A comparison of Prx- and OxyR-based H2O2 probes expressed in S. cerevisiae

Genetically encoded fluorescent H₂O₂ probes continue to advance the field of redox biology. Here, we compare the previously established peroxiredoxin-based H₂O₂ probe roGFP2-Tsa2ΔC_R with the newly described OxyR-based H₂O₂ probe HyPer7, using yeast as the model system. Although not as sensitive as roGFP2-Tsa2ΔC_R, HyPer7 is much improved relative to earlier HyPer versions, most notably by ratiometric pH stability. The most striking difference between the two probes is the dynamics of intracellular probe reduction. HyPer7 is rapidly reduced, predominantly by the thioredoxin system, whereas roGFP2-Tsa2ΔC_R is reduced more slowly, predominantly by the glutathione system. We discuss the pros and cons of each probe and suggest that future side-by-side measurements with both probes may provide information on the relative activity of the two major cellular reducing systems.     

Response properties of the genetically encoded optical H2O2 sensor HyPer

Reactive oxygen species mediate cellular signaling and neuropathologies. Hence, there is tremendous interest in monitoring (sub)cellular redox conditions. We evaluated the genetically engineered redox sensor HyPer in mouse hippocampal cell cultures. Two days after lipofection, neurons and glia showed sufficient expression levels, and H₂O₂ reversibly and dose-dependently increased the fluorescence ratio of cytosolic HyPer. Yet, repeated H₂O₂ treatment caused progressively declining responses, and with millimolar doses an apparent recovery started while H₂O₂ was still present. Although HyPer should be H₂O₂ specific, it seemingly responded also to other oxidants and altered cell-endogenous superoxide production. Control experiments with the SypHer pH sensor confirmed that the HyPer ratio responds to pH changes, decreasing with acidosis and increasing during alkalosis. Anoxia/reoxygenation evoked biphasic HyPer responses reporting apparent reduction/oxidation; replacing Cl⁻ exerted only negligible effects. Mitochondria-targeted HyPer readily responded to H₂O₂—albeit less intensely than cytosolic HyPer. With ratiometric two-photon excitation, H₂O₂ increased the cytosolic HyPer ratio. Time-correlated fluorescence-lifetime imaging microscopy (FLIM) revealed a monoexponential decay of HyPer fluorescence, and H₂O₂ decreased fluorescence lifetimes. Dithiothreitol failed to further reduce HyPer or to induce reasonable FLIM and two-photon responses. By enabling dynamic recordings, HyPer is superior to synthetic redox-sensitive dyes. Its feasibility for two-photon excitation also enables studies in more complex preparations. Based on FLIM, quantitative analyses might be possible independent of switching excitation wavelengths. Yet, because of its pronounced pH sensitivity, adaptation to repeated oxidation, and insensitivity to reducing stimuli, HyPer responses have to be interpreted carefully. For reliable data, side-by-side pH monitoring with SypHer is essential.     

NADPH oxidase controls EGF-induced proliferation via an ERK1/2-independent mechanism

We have used HyPer, a ratiometric GFP-based biosensor, to follow H₂O₂ dynamics in live cells. We have found that activation of the EGF receptor in epithelial cells leads to sustained generation of intracellular H₂O₂, which is blocked by apocynin, an inhibitor of the plasma membrane NADPH oxidase assembly. Apocynin also blocked HeLa cell proliferation induced by EGF, indicating that NADPH oxidase is critically involved. However, apocynin failed to alter the kinetics of EGF-stimulated ERK1/2 activation. We conclude that NADPH oxidase and intracellular H₂O₂ are important downstream targets of EGF receptor that mediate the proliferation response by mechanisms distinct from activation of the classical ERK1/2 MAP-kinase pathway.     

Methods for detection and measurement of hydrogen peroxide inside and outside of cells

Hydrogen peroxide (H₂O₂) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H₂O₂ requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H₂O₂ in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3’5’-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H₂O₂, methods based on dihydro compounds such as 2’,7’-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H₂O₂. Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H₂O₂-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H₂O₂ in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H₂O₂ can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H₂O₂, they cannot be used to monitor transient changes in H₂O₂ concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H₂O₂ and can be targeted to various compartments of the cell, but they are not selective for H₂O₂ because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H₂O₂-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.     

Interleukin-17F attenuates H2O2-induced cell cycle arrest

Interleukin IL-17F was expressed in colon epithelial cells and showed multiple functions in colon tumorigenesis. However, the role of IL-17F in colon cancer cell cycle progression remains unclear. In this study, we analyzed the effects of IL-17F on oxidant-induced cell cycle shift in human colon cancer cells. IL-17F overexpressing and wildtype HCT116 cells were challenged with H₂O₂. Cell cycle distribution analysis showed IL-17F attenuated H₂O₂-induced G2/M phase arrest by inhibiting S to G2/M transition. We further checked expression levels of two critical cell cycle regulators p21 and p27. The results showed that IL-17F could inhibit H₂O₂ induced p27 up-regulation. Meanwhile, IL-17F could increase the phosphorylation of p38 after H₂O₂ treatment. The regulations of p27 level and p38 activity may contribute to the impaired G2/M phase arrest by IL-17F. Taken together, our findings extend IL-17F as an important factor in colon cancer development and provide new insight into the signaling pathway.     

Spatial and temporal control of mitochondrial H2O2 release in intact human cells

Hydrogen peroxide (H₂O₂) has key signaling roles at physiological levels, while causing molecular damage at elevated concentrations. H₂O₂ production by mitochondria is implicated in regulating processes inside and outside these organelles. However, it remains unclear whether and how mitochondria in intact cells release H₂O₂. Here, we employed a genetically encoded high‐affinity H₂O₂ sensor, HyPer7, in mammalian tissue culture cells to investigate different modes of mitochondrial H₂O₂ release. We found substantial heterogeneity of HyPer7 dynamics between individual cells. We further observed mitochondria‐released H₂O₂ directly at the surface of the organelle and in the bulk cytosol, but not in the nucleus or at the plasma membrane, pointing to steep gradients emanating from mitochondria. Gradient formation is controlled by cytosolic peroxiredoxins, which act redundantly and with a substantial reserve capacity. Dynamic adaptation of cytosolic thioredoxin reductase levels during metabolic changes results in improved H₂O₂ handling and explains previously observed differences between cell types. Our data suggest that H₂O₂‐mediated signaling is initiated only in close proximity to mitochondria and under specific metabolic conditions.     

Determination of H2O2-dependent generation of singlet oxygen from human saliva with a novel chemiluminescence probe

Singlet oxygen (¹O₂) has been shown to play an important role in salivary defense system, but its generation process and level from human saliva remain uncertain due to the lack of a reliable detection method. We have previously reported 4,4′(5′)-bis[2-(9-anthryloxy)ethylthio]tetrathiafulvalene (BAET) as a novel chemiluminescence probe for ¹O₂. In this work, the probe is successfully used to characterize H₂O₂-dependent generation of ¹O₂ from saliva in real time. However, the yield of ¹O₂ is found to be very low, for example, being about 0.13 nmol from 200 μL saliva in the presence of 1 mM of hydrogen peroxide over a 5-s reaction period. The result is also compared with that obtained with another ¹O₂ probe 2-methyl-6-phenyl-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (CLA), demonstrating that, besides ¹O₂, the other reactive oxygen species such as hydroxyl radical may also be involved in the reaction of saliva with H₂O₂. Furthermore, the present study shows that the selectivity of BAET for ¹O₂ is much higher than that of CLA and thus BAET is highly suited for the detection of ¹O₂ in the presence of other reactive oxygen species in biological systems.     

OxyR介导的细菌氧化胁迫应答调控机制研究进展

OxyR属于LysR型转录因子家族的氧化胁迫调控蛋白,是细菌抵抗氧化胁迫压力的重要调控因子。OxyR能够通过调控过氧化氢酶和过氧化物酶等抗氧化基因的表达清除H₂O₂、参与铁代谢控制胞内过氧化物的产生以及修复生物大分子氧化损伤,从而抵抗氧化胁迫。OxyR的基因表达调控功能依赖于其还原态和氧化态之间的转变,改变调控蛋白对下游基因调控区的亲和能力。氧化态OxyR识别启动子区的结合序列,激活或抑制过氧化氢酶等基因的表达。还原态和氧化态的转换依赖于在氧化状态下分子间二硫键的形成。本文综述了近年来细菌OxyR调控基因表达的最新研究进展,有助于深入理解OxyR在细菌抵抗氧化胁迫的作用方式,为相关致病菌的防治奠定分子基础。     

Synthesis, crystal structure and vibrational spectroscopy of a novel mixed ligands Ni(II) pentaborate: [Ni(C4H10N2)(C2H8N2)2][B5O6(OH)4]2

A novel organic-inorganic hybrid pentaborate [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂][B₅O₆(OH)₄]₂ has been synthesized by hydrothermal reaction and characterized by FT-IR, Raman spectroscopy, elemental analyses and DTA-TGA. Its crystal structure was determined from single crystal X-ray diffraction. The structure consists of isolated polyborate anion [B₅O₆(OH)₄]⁻ and nickel complex cation of [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺, in which the two kinds of ligands come from the decomposition of triethylenetriamine material. The [B₅O₆(OH)₄]⁻ units are connected to one another through hydrogen bonds, forming a three-dimensional framework with large channel along the a and c axes, in which the templating [Ni(C₄H₁₀N₂)(C₂H₈N₂)₂]²⁺ cations are located. The assignments of the record FT-IR absorption frequencies and Raman shifts were given.     

Selective synthesis of triangular cluster oxido-sulfidocomplexes of Mo and W: High yield preparations of [Mo3O2S2(H2O)9], [W3O2S2(H2O)9], [W2MoO2S2(H2O)9] and their derivatization

Reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ or metallic Mo under hydrothermal conditions (140 °C, 4 M HCl) gives oxido-sulfido cluster aqua complex [Mo₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (1). Similarly, [W₃(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (2) is obtained from [W₂O₂(μ-S)₂(H₂O)₆]²⁺ and W(CO)₆. While reaction of [Mo₂O₂(μ-S)₂(H₂O)₆]²⁺ with W(CO)₆ mainly proceeds as simple reduction to give 1, [W₂O₂(μ-S)₂(H₂O)₆]²⁺ with Mo(CO)₆ produces new mixed-metal cluster [W₂Mo(μ₃-S)(μ-O)₂(μ-S)(H₂O)₉]⁴⁺ (3) as main product. From solutions of 1 in HCl supramolecular adduct with cucurbit[6]uril (CB[6]) {[Mo₃O₂S₂(H₂O)₆Cl₃]₂CB[6]}Cl₂⋅18H₂O (4) was isolated and structurally characterized. The aqua complexes were converted into acetylacetonates [M₃O₂S₂(acac)₃(py)₃]PF₆ (M₃ = Mo₃, W₃, W₂Mo; 5a-c), which were characterized by X-ray single crystal analysis, electrospray ionization mass spectrometry and ¹H NMR spectroscopy. Crystal structure of (H₅O₂)(Me₄N)₄[W₃(μ₃-S)(μ₂-S)(μ₂-O)₂(NCS)₉] (6), obtained from 2, is also reported.     

Hydrogen peroxide in the reactions of cancer cells to cisplatin

BackgroundHydrogen peroxide (H₂O₂) is thought to be one of the key components involved in the responses of tumor cells to chemotherapy. The aim of this study was to reveal the pathways and the phases of cisplatin-induced cell death that are characterized by changes of H₂O₂ level.MethodsThe genetically encoded cytosolic fluorescent sensor HyPer2 was used for flow cytometric analysis of the cisplatin-induced changes in H₂O₂ level in HeLa Kyoto cells. Using a vital dye and the apoptotic markers PE Annexin V or TMRE the pathways and stages of cell death were investigated simultaneously with HyPer2 response. The H₂O₂ level was studied separately in viable and early apoptotic cells after 12, 18, 24 h's incubation with cisplatin at several concentrations with or without the scavenger of reactive oxygen species NAC.ResultsCisplatin causes dose- and time-dependent increase of H₂O₂ level in TMRE-positive and PE Annexin V-negative cancer cells. The scavenging of ROS by NAC decreased the H₂O₂ level and restored cell viability.ConclusionН₂О₂ generation begins in cells that have already lost mitochondrial membrane potential but have not yet externalized phosphatidylserine. Prevention of apoptosis by NAC confirmed the role of H₂O₂ in apoptosis induction.General significanceThis is the first time that the sensor HyPer2 has been used in parallel with apoptotic markers and vital dye to demonstrate the role of H₂O₂ in different stages and types of tumor cell death under chemotherapeutic action.     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H₂O₂) through the plasma membrane decreases during adaptation to H₂O₂ by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H₂O₂ the anisotropy of the plasma membrane increases. Adaptation to H₂O₂ was studied at several times (15min up to 90min) by applying the steady-state H₂O₂ delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H₂O₂ was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H₂O₂ adaptation. H₂O₂ diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H₂O₂ permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H₂O₂ is mediated by modulation of the biophysical properties of the plasma membrane.     

H2O2 in plant peroxisomes: an in vivo analysis uncovers a Ca2+‐dependent scavenging system

Oxidative stress is a major challenge for all cells living in an oxygen‐based world. Among reactive oxygen species, H₂O₂, is a well known toxic molecule and, nowadays, considered a specific component of several signalling pathways. In order to gain insight into the roles played by H₂O₂ in plant cells, it is necessary to have a reliable, specific and non‐invasive methodology for its in vivo detection. Hence, the genetically encoded H₂O₂ sensor HyPer was expressed in plant cells in different subcellular compartments such as cytoplasm and peroxisomes. Moreover, with the use of the new green fluorescent protein (GFP)‐based Cameleon Ca²⁺ indicator, D3cpv–KVK–SKL, targeted to peroxisomes, we demonstrated that the induction of cytoplasmic Ca²⁺ increase is followed by Ca²⁺ rise in the peroxisomal lumen. The analyses of HyPer fluorescence ratios were performed in leaf peroxisomes of tobacco and pre‐ and post‐bolting Arabidopsis plants. These analyses allowed us to demonstrate that an intraperoxisomal Ca²⁺ rise in vivo stimulates catalase activity, increasing peroxisomal H₂O₂ scavenging efficiency.     

Study on the heterogeneous degradation of chitosan with H2O2 catalyzed by a new supermolecular assembly crystal: [C6H8N2]6H3[PW12O40]·2H2O

A new supermolecular assembly crystal, [C₆H₈N₂]₆H₃[PW₁₂O₄₀]·2H₂O (DMB-PWA), was synthesized with phosphotungstic acid (PWA) and 1,2-diaminobenzene (DMB) under hydrothermal conditions and was characterized by Fourier-transform infrared spectra (FTIR) and single-crystal X-ray diffraction analysis. DMB-PWA could effectively catalyze oxidative degradation of chitosan with H₂O₂ in the heterogeneous phase. The optimum degradation conditions were determined by orthogonal tests as follows: amount of chitosan 1.00 g, 30% (wt %); H₂O₂, 3.0 mL; dosage of catalyst, 0.06 g; reaction temperature, 85 °C; and reaction time, 30 min. The water-soluble chitosan with a viscosity-average molecular weight (M_v) of 4900 was obtained under the optimum degradation conditions and was characterized by FTIR, ultraviolet-visible diffuse reflection spectra (UV-vis DRS), and X-ray powder diffraction analysis.     

Catalase evolved to concentrate H2O2 at its active site

Catalase is a homo-tetrameric enzyme that has its heme active site deeply buried inside the protein. Its only substrate, hydrogen peroxide (H₂O₂), reaches the heme through a 45 Å-long channel. Large-subunit catalases, but not small-subunit catalases, have a loop (gate loop) that interrupts the major channel. Two accesses lead to a gate that opens the final section of the channel to the heme; gates from the R-related subunits are interconnected. Using molecular dynamic simulations of the Neurospora crassa catalase-1 tetramer in a box of water (48,600 molecules) or 6 M H₂O₂, it is shown that the number of H₂O₂ molecules augments at the surface of the protein and in the accesses to the gate and the final section of the channel. Increase in H₂O₂ is due to the prevalence and distribution of amino acids that have an increased residency for H₂O₂ (mainly histidine, proline and charged residues), which are localized at the protein surface and the accesses to the gate. In the section of the channel from the heme to the gate, turnover rate of water molecules was faster than for H₂O₂ and increased residence sites for water and H₂O₂ were determined. In the presence of H₂O₂, the exclusion of water molecules from a specific site suggests a mechanism that could contend with the competing activity of water, allowing for catalase high kinetic efficiency.     

NADPH oxidase inhibitor diphenyleneiodonium and reduced glutathione mitigate ethephon-mediated leaf senescence,H2O2 elevation and senescence-associated gene expression in sweet potato (Ipomoea batatas)

Ethephon, an ethylene releasing compound, promoted leaf senescence, H₂O₂ elevation, and senescence-associated gene expression in sweet potato. It also affected the glutathione and ascorbate levels, which in turn perturbed H₂O₂ homeostasis. The decrease of reduced glutathione and the accumulation of dehydroascorbate correlated with leaf senescence and H₂O₂ elevation at 72 h in ethephon-treated leaves. Exogenous application of reduced glutathione caused quicker and significant increase of its intracellular level and resulted in the attenuation of leaf senescence and H₂O₂ elevation. A small H₂O₂ peak produced within the first 4 h after ethephon application was also eliminated by reduced glutathione. Diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, delayed leaf senescence and H₂O₂ elevation at 72 h, and its influence was effective only within the first 4 h after ethephon treatment. Ethephon-induced senescence-associated gene expression was repressed by DPI and reduced glutathione at 72 h in pretreated leaves. Leaves treated with l-buthionine sulfoximine, an endogenous glutathione synthetase inhibitor, did enhance senescence-associated gene expression, and the activation was strongly repressed by reduced glutathione. In conclusion, ethephon-mediated leaf senescence, H₂O₂ elevation and senescence-associated gene expression are all alleviated by reduced glutathione and NADPH oxidase inhibitor DPI. The speed and the amount of intracellular reduced glutathione accumulation influence its effectiveness of protection against ethephon-mediated effects. Reactive oxygen species generated from NADPH oxidase likely serves as an oxidative stress signal and participates in ethephon signaling. The possible roles of NADPH oxidase and reduced glutathione in the regulation of oxidative stress signal in ethephon are discussed.     

How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway

When Escherichia coli grows on conventional substrates, it continuously generates 10 to 15 μM/s intracellular H₂O₂ through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H₂O₂ below toxic levels. Therefore, it seemed potentially problematic that E. coli can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H₂O₂. This study was undertaken to understand how E. coli tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H₂O₂ at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H₂O₂ stress response. Null mutants (ΔoxyR) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H₂O₂. The critical element of the OxyR response was the induction of H₂O₂ scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H₂O₂ will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H₂O₂-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.     

Prooxidant capacity of phenolic acids defines antioxidant potential

Phenolic acids derived from vegetables, fruits and beverages are considered abundant sources of natural antioxidants consumed in the human diet. In addition to having well-known antioxidant activity, phenolic acids also exhibit pro-oxidant activity under selected conditions. We hypothesized that the availability of extracellular H₂O₂ derived from phenolic acid autoxidation will diffuse across cell membranes to participate as a messenger molecule to activate intracellular redox signaling in response to oxidative stress. We report on the relative activity of structurally different phenolic acids to generate specific changes in the extracellular - intracellular H₂O₂ flux that induces intracellular redox signaling corresponding to a function to reduce intracellular oxidative stress. HyPer-3 methodology was used to measure increases in intracellular H₂O₂ in differentiated Caco-2 intestinal cells in response to phenolic acid autoxidation and changes in extracellular H₂O₂ production. The potential for different phenolic acids to autoxidize and generate H₂O₂ was dependent on the structure and concentration of phenolic acid. Activation of nuclear factor erythroid 2-related factor (Nrf2) cell signaling was enhanced (p < 0.05) by phenolic acid induced H₂O₂ production, and mitigated when present along with catalase (p < 0.05), or, alternatively by blocking aquaporin 3 (AQP3) function (p < 0.05) using DFP00173 as the AQP3 inhibitor. The relative capacity of phenolic acids to generate H₂O₂ via autoxidation was structure specific and corresponded to the level of Nrf2 cell signaling in differentiated Caco-2 epithelial cells. The Nrf2-Keap1 response paralleled the extent of reduced oxidative stress observed in differentiated Caco-2 cells determined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.     

Preparation and structure of [Ru2(O2CMe)4]2[Fe(CN)5NO] and magnetically ordered Hx[Ru2(O2CMe)4]3−x[Cr(CN)5NO] possessing interpenetrating lattices

Reaction of [Ru₂(O₂CMe)₄]Cl with K₃[Cr(CN)₅NO] in water forms H_x[Ru^II/III₂(O₂CMe)₄]_3−x-[Cr(CN)₅NO]·zH₂O (x = 0.2) that magnetically orders at 4.0 K and possesses an interpenetrating body centered cubic [a = 13.2509(2) Å] structure with random locations of the bridging nitrosyl ligands, and x/3 vacant cation sites. Similarly, the aqueous reaction of [Ru₂(O₂CMe)₄]Cl with Na₂[Fe(CN)₅NO] forms paramagnetic [Ru₂(O₂CMe)₄]₂[Fe(CN)₅NO]·H₂O, which has a similar tetragonal interpenetrating structure [a = 13.0186(1) Å, c = 13.0699(2) Å] where the NO ligands are presumably nonbridging and 1/3 of the expected cation sites are unoccupied. The presence of uncoordinated NO sites in addition to missing neighboring [Ru₂(O₂CMe)₄]⁺ units, results in significant vacancies (or holes) in the lattice.