Evidence for a pathway that facilitates nitric oxide diffusion in the brain

Nitric oxide (NO) is a diffusible messenger that conveys information based on its concentration dynamics, which is dictated by the interplay between its synthesis, inactivation and diffusion. Here, we characterized NO diffusion in the rat brain in vivo. By direct sub-second measurement of NO, we determined the diffusion coefficient of NO in the rat brain cortex. The value of 2.2 × 10⁻⁵ cm²/s obtained in vivo was only 14% lower than that obtained in agarose gel (used to evaluate NO free diffusion). These results reinforce the view of NO as a fast diffusing messenger but, noticeably, the data indicates that neither NO diffusion through the brain extracellular space nor homogeneous diffusion in the tissue through brain cells can account for the similarity between NO free diffusion coefficient and that obtained in the brain. Overall, the results support that NO diffusion in brain tissue is heterogeneous, pointing to the existence of a pathway that facilitates NO diffusion, such as cell membranes and other hydrophobic structures.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH) and singlet oxygen (¹O₂), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH, evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH, whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of ¹O₂ is not closely linked to PSII donor side.     

The formation of peroxynitrite in the applied physiology of mitochondrial nitric oxide

Mitochondria require nitric oxide (NO) to exert a delicate control of metabolic rate as well as to regulate life functions, cell cycle activation and arrest, and apoptosis. All activities depend on the matrical NO steady state concentration as provided by mitochondrial (mtNOS) and cytosolic sources (eNOS) and reduced by forming superoxide anion and H₂O₂ and a low peroxynirite (ONOO⁻) yield. We review herein the biochemical pathways involved in the control of NO mitochondrial level and its biological and physiological significance in hormone effects and aging. At high NO, the cost of this physiological regulation is that ONOO⁻ excess will lead to nitrosation/nitration and oxidization of mitochondrial and cell proteins and lipids. The disruption of NO modulation of mitochondrial respiration supports then, a platform for prevalent neurodegenerative and metabolic diseases.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

H2O2 and (.)NO scavenging by Mycobacterium leprae truncated hemoglobin O

Kinetics of ferric Mycobacterium leprae truncated hemoglobin O (trHbOFe(III)) oxidation by H₂O₂ and of trHbOFe(IV)O reduction by NO and NO₂⁻ are reported. The value of the second-order rate constant for H₂O₂-mediated oxidation of trHbOFe(III) is 2.4 × 10³ M⁻¹ s⁻¹. The value of the second-order rate constant for NO-mediated reduction of trHbOFe(IV)O is 7.8 × 10⁶ M⁻¹ s⁻¹. The value of the first-order rate constant for trHbOFe(III)ONO decay to the resting form trHbOFe(III) is 2.1 × 10¹ s⁻¹. The value of the second-order rate constant for NO₂⁻-mediated reduction of trHbOFe(IV)O is 3.1 × 10³ M⁻¹ s⁻¹. As a whole, trHbOFe(IV)O, generated upon reaction with H₂O₂, catalyzes NO reduction to NO₂⁻. In turn, NO and NO₂⁻ act as antioxidants of trHbOFe(IV)O, which could be responsible for the oxidative damage of the mycobacterium. Therefore, Mycobacterium leprae trHbO could be involved in both H₂O₂ and NO scavenging, protecting from nitrosative and oxidative stress, and sustaining mycobacterial respiration.     

Oxidative decarboxylation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: Intermediate formation and characterization of the corresponding cations

Tris(p-carboxyltetrathiaaryl)methyl radicals (TAM) are good EPR probes for measurement of dioxygen concentration in biological systems and for EPR imaging. It has been previously reported that these radicals are efficiently oxidized by superoxide, O₂⁻, or alkylperoxyl radicals, ROO, and by liver microsomes via an oxidative decarboxylation mechanism leading to the corresponding quinone-methides (QM). This article shows that peroxidases, such as horseradish peroxidase (HRP), lactoperoxidase (LPO) and prostaglandin synthase (PGHS), and other hemeproteins, such as methemoglobin (metHb), metmyoglobin (metMb) and catalase, also efficiently catalyze the oxidation of TAM radicals to QM by H₂O₂ or alkylhydroperoxides. These reactions involve the intermediate formation of the corresponding cations TAM⁺ that have also been cleanly generated by K₂Ir(IV)Cl₆ and characterized by UV-Visible spectroscopy and mass spectrometry, and through their reactions with ascorbate or H₂O₂. Labelling experiments on HRP-catalyzed oxidation of TAM to QM using H₂¹⁸O or ¹⁸O₂ in the presence of glucose and glucose oxidase (GOX) showed that the oxygen atom incorporated into QM came both from O₂ and from H₂O. Mechanisms for these reactions in agreement with those data were proposed. Oxidative decarboxylation of TAM to QM is a new reaction catalyzed by peroxidases. Such reactions should be considered when using TAM as EPR oximetry probes invivo or in vitro in complex biological media.     

Closure of VDAC causes oxidative stress and accelerates the Ca-induced mitochondrial permeability transition in rat liver mitochondria

The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca²⁺-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O₂⁻) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O₂⁻ from the IMS and sensitizing to the Ca²⁺-induced MPT. Treatment of isolated rat liver mitochondria with 5 μM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8 ± 1.4 min within a range of 100-250 μM Ca²⁺. G3139-mediated acceleration of the MPT was reversed by 20 μM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O₂⁻ in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O₂⁻ accumulation. Mathematical modeling of generation and turnover of O₂⁻ within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O₂⁻. In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O₂⁻, which causes an internal oxidative stress and sensitizes mitochondria toward the Ca²⁺-induced MPT.     

Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical

The hydroxyl radical (OH) has detrimental biological activity due to its very high reactivity. Our experiments were designed to determine the effects of equimolar concentrations of glucose, fructose and mannitol and three phosphorylated forms of fructose (fructose-1-phosphate (F1P); fructose-6-phosphate (F6P); and fructose-1,6-bis(phosphate) (F16BP)) on OH radical production via the Fenton reaction. EPR spectroscopy using spin-trap DEPMPO was applied to detect radical production. We found that the percentage inhibition of OH radical formation decreased in the order F16BP > F1P > F6P > fructose > mannitol = glucose. As ketoses can sequester redox-active iron thus preventing the Fenton reaction, the Haber-Weiss-like system was also employed to generate OH, so that the effect of iron sequestration could be distinguished from direct OH radical scavenging. In the latter system, the rank order of OH scavenging activity was F16BP > F1P > F6P > fructose = mannitol = glucose. Our results clearly demonstrate that intracellular phosphorylated forms of fructose have more scavenging properties than fructose or glucose, leading us to conclude that the acute administration of fructose could overcome the body’s reaction to exogenous antioxidants during appropriate therapy in certain pathophysiological conditions related to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, malignancy, and some complications of pregnancy.     

Spin-spin interactions in iron(III) porphyrin radical cations with ruffled and saddled structure

Oxidation of essentially pure intermediate-spin iron(III) porphyrinates such as ruffled Fe(TⁱPrP)ClO₄ and saddled Fe(OETPP)ClO₄ produces the corresponding six-coordinate iron(III) porphyrin(por) radical cations [Fe(Por)(ClO₄)₂], where TⁱPrP and OETPP are dianions of 5,10,15,20-tetraisopropylporphyrin and 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin, respectively.Spin-spin interactions in these complexes are very much different; while ruffled [Fe(TⁱPrP)(ClO₄)₂] exhibits no antiferromagnetic coupling, saddled [Fe(OETPP)(ClO₄)₂] does exhibit it. The difference in magnetic behaviors has been explained in terms of the deformation mode and electron configuration of these complexes.     

Complexes of [Re(CO)3Cl] with different oxidation states of the polyfunctional bmtz/H2bmtz ligand system (bmtz=3,6-bis(2-pyrimidyl)-1,2,4,5-tetrazine)

Combining fac-[Re(CO)₃Cl] with components of the ligand redox system bmtz/bmtz⁻/H₂bmtz/H₂bmtz⁻ (bmtz=3,6-bis(2-pyrimidyl)-1,2,4,5-tetrazine) has led to the isolation of the complexes (H₂bmtz)Re(CO)₃Cl, (μ-H₂bmtz)[Re(CO)₃Cl]₂ and (μ-bmtz)[Re(CO)₃Cl]₂. Other species characterized were (bmtz)Re(CO)₃Cl (UV/Vis, IR), [(H₂bmtz)Re(CO)₃Cl]⁻ (UV/Vis, IR, EPR), {(μ-H₂bmtz)[Re(CO)₃Cl]₂}⁻ (UV/Vis, IR, EPR) and {(μ-bmtz)[Re(CO)₃Cl]₂}⁻ (UV/Vis, IR, X band and high-field EPR). The results confirm bmtz as very strong and H₂bmtz as moderate π acceptor ligand versus one or two chelate-bonded low-valent metal centers. Reactivity is observed in terms of oxidative proton and reductive chloride dissociation.