2‐Propanol with a Cesium Peroxidotitanate Complex

AbstractThe mechanism of the photocatalytic degradation of 2‐propanol under anaerobic condition was investigated in the presence of the novel cesium peroxoidotitanate complex Cs2(NH4)6[Ti4(O2)4(Hcit)2(cit)2]Cl2·8H2O (1) (cit = citrate). This complex contains two different kinds of peroxido units. Two peroxido groups bind to the titanium atom in a side‐on η2‐fashion, the other peroxido units are directly bonded to the titanium and cesium atoms. These bridging peroxido groups are responsible for the inhibited activity in the photooxidation in contrast to what was found in previous studies about unbridged peroxidotitanate complexes. The decelerated oxidation of the alcohol is useful for the determination of the mechanism during the photocatalytic oxidation process. Therefore, ESI mass spectrometry and 13C NMR spectroscopy were applied to obtain a deeper insight into the mechanism in the alcohol oxidation. Furthermore, an influence of the metal salt (Cs vs. Li) with respect of the photocatalysis was detected. The cesium peroxido complex 1 shows significant results in the photochemical oxidation of 2‐propanol to acetone. More than 50 % acetone was obtained without detection of any side products. The conversion yield from 2‐propanol to acetone was monitored by 1H and 13C NMR spectroscopy and by Raman spectroscopy. The catalytic activity is not limited to 2‐propanol; 1‐phenylethanol could also be oxidized by 1. An intermediate of the photooxidation could be isolated and was characterized. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Cellular defenses against superoxide and hydrogen peroxide.

Life evolved in an anaerobic world; therefore, fundamental enzymatic mechanisms and biochemical pathways were refined and integrated into metabolism in the absence of any selective pressure to avoid reactivity with oxygen. After photosystem II appeared, environmental oxygen levels rose very slowly. During this time, microorganisms acquired oxygen tolerance by jettisoning enzymes that use glycyl radicals and exposed low-potential iron-sulfur clusters, which can be directly poisoned by oxygen. They also developed mechanisms to defend themselves against superoxide (O(2)()) and hydrogen peroxide, partially reduced oxygen species that are generated as inadvertent by-products of aerobic metabolism. Contemporary organisms have inherited both the vulnerabilities and the defenses of these ancestral microbes. Current research seeks to identify these, and bacteria comprise an exceptionally accessible experimental system that has provided many of the answers. This manuscript reviews recent developments and identifies remaining puzzles.

Modeling the Kinetics of Fe(II) Oxidation in the Presence of Citrate and Salicylate in Aqueous Solutions at pH 6.0−8.0 and 25 °C

The kinetics of Fe(II) oxidation in the presence of low concentrations of citrate and salicylate have been investigated in aqueous solutions over the pH range 6.0-8.0 using colorimetry. A kinetic model has been developed to describe the oxidation of Fe(II) with specific attention given to the oxidation of inorganic Fe(II), formation and dissociation of Fe(II) complexes and the oxidation of these complexes. At low concentrations of salicylate, both experimental data and model show that the common approach to modeling Fe(II) oxidation that assumes pre-equilibrium between metal and ligand prior to their oxidation is not valid. Complexation of Fe(II) by salicylate is found to be relatively slow, and oxidation of the complex formed occurs rapidly. Citrate, on the other hand was found to be in rapid equilibrium with Fe(II) but the complex formed was oxidized slowly. Both citrate and salicylate complexes are found to dissociate at a rate much faster than previously thought. A model of the oxidation kinetics of Fe(II) species that incorporates the formation and dissociation kinetics of Fe(II) and Fe(III) complexes of citrate and salicylate as well as the reactions of these species with oxygen and reduced oxygen species including superoxide and hydrogen peroxide provides an excellent description of data obtained over a wide range of concentration and pH conditions.

Preparation and characterization of Ce 1- xFe xO 2 complex oxides and its catalytic activity for methane selective oxidation

A series of Ce 1- x Fe x O 2 ( x=0, 0.2, 0.4, 0.6, 0.8, 1) complex oxide catalysts were prepared using the coprecipitation method. The catalysts were characterized by means of XRD and H 2-TPR. The reactions between methane and lattice oxygen from the complex oxides were investigated. The characteristic results revealed that the combination of Ce and Fe oxide in the catalysts could lower the temperature necessary to reduce the cerium oxide. The catalytic activity for selective CH 4 oxidation was strongly influenced by dropped Fe species. Adding the appropriate amount of Fe 2O 3 to CeO 2 could promote the action between CH 4 and CeO 2. Dispersed Fe 2O 3 first returned to the original state and would then virtually form the Fe species on the catalyst, which could be considered as the active site for selective CH 4 oxidation. The appearance of carbon formation was significant and the oxidation of carbon appeared to be the rate-determining step; the amounts of surface reducible oxygen species in CeO 2 were also relevant to the activity. Among all the catalysts, Ce 0.6Fe 0.4O 2 exhibited the best activity, which converted 94.52% of CH 4 at 900 °C.

The PsaE subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803

The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the ΔpsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase ( katG) and iron superoxide dismutase ( sodB) was upregulated in the ΔpsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in ΔpsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the ΔpsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the ΔpsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.

The chemistry and biochemistry of partially reduced oxygen species

The chemistry and biochemistry of partially reduced oxygen species

Redox Potential Control by Drug Binding to Cytochrome P450 3A4

The cytochrome P450s are ubiquitous heme proteins that utilize two reducing equivalents to cleave a ferrous iron - dioxygen complex to produce a single water molecule with the insertion of one oxygen atom into a bound substrate. For the case of soluble cytochrome P450 CYP101, it has been shown that there is a linear free energy relationship between heme redox potential and the spin state of the ferric protein. However, the universality of this relationship has been challenged in the case of mammalian enzymes. Most cytochrome P450s are integral membrane proteins, and detailed redox potential measurements have proved difficult due protein aggregation or the necessary presence of detergent. In this communication we utilize a soluble nanometer scale membrane bilayer disc (Nanodisc) to stabilize monomeric human cytochrome P450 CYP3A4. The Nanodisc system allows facile redox potential measurements to be made on substrate-free CYP3A4 as well as with several drug molecules bound at the active site. We show that substrate binding can dramatically effect the redox potential of the heme protein through modulation of the ferric spin state. A linear free energy relationship is observed, analogous to that noted for the soluble P450s, indicating a common mechanism for this linkage and providing a means for control of electron input in response to the presence of a metabolizable substrate, this potentially limiting the unwanted production of reduced oxygen species.

A Cytochrome c Oxidase Model Catalyzes Oxygen to Water Reduction Under Rate-Limiting Electron Flux

We studied the selectivity of a functional model of cytochrome c oxidase's active site that mimics the coordination environment and relative locations of Fe(a3), Cu(B), and Tyr(244). To control electron flux, we covalently attached this model and analogs lacking copper and phenol onto self-assembled monolayer-coated gold electrodes. When the electron transfer rate was made rate limiting, both copper and phenol were required to enhance selective reduction of oxygen to water. This finding supports the hypothesis that, during steady-state turnover, the primary role of these redox centers is to rapidly provide all the electrons needed to reduce oxygen by four electrons, thus preventing the release of toxic partially reduced oxygen species.