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Chow diet is used in the majority of rodent studies and, although assumed to be standardized for dietary source and nutritional contents, it varies widely across commercial formulations. Similarly, current approaches to study aging in rodents involve a single-diet formulation across the lifespan and overlook age-specific nutritional requirements, which may have long-term effects on aging processes. Together, these nutrition-based disparities represent major gaps in geroscience research, affecting the interpretation and reproducibility of the studies. This perspective aims to raise awareness on the importance of rodent diet formulation and proposes that geroscientists include detailed descriptions of all experimental diets and feeding protocols. Detailed reporting of diets will enhance rigor and reproducibility of aging rodent studies and lead to more translational outcomes in geroscience research.

The catalytically competent Co(II)-loaded form of the chlorothalonil dehalogenase from Pseudomonas sp. CTN-3 (Chd, EC 3.8.1.2) was characterized by kinetic and spectroscopic methods. Maximum chlorothalonil (TPN; 2,4,5,6-tetrachloroisophtalonitrile) dehalogenase activity was observed in the presence of one Co(II) ion per monomer with kcat and Km values of 12 ± 3 s−1 and 130 ± 10 μM, respectively, providing a catalytic efficiency (kcat/Km) of ∼9.2 × 104 M−1s−1. The dissociation constant (Kd) for Co(II) was determined to be 0.29 µM, and UV-Vis spectroscopy indicated the active site Co(II) ion resides in a penta-coordinate environment. EPR spectra of Co1-Chd contain at least three distinct signals, an MS = ± 1/2 signal with a ∼94 G 59Co hyperfine pattern centered at g1’ ≅ 6.7, a broader MS = ± 1/2 signal with g1’ ≅ 5.7, an MS = ± 3/2 signal with tentatively estimated parameters of g1’ ≅ 10.5 (gz = 2.75), A1(59Co) ≅ 110 G, and a high-field broad resonance at g3’ ≅ 1.8. Four substrate-analog inhibitors with IC50 values ranging from 110 μM to 19 mM were also identified and characterized. Upon the addition of each of the substrate-like inhibitors to Co1-Chd, changes in the EPR spectrum were observed that, in all cases, were simpler than that of Co1-Chd in the absence of inhibitors and could be simulated as either a single species or a mixture of two. Simulation of these data indicate that the corresponding EPR signals are each due to a ground state MS = 1/2 Kramers’ doublet and are consistent with pentacoordinate Co(II) with a relatively constrained coordination sphere. These data suggest that the nitrile moiety of TPN may not directly coordinate to the active site metal ion, providing new insight into the catalytic mechanism for Chd.

An operator within the North Sea had historically been using seawater injection for pressure support, with produced water being discharged to sea. Water injection was via subsea injection wells, with a subsea flowline transporting water from the host platform to these wells. For the last 6 years, this strategy has changed – seawater injection has been halted and the objective has been to reinject all produced water. During this period of time, the field had experienced rising H2S levels within the produced water and increasing oil in produced water, due to problems with separation that were controlled by application of flocculants and monthly cleaning of separation equipment. The solids found within the hydrocyclones were determined to be iron sulphide, reservoir silicates, barium sulphate scale, and biomass. Over time it became clear that the subsea water injection pipeline was also facing challenges with increased pressure drop resulting in lower injection capacity. Injection line fouling Cleaning the subsea water injection line was evaluated initially via batch application of chemicals such as hydrocarbon solvents, mutual solvents (hydrocarbon removal and water wetting), dilute acid solutions and THPS solutions (sulphide dissolver), chelants (sulphate dissolvers) and oxidising agents (biomass dissolvers). Evaluation of the volumes of chemical required to treat the flowline, in addition to the aggressive nature of these materials and the logistic issues associated with the application of such large chemical volumes resulted in another cleaning program option being investigated. The laboratory testing of a novel dissolver/dispersant not based on THPS (tetrakis (hydroxymethyl) phosphonium sulphate) was carried out leading to a field application of the cleaning chemical at treatment rates between 5 and 40 ppm into the produced water for a trial period of 3 months. The application of the continual injection cleaning chemical program to remove the fouling in the produced water system using the novel dissolver/dispersant and control of additional biofilm formation with the biocide program applied to the produced water resulted in the following changes to the produced water injection system. Produced water injection rate increased from 729 m3/h to 777 m3/hour (7% increase). Injection pressure reduction from ∼50 bar to 40 bar (a 20% decrease despite injection rate increasing as above). Increased production as the injection line fouling was reducing reservoir voidage replacement. This paper outlines the development/deployment of this novel non THPS based iron sulphide cleaning chemical package which is suitable for application within the North Sea.

Linkage isomers are coordination compounds with the same composition but different donor atoms, resulting in distinct physical and electronic structures. A pair of linkage isomers

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), catalyzed by dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR is made of electron donor (BchL) and acceptor (BchNB) component proteins. BchNB is further composed of two subunits each of BchN and BchB arranged as an α(2)β(2) heterotetramer with two active sites for substrate reduction. Such oligomeric architectures are found in several other electron transfer (ET) complexes, but how this architecture influences activity is unclear. Here, we describe allosteric communication between the two identical active sites in Rhodobacter sphaeroides BchNB that drives sequential and asymmetric ET. Pchlide binding to one BchNB active site initiates ET from the pre-reduced [4Fe-4S] cluster of BchNB, a process similar to the deficit spending mechanism observed in the structurally related nitrogenase complex. Pchlide binding in one active site is recognized in trans by an Asp-274 from the opposing half, which is positioned to serve as the initial proton donor. A D274A variant DPOR binds to two Pchlide molecules in the BchNB complex, but only one is bound productively, stalling Pchlide reduction in both active sites. A half-active complex combining one WT and one D274A monomer also stalled after one electron was transferred in the WT half. We propose that such sequential electron transfer in oligomeric enzymes serves as a regulatory mechanism to ensure binding and recognition of the correct substrate. The findings shed light on the functional advantages imparted by the oligomeric architecture found in many electron transfer enzymes.

An Fe-type nitrile hydratase α(ɛ) protein complex from Rhodococcus equi TG328-2 (ReNHase) was discovered and shown by MALDI-TOF to form a 1:1 complex. As isolated, the α(ɛ) protein complex exhibited no detectable NHase activity even in the presence of iron. The addition of the ReNHase β-subunit and Fe(II) to the ReNHase apo-α(e) complex, provided an enzyme with a kcat value of 0.7 ± 0.1 s−1 using acrylonitrile as the substrate, indicating that the β-subunit is important for the reconstitution of NHase activity. The addition of the reducing agent TCEP enhanced the activity by more than 50% (kcat of 1.7 ± 0.2 s−1). As the (ɛ) protein was previously shown to bind and hydrolyze GTP, the addition of GTP to the as-purified α(e) complex provided a kcat value of 1.1 ± 0.2 s−1, in the presence of Fe(II) and β-subunit. The addition of TCEP to this combination further enhanced the activity (kcat of 2.1 ± 0.3 s−1). Apo α-subunit was expressed in purified and added to the (ɛ) protein and β-subunits plus Fe(II) and TCEP resulting in a kcat value of 0.7 ± 0.2 s−1 suggesting an α(ɛ) complex can form in vitro. The addition of GTP to this sample increased the observed rate of nitrile hydration by ~ 30%, while TCEP free samples exhibited no activity. Taken together, these data provide insight into the role of the (ɛ) protein and the newly discovered α(ɛ) complex in NHase metallocenter assembly.

Background Magnolia extract (ME) is known to inhibit cancer growth and metastasis in several cell types in vitro and in animal models. However, there is no detailed study on the preventive efficacy of ME for oral cancer, and the key components in ME and their exact mechanisms of action are not clear. The overall goal of this study is to characterize ME preclinically as a potent oral cancer chemopreventive agent and to determine the key components and their molecular mechanism(s) that underlie its chemopreventive efficacy. Methods The antitumor efficacy of ME in oral cancer was investigated in a 4-nitroquinoline-1-oxide (4NQO)-induced mouse model and in two oral cancer orthotopic models. The effects of ME on mitochondrial electron transport chain activity and ROS production in mouse oral tumors was also investigated. Results ME did not cause detectable side effects indicating that it is a promising and safe chemopreventive agent for oral cancer. Three major key active compounds in ME (honokiol, magnolol and 4-O-methylhonokiol) contribute to its chemopreventive effects. ME inhibits mitochondrial respiration at complex I of the electron transport chain, oxidizes peroxiredoxins, activates AMPK, and inhibits STAT3 phosphorylation, resulting in inhibition of the growth and proliferation of oral cancer cells. Conclusion Our data using highly relevant preclinical oral cancer models, which share histopathological features seen in human oral carcinogenesis, suggest a novel signaling and regulatory role for mitochondria-generated superoxide and hydrogen peroxide in suppressing oral cancer cell proliferation, progression, and metastasis.

In oil and gas production iron sulfide and/or inorganic scales can be problematic and can result in loss of production due to issues such as plugging, corrosion, flow restriction and emulsions. Efficient ways to handle the oil wet solids is a challenge. Scale inhibitors, THPS (Tetrakishydroxymethylphosphonium sulfate) or acids have historically been used for remediation purposes, but either they were found to be ineffective, or can lead to a variety of additional problems such as corrosivity or hydrogen sulfide generation upon use. The focus of this paper is on the laboratory development of a series of products that act as cleaners, corrosion inhibitors, iron sulfide dissolvers and scale inhibitors. The field deployment of one of these products is also summarized herein.

A new method to trap catalytic intermediate species was employed with Fe-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase). ReNHase was incubated with substrates in a 23% (w/w) NaCl/H(2)O eutectic system that remained liquid at −20 °C, thereby permitting the observation of transient species that were present at electron paramagnetic resonance (EPR)-detectable levels in samples frozen while in the steady state. Fe(III)-EPR signals from the resting enzyme were unaffected by the presence of 23% NaCl, and the catalytic activity was ~55% that in the absence of NaCl at the optimum pH of 7.5. The reaction of ReNHase in the eutectic system at −20 °C with the substrates acetonitrile or benzonitrile induced significant changes in the EPR spectra. A previously unobserved signal with highly rhombic g-values (g(1) = 2.31) was observed during the steady state but did not persist beyond the exhaustion of the substrate, indicating that it arises from a catalytically competent intermediate. Distinct signals due to product complexes provide a detailed mechanism for product release, the rate-limiting step of the reaction. Assignment of the observed EPR signals was facilitated by density functional theory calculations, which provided candidate structures and g-values for various proposed ReNHase intermediates. Collectively, these results provide new insights into the catalytic mechanism of NHase and offer a new approach for isolating and characterizing EPR-active intermediates in metalloenzymes.

Nitrile hydratase (NHase) is a non-heme iron-containing enzyme that has applications in commodity chemical synthesis, pharmaceutical intermediate synthesis, and reclamation of nitrile-(bromoxynil) contaminated land. Mechanistic study of the enzyme has been complicated by the expression of multiple overlapping Fe(III) EPR signals. The individual signals were recently assigned to distinct chemical species with the assistance of DFT calculations. Here, the origins and evolution of the EPR signals from cells overexpressing the enzyme were investigated, with the aims of optimizing the preparation of homogeneous samples of NHase for study and investigating the application of E. coli overexpressing the enzyme for "green" chemistry. It was revealed that nitrile hydratase forms two sets of inactive complexes in vivo over time. One is due to reversible complexation with endogenous carboxylic acids, while the second is due to irreversibly inactivating oxidation of an essential cysteine sulfenic acid. It was shown that the homogeneity of preparations can be improved by employing an anaerobic protocol. The ability of the substrates acrylonitrile and acetonitrile to be taken up by cells and hydrated to the corresponding amides by NHase was demonstrated by EPR identification of the product complexes of NHase in intact cells. The inhibitors butyric acid and butane boronic acid were also taken up by E. coli and formed complexes with NHase in vivo, indicating that care must be taken with environmental variables when attempting microbially assisted synthesis and reclamation.

Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Given TPN′s widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters k(cat), K(m), and k(cat)/K(m); and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a K(d) of 0.17 μm, consistent with inductively coupled plasma MS data for the as-isolated Chd dimer. We observed that Chd was maximally active toward chlorothalonil in the pH range 7.0–9.0, and fits of these data yielded a pK(ES1) of 5.4 ± 0.2, a pK(ES2) of 9.9 ± 0.1 (k′(cat) = 24 ± 2 s(−1)), a pK(E1) of 5.4 ± 0.3, and a pK(E2) of 9.5 ± 0.1 (k′(cat)/k′(m) = 220 ± 10 s(−1) mm(−1)). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-visible stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation (i.e. a k′(2) of 35.2 ± 0.1 s(−1)) and product release (i.e. a k′(3) of 1.1 ± 0.2 s(−1)), indicating that product release is the slow step in catalysis. On the basis of these results, along with those previously reported, we propose a mechanism for Chd catalysis.

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

The metal binding motif of all nitrile hydratases (NHases, EC 4.2.1.84) is highly conserved (CXXCSCX) in the α-subunit. Accordingly, an eight amino acid peptide (VCTLCSCY), based on the metal binding motif of the Co-type NHase from Pseudonocardia thermophilia (PtNHase), was synthesized and shown to coordinate Fe(II) under anaerobic conditions. Parallel-mode EPR data on the mononuclear Fe(II)-peptide complex confirmed an integer-spin signal at g' ∼ 9, indicating an S = 2 system with unusually small axial ZFS, D = 0.29 cm-1 Exposure to air yielded a transient high-spin EPR signal most consistent with an intermediate/admixed S = 3/2 spin state, while the integer-spin signal was extinguished. Prolonged exposure to air resulted in the observation of EPR signals at g = 2.04, 2.16, and 2.20, consistent with the formation of a low-spin Fe(III)-peptide complex with electronic and structural similarity to the NHase from Rhodococcus equi TG328-2 (ReNHase). Coupled with MS data, these data support a progression for iron oxidation in NHases that proceeds from a reduced high spin to an oxidized high spin followed by formation of an oxidized low-spin iron center, something that heretofore has not been observed.

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]–containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen–deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the ‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.

Test and evaluation (T&E) of bioaerosol detectors presents unique measurement challenges, especially when evaluating systems designed to operate in complex battlefield environments. Equipment used to referee such T&E operations must be able to characterize the operating environment, including complex bioaerosol backgrounds, and the bioaerosol challenge presented to the detector. Test aerosols often represent only a fraction of the total atmospheric aerosol load. Therefore, selection of effective measurement equipment involves first developing an understanding of the natural background in environments where bioaerosol detectors may be used. It is also helpful to have a single platform for common sampling, so that multiple technologies may be evaluated under the same conditions for effective comparison. Such a platform was designed, characterized, and deployed under a cooperative development effort between the United States and Norway executed by the US Army Chemical Biological Center (CBC) and the Norwegian Defence Research Establishment (FFI). The testbed design is presented along with results from aerosol tests performed to document the sampling efficiency of the common inlet and isokinetic sampling manifold across a wide particle size range. The current testbed implementation hosts one Aerodynamic Particle Sizer (APS) and two ultraviolet light induced fluorescence (UVLIF) based bioaerosol particle counting and sizing instruments: the Ultra-Violet Aerodynamic Particle Sizer (UV-APS), and the Wideband Integrated Bioaerosol Sensor (WIBS). A comparative performance analysis of the two UV-LIF systems will be presented, as well as results describing the natural bioaerosol background at locations in both the US and Norway, including particle concentration, size distribution and fluorescence trends.

Cleavage of aromatic carbon–chlorine bonds is critical for the degradation of toxic industrial compounds. Here, we solved the X-ray crystal structure of chlorothalonil dehalogenase (Chd) from Pseudomonas sp. CTN-3, with 15 of its N-terminal residues truncated (Chd(T)), using single-wavelength anomalous dispersion refined to 1.96 Å resolution. Chd has low sequence identity (

An Fe-type nitrile hydratase α(ɛ) protein complex from Rhodococcus equi TG328-2 (ReNHase) was discovered and shown by MALDI-TOF to form a 1:1 complex. As isolated, the α(ɛ) protein complex exhibited no detectable NHase activity even in the presence of iron. The addition of the ReNHase β-subunit and Fe(II) to the ReNHase apo-α(ε) complex, provided an enzyme with a k

The functional roles of the (His)(17) region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHase(Δ238−257) and MbNHase(Δ219−272), were prepared in which the (His)(17) sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α(2)β(2) heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)(17) motif provides an MbNHase enzyme that is ~55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHase(act)) but ~28% more active when expressed in the presence of PtNHase(act). MbNHase(Δ219−272) exhibits ~55% and ~89% of WT activity, respectively, when expressed in the absence or presence of PtNHase(act). Proteolytic cleavage of MbNHase provides an α(2)β(2) heterotetramer that is modestly more active compared to WT MbNHase (k(cat) = 163 ± 4 vs 131 ± 3 s(−1)). Combination of these data establish that neither the (His)(17) nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.

WOS:000436226600032; Reactive oxygen species (ROS) have been implicated in tumorigenesis (tumor initiation, tumor progression, and metastasis). Of the many cellular sources of ROS generation, the mitochondria and the NADPH oxidase family of enzymes are possibly the most prevalent intracellular sources. In this article, we discuss the methodologies to detect mitochondria-derived superoxide and hydrogen peroxide using conventional probes as well as newly developed assays and probes, and the necessity of characterizing the diagnostic marker products with HPLC and LC-MS in order to rigorously identify the oxidizing species. The redox signaling roles of mitochondrial ROS, mitochondrial thiol peroxidases, and transcription factors in response to mitochondria-targeted drugs are highlighted. ROS generation and ROS detoxification in drug-resistant cancer cells and the relationship to metabolic reprogramming are discussed. Understanding the subtle role of ROS in redox signaling and in tumor proliferation, progression, and metastasis as well as the molecular and cellular mechanisms (e.g., autophagy) could help in the development of combination therapies. The paradoxical aspects of antioxidants in cancer treatment are highlighted in relation to the ROS mechanisms in normal and cancer cells. Finally, the potential uses of newly synthesized exomarker probes for in vivo superoxide and hydrogen peroxide detection and the low-temperature electron paramagnetic resonance technique for monitoring oxidant production in tumor tissues are discussed.

Two homobimetallic tricarbonylrhenium(I) complexes of new redox-active dinucleating pincer ligands have been prepared to assess the impact of spacer size on the first oxidation potentials with respect to mononucleating analogues and on intramolecular electronic communication. The new pincer ligands feature two tridentate NNN- sites each composed of two pyrazolyl flanking donors and a diarylamido anchor that are either directly linked (to form a central benzidene core, H2(L1)) or linked via a para-phenylene group (to form a para-terphenyldiamine core, H2(L2)). The bimetallic complexes of the deprotonated ligands, [fac-Re(CO)3]2(μ-L1), 1, and [fac-Re(CO)3]2(μ-L2), 2, were fully characterized in solution and the solid state including by single-crystal X-ray diffraction for 1. The electrochemical properties of each depended strongly on solvent and electrolyte. Complex 1 exhibits two one-electron oxidations in all electrolyte-containing solutions but with separations between first and second oxidation potentia...

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 ( Re NHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a k cat value of 1.2 × 10−3 s−1 and a K m value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the Re NHase TG328-2 activator protein with a K d of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the Re NHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the Re NHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes. * ATP, : adenosine triphosphate; CD, : circular dichroism; EDTA, : ethylenediaminetetraacetic acid; GTP, : guanosine triphosphate; HEPES, : 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; ICP-AES, : inductively coupled plasma atomic emission spectroscopy; ITC, : isothermal titration calorimetry; MBP, : maltose-binding protein; NHase, : nitrile hydratase; NTP, : nucleoside triphosphate; PDB, : protein database; TCEP, : tris(2-carboxyethyl)phosphine; TEV, : tobacco etch virus; UTP, : uridine triphosphate.

Previous studies have shown that reactive oxygen species (ROS) such as superoxide or hydrogen peroxide generated at low levels can exert a tumor-promoting role via a redox-signaling mechanism. Reports also suggest that both tumorigenesis and tumor growth are associated with enhanced ROS formation. However, whether ROS levels or ROS-derived oxidative marker levels increase during tumor growth remains unknown. In this study, in vivo bioluminescence imaging with a boronate-based pro-luciferin probe was used to assess ROS formation. Additionally, probe-free cryogenic electron paramagnetic resonance was used to quantify a characteristic aconitase [3Fe4S](+) center that arises in the tumor tissue of mouse xenografts from the reaction of the native [4Fe4S](2+) cluster with superoxide. Results indicated that tumor growth is accompanied by increased ROS formation, and revealed differences in oxidant formation in the inner and outer sections of tumor tissue, respectively, demonstrating redox heterogeneity. Studies using luciferin and pro-luciferin probes enabled the assessment of tumor size, ROS formation, and bioenergetic status (e.g., ATP) in luciferase-transfected mice tumor xenografts. Probe-free ex vivo low-temperature electron paramagnetic resonance can also be translated to clinical studies.

The dark-operative protochlorophyllide oxidoreductase (DPOR) enzyme contains two [4Fe-4S]- containing component proteins (BchL and BchNB) that assemble in an ATP-dependent fashion to coordinate electron transfer and reduction of protochlorophyllide to chlorophyllide. Photosynthesis generates an oxygenic environment that is non-optimal for [Fe-S] clusters and we here present an elegant evolutionarily conserved mechanism in BchL to protect its [4Fe-4S] cluster. We present a crystal structure of BchL in the nucleotide-free form with an ordered N-terminus that shields the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions that perturb the shielding of the [4Fe-4S] cluster produce an unstable, but hyper-active enzyme complex, suggesting a role for the N-terminus in both auto-inhibition and enzyme stability. Upon ATP binding, a patch of amino acids, Asp-Phe-Asp (‘DFD patch’), situated at the mouth of the BchL ATP-binding pocket promotes inter-subunit cross stabilization of the two subunits and relieves the auto-inhibition by the N-terminus. A linked BchL dimer with one defective ATP-binding site does not support substrate reduction, illustrating that nucleotide binding to both subunits is a prerequisite for the inter-subunit cross stabilization. We propose that ATP-binding produces a conformational compaction of the BchL homodimer leading to a release of the flexible N-terminus from blocking the [4Fe-4S] cluster and promotes complex formation with BchNB to drive electron transfer. The auto-inhibitive feature and release mechanism appear unique to DPOR and is not found in the structurally similar nitrogenase.

Cytochrome

Tetrahydrobiopterin (BH4) represents a potential strategy for the treatment of cardiac remodeling, fibrosis and/or diastolic dysfunction. The effects of oral treatment with BH4 (Sapropterin™ or Kuvan™) are however dose-limiting with high dose negating functional improvements. Cardiomyocyte-specific overexpression of GTP cyclohydrolase I (mGCH) increases BH4 several-fold in the heart. Using this model, we aimed to establish the cardiomyocyte-specific responses to high levels of BH4. Quantification of BH4 and BH2 in mGCH transgenic hearts showed age-based variations in BH4:BH2 ratios. Hearts of mice (

Introduction Melanoma is an aggressive form of skin cancer for which there are no effective drugs for prolonged treatment. The existing kinase inhibitor antiglycolytic drugs (B-Raf serine/threonine kinase or BRAF inhibitors) are effective for a short time followed by a rapid onset of drug resistance. Presentation of case Here, we show that a mitochondria-targeted analog of magnolol, Mito-magnolol (Mito-MGN), inhibits oxidative phosphorylation (OXPHOS) and proliferation of melanoma cells more potently than untargeted magnolol. Mito-MGN also inhibited tumor growth in murine melanoma xenografts. Mito-MGN decreased mitochondrial membrane potential and modulated energetic and mitophagy signaling proteins. Discussion Results indicate that Mito-MGN is significantly more potent than the FDA-approved OXPHOS inhibitor in inhibiting proliferation of melanoma cells. Conclusion These findings have implications in the treatment of melanomas with enhanced OXPHOS status due to metabolic reprogramming or drug resistance.

Mitochondrial diseases (MDs) result from alteration of the mitochondrial respiratory chain (MRC) function. Despite the prevalence of MDs in the population, the paucity of animal models available limits the understanding of these disorders. Mutations in SDHA, a gene that codes for the alpha subunit of succinate dehydrogenase (SDH), can cause some forms of MD. SDHA is a crucial contributor to MRC function. In order to expand the range of MD animal models available, we attempted to generate a Sdha knockout rat. Since homozygous Sdha(-/-) rats could neither be identified in newborn litters, nor as early as embryonic day 14, we evaluated wild-type (WT) and heterozygous Sdha(+)(/-) genotypes. No differences in behavioral, biochemical, or molecular evaluations were observed between WT and Sdha(+/-) rats at 6 weeks or 6 months of age. However, 30% of Sdha(+/-) rats displayed mild muscle fiber atrophy with rare fibers negative for cytochrome oxidase and SDH on histochemical staining. Collectively, our data provide additional evidence that modeling SDH mutations in rodents may be challenging due to animal viability, and heterozygous rats are insufficiently symptomatic at a phenotypic and molecular level to be of significant use in the study of SDH deficiency.

WOS:000437096700034; Reactive oxygen and nitrogen species (ROS/RNS) such as superoxide (O), hydrogen peroxide, lipid hydroperoxides, peroxynitrite, and hypochlorous and hypobromous acids play a key role in many pathophysiological processes. Recent studies have focused on mitochondrial ROS as redox signaling species responsible for promoting cell division, modulating and regulating kinases and phosphatases, and activating transcription factors. Many ROS also stimulate cell death and senescence. The extent to which these processes occur is attributed to ROS levels (low or high) in cells. However, the exact nature of ROS remains unknown. Investigators have used redox-active probes that, upon oxidation by ROS, yield products exhibiting fluorescence, chemiluminescence, or bioluminescence. Mitochondria-targeted probes can be used to detect ROS generated in mitochondria. However, because most of these redox-active probes (untargeted and mitochondria-targeted) are oxidized by several ROS species, attributing redox probe oxidation to specific ROS species is difficult. It is conceivable that redox-active probes are oxidized in common one-electron oxidation pathways, resulting in a radical intermediate that either reacts with another oxidant (including oxygen to produce O) and forms a stable fluorescent product or reacts with O to form a fluorescent marker product. Here, we propose the use of multiple probes and complementary techniques (HPLC, LC-MS, redox blotting, and EPR) and the measurement of intracellular probe uptake and specific marker products to identify specific ROS generated in cells. The low-temperature EPR technique developed to investigate cellular/mitochondrial oxidants can easily be extended to animal and human tissues.

Copper in various forms has been known to have bactericidal activity. Challenges to its application include preventing mobilization of the copper, to both extend activity and avoid toxicity, and bioincompatibility of many candidate substrates for copper immobilization. Using a simple ionic liquid, butylmethylimmidazolium chloride as the solvent, we developed a facile and green method to synthesize biocompatible composites containing copper oxide nanoparticles (CuONPs) from cellulose (CEL) and chitosan (CS) or CEL and keratin (KER). Spectroscopy and imaging results indicate that CEL, CS, and KER remained chemically intact and were homogeneously distributed in the composites with CuONPs with size of 22 ± 1 nm. Electron paramagnetic resonance (EPR) suggests that some 25% of the EPR-detectable Cu(II) is present as a monomeric species, chemically anchored to the substrate by two or more nitrogen atoms, and, further, adopts a unique spatially oriented conformation when incorporated into the [CEL + CS] composite but not in the [CEL + KER] composite. The remaining 75% of EPR-detectable Cu(II) exhibited extensive spin-spin interactions, consistent with Cu(II) aggregates and Cu(II) on the surface of CuONPs. At higher levels of added copper (>59 nmol/mg), the additional copper was EPR-silent, suggesting an additional phase in larger CuONPs, in which S > 0 spin states are either thermally inaccessible or very fast-relaxing. These data suggest that Cu(II) initially binds substrate via nitrogen atoms, from which CuONPs develop through aggregation of copper. The composites exhibited excellent antimicrobial activity against a wide range of bacteria and fungi, including methicillin-resistant Staphylococcus aureus; vancomycin-resistant Enterococcus; and highly resistant Escherichia coli, Streptococcus agalactiae, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Candida albicans. Expectedly, the antibacterial activity was found to be correlated with the CuONPs content in the composites. More importantly, at CuONP concentration of 35 nmol/mg or lower, bactericidal activity of the composite was complemented by its biocompatibility with human fibroblasts.