pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

S-nitrosocysteamine-functionalised porous graphene oxide nanosheets as nitric oxide delivery vehicles for cardiovascular applications.

Nitric oxide (NO) is a key signalling molecule released by vascular endothelial cells that is essential for vascular health. Low NO bioactivity is associated with cardiovascular diseases, such as hypertension, atherosclerosis, and heart failure and NO donors are a mainstay of drug treatment. However, many NO donors are associated with the development of tolerance and adverse effects, so new formulations for controlled and targeted release of NO would be advantageous. Herein, we describe the design and characterisation of a novel NO delivery system via the reaction of acidified sodium nitrite with thiol groups that had been introduced by cysteamine conjugation to porous graphene oxide nanosheets, thereby generating S-nitrosated nanosheets. An NO electrode, ozone-based chemiluminescence and electron paramagnetic resonance spectroscopy were used to measure NO released from various graphene formulations, which was sustained at >5 × 10-10 mol cm-2 min-1 for at least 3 h, compared with healthy endothelium (cf. 0.5-4 × 10-10 mol cm-2 min-1). Single cell Raman micro-spectroscopy showed that vascular endothelial and smooth muscle cells (SMCs) took up graphene nanostructures, with intracellular NO release detected via a fluorescent NO-specific probe. Functionalised graphene had a dose-dependent effect to promote proliferation in endothelial cells and to inhibit growth in SMCs, which was associated with cGMP release indicating intracellular activation of canonical NO signalling. Chemiluminescence detected negligible production of toxic N-nitrosamines. Our findings demonstrate the utility of porous graphene oxide as a NO delivery vehicle to release physiologically relevant amounts of NO in vitro, thereby highlighting the potential of these formulations as a strategy for the treatment of cardiovascular diseases.

Plant Cysteine Oxidase Oxygen-Sensing Function Is Conserved in Early Land Plants and Algae.

All aerobic organisms require O2 for survival. When their O2 is limited (hypoxia), a response is required to reduce demand and/or improve supply. A hypoxic response mechanism has been identified in flowering plants: the stability of certain proteins with N-terminal cysteine residues is regulated in an O2-dependent manner by the Cys/Arg branch of the N-degron pathway. These include the Group VII ethylene response factors (ERF-VIIs), which can initiate adaptive responses to hypoxia. Oxidation of their N-terminal cysteine residues is catalyzed by plant cysteine oxidases (PCOs), destabilizing these proteins in normoxia; PCO inactivity in hypoxia results in their stabilization. Biochemically, the PCOs are sensitive to O2 availability and can therefore act as plant O2 sensors. It is not known whether oxygen-sensing mechanisms exist in other phyla from the plant kingdom. Known PCO targets are only conserved in flowering plants, however PCO-like sequences appear to be conserved in all plant species. We sought to determine whether PCO-like enzymes from the liverwort, Marchantia polymorpha (MpPCO), and the freshwater algae, Klebsormidium nitens (KnPCO), have a similar function as PCO enzymes from Arabidopsis thaliana. We report that MpPCO and KnPCO show O2-sensitive N-terminal cysteine dioxygenase activity toward known AtPCO ERF-VII substrates as well as a putative endogenous substrate, MpERF-like, which was identified by homology to the Arabidopsis ERF-VIIs transcription factors. This work confirms functional and O2-dependent PCOs from Bryophyta and Charophyta, indicating the potential for PCO-mediated O2-sensing pathways in these organisms and suggesting PCO O2-sensing function could be important throughout the plant kingdom.

Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase.

Isopenicillin N synthase (IPNS) catalyzes formation of the ß-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O2)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O2 to the IPNS-Fe(II)-ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O2 analog nitric oxide, using the less mobile α6 for comparison. 19F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron-electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.

Hydrodeoxygenation of water-insoluble bio-oil to alkanes using a highly dispersed Pd-Mo catalyst.

Bio-oil, produced by the destructive distillation of cheap and renewable lignocellulosic biomass, contains high energy density oligomers in the water-insoluble fraction that can be utilized for diesel and valuable fine chemicals productions. Here, we show an efficient hydrodeoxygenation catalyst that combines highly dispersed palladium and ultrafine molybdenum phosphate nanoparticles on silica. Using phenol as a model substrate this catalyst is 100% effective and 97.5% selective for hydrodeoxygenation to cyclohexane under mild conditions in a batch reaction; this catalyst also demonstrates regeneration ability in long-term continuous flow tests. Detailed investigations into the nature of the catalyst show that it combines hydrogenation activity of Pd and high density of both Brønsted and Lewis acid sites; we believe these are key features for efficient catalytic hydrodeoxygenation behavior. Using a wood and bark-derived feedstock, this catalyst performs hydrodeoxygenation of lignin, cellulose, and hemicellulose-derived oligomers into liquid alkanes with high efficiency and yield.Bio-oil is a potential major source of renewable fuels and chemicals. Here, the authors report a palladium-molybdenum mixed catalyst for the selective hydrodeoxygenation of water-insoluble bio-oil to mixtures of alkanes with high carbon yield.

Natural Conformational Sampling of Human TNFα Visualized by Double Electron-Electron Resonance.

Double electron-electron resonance in conjunction with site-directed spin labeling has been used to probe natural conformational sampling of the human tumor necrosis factor α trimer. We suggest a previously unreported, predeoligomerization conformation of the trimer that has been shown to be sampled at low frequency. A model of this trimeric state has been constructed based on crystal structures using the double-electron-electron-resonance distances. The model shows one of the protomers to be rotated and tilted outward at the tip end, leading to a breaking of the trimerous symmetry and distortion at a receptor-binding interface. The new structure offers opportunities to modulate the biological activity of tumor necrosis factor α through stabilization of the distorted trimer with small molecules.

Ribonucleotide Reductase Requires Subunit Switching in Hypoxia to Maintain DNA Replication.

Cells exposed to hypoxia experience replication stress but do not accumulate DNA damage, suggesting sustained DNA replication. Ribonucleotide reductase (RNR) is the only enzyme capable of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs). However, oxygen is an essential cofactor for mammalian RNR (RRM1/RRM2 and RRM1/RRM2B), leading us to question the source of dNTPs in hypoxia. Here, we show that the RRM1/RRM2B enzyme is capable of retaining activity in hypoxia and therefore is favored over RRM1/RRM2 in order to preserve ongoing replication and avoid the accumulation of DNA damage. We found two distinct mechanisms by which RRM2B maintains hypoxic activity and identified responsible residues in RRM2B. The importance of RRM2B in the response to tumor hypoxia is further illustrated by correlation of its expression with a hypoxic signature in patient samples and its roles in tumor growth and radioresistance. Our data provide mechanistic insight into RNR biology, highlighting RRM2B as a hypoxic-specific, anti-cancer therapeutic target.

Biochemical and Spectroscopic Studies of Epoxyqueuosine Reductase: A Novel Iron-Sulfur Cluster- and Cobalamin-Containing Protein Involved in the Biosynthesis of Queuosine.

Queuosine is a hypermodified nucleoside present in the wobble position of tRNAs with a 5'-GUN-3' sequence in their anticodon (His, Asp, Asn, and Tyr). The 7-deazapurine core of the base is synthesized de novo in prokaryotes from guanosine 5'-triphosphate in a series of eight sequential enzymatic transformations, the final three occurring on tRNA. Epoxyqueuosine reductase (QueG) catalyzes the final step in the pathway, which entails the two-electron reduction of epoxyqueuosine to form queuosine. Biochemical analyses reveal that this enzyme requires cobalamin and two [4Fe-4S] clusters for catalysis. Spectroscopic studies show that the cobalamin appears to bind in a base-off conformation, whereby the dimethylbenzimidazole moiety of the cofactor is removed from the coordination sphere of the cobalt but not replaced by an imidazole side chain, which is a hallmark of many cobalamin-dependent enzymes. The bioinformatically identified residues are shown to have a role in modulating the primary coordination sphere of cobalamin. These studies provide the first demonstration of the cofactor requirements for QueG.

How Formaldehyde Inhibits Hydrogen Evolution by [FeFe]-Hydrogenases: Determination by ¹³C ENDOR of Direct Fe-C Coordination and Order of Electron and Proton Transfers.

Formaldehyde (HCHO), a strong electrophile and a rapid and reversible inhibitor of hydrogen production by [FeFe]-hydrogenases, is used to identify the point in the catalytic cycle at which a highly reactive metal-hydrido species is formed. Investigations of the reaction of Chlamydomonas reinhardtii [FeFe]-hydrogenase with formaldehyde using pulsed-EPR techniques including electron-nuclear double resonance spectroscopy establish that formaldehyde binds close to the active site. Density functional theory calculations support an inhibited super-reduced state having a short Fe-(13)C bond in the 2Fe subsite. The adduct forms when HCHO is available to compete with H(+) transfer to a vacant, nucleophilic Fe site: had H(+) transfer already occurred, the reaction of HCHO with the Fe-hydrido species would lead to methanol, release of which is not detected. Instead, Fe-bound formaldehyde is a metal-hydrido mimic, a locked, inhibited form analogous to that in which two electrons and only one proton have transferred to the H-cluster. The results provide strong support for a mechanism in which the fastest pathway for H2 evolution involves two consecutive proton transfer steps to the H-cluster following transfer of a second electron to the active site.

Paramagnetic intermediates generated by radical S-adenosylmethionine (SAM) enzymes.

A [4Fe-4S](+) cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5'-deoxyadenosyl radical (5'-dA(•)). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5'-dA(•) is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, L-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5'-dA(•) to the product radical L-ß-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the L-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN(-) ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp(2)-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.

The HydG enzyme generates an Fe(CO)2(CN) synthon in assembly of the FeFe hydrogenase H-cluster.

Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.

A radical intermediate in tyrosine scission to the CO and CN- ligands of FeFe hydrogenase.

The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN(-) for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of (2)H, (13)C, and (15)N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cß bond forms a transient 4-oxidobenzyl (4OB(•)) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB(•) radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN(-), a key intermediate in the assembly of the 2Fe subunit of the H cluster.

Double electron-electron resonance probes Ca²âº-induced conformational changes and dimerization of recoverin.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca²âº-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca²âº-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron-electron resonance (DEER) experiments on recoverin that probe Ca²âº-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca²âº-free recoverin and 3.7 ± 0.1 nm for Ca²âº-bound recoverin. An additional DEER distance (5-6 nm) observed for Ca²âº-bound recoverin may represent an intermolecular distance between C39 and N120. ¹5N NMR relaxation analysis and CW-EPR experiments both confirm that Ca²âº-bound recoverin forms a dimer at protein concentrations above 100 µM, whereas Ca²âº-free recoverin is monomeric. We propose that Ca²âº-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca²âº-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca²âº-induced dimerization is functionally important but not well understood.

9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli.

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5'-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the µ-sulfide of the nearby [2Fe-2S](2+) cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe-2S](+) cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe-2S](+) cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe-2S](+) cluster.