Hydroxyl Radical Probing Research Articles

Oxidative and Reductive Pathways in Iron-Ethylenediaminetetraacetic Acid–Activated Persulfate Systems

The iron (II)-ethylenediaminetetraacetic acid (EDTA) and iron (III)-EDTA activation of persulfate was compared using reactant-specific probe compounds: the combined sulfate radical and hydroxyl radical probe anisole, the hydroxyl radical probe nitrobenzene, and the reductant/nucleophile probe hexachloroethane. Iron (II)-EDTA and iron (III)-EDTA were equally effective for activating persulfate decomposition and for generating reductants/nucleophiles at pH 5, while iron (III)-EDTA was a more effective activator than iron (II)-EDTA for generating oxidants. Use of the combined sulfate radical and hydroxyl radical scavenger isopropanol and the hydroxyl radical scavenger tert-butanol demonstrated that approximately 86 and 73% of the oxidation activity in iron (II)-EDTA and iron (III)-EDTA-activated persulfate systems, respectively, was attributable to hydroxyl radical activity. The generation of hydroxyl radical in iron-EDTA-activated persulfate systems at pH 5 was confirmed using electron spin resonance spectroscopy. The results of these pathway analyses, in conjunction with confirmation of the results using the common groundwater contaminant trichloroethylene (TCE), show that iron (II)-EDTA and iron (III)-EDTA-activated persulfate may be an effective system for the in situ remediation of contaminated groundwater.

DNA binding hydroxyl radical probes

The hydroxyl radical is the primary mediator of DNA damage by the indirect effect of ionizing radiation. It is a powerful oxidizing agent produced by the radiolysis of water and is responsible for a significant fraction of the DNA damage associated with ionizing radiation. There is therefore an interest in the development of sensitive assays for its detection. The hydroxylation of aromatic groups to produce fluorescent products has been used for this purpose. We have examined four different chromophores, which produce fluorescent products when hydroxylated. Of these, the coumarin system suffers from the fewest disadvantages. We have therefore examined its behavior when linked to a cationic peptide ligand designed to bind strongly to DNA.

Use of a Coumarin-Labeled Hexa-Arginine Peptide as a Fluorescent Hydroxyl Radical Probe in a Nanoparticulate Plasmid DNA Condensate

Coumarin derivatives have found application as probes for the hydroxyl radical because one of the products of the reaction between them is a highly fluorescent umbelliferone. We have examined the interaction in aqueous solution between a cationic coumarin-labeled hexa-arginine peptide ligand and plasmid DNA, and compared after gamma irradiation the yields of products derived from both of them. At low ionic strengths, the ligand binds very tightly to the plasmid. Compared with the structurally similar 4-methylumbelliferone (phenolic pK(a) = 7.8), the fluorescent product derived from gamma irradiation of the coumarin labeled cationic peptide is significantly more acidic (pK(a) = 6.1), making it a very convenient probe for solutions of pH in the physiological range. The yield of this product is generally in excellent agreement over a wide range of conditions with that of the single strand break product produced by the reaction of the hydroxyl radical with the plasmid. Thus coumarin-labeled peptide ligands offer promise as hydroxyl radical probes for locations in close proximity to DNA.

The TFIIF-Like Rpc37/53 Dimer Lies at the Center of a Protein Network To Connect TFIIIC, Bdp1, and the RNA Polymerase III Active Center

Eukaryotic RNA polymerase III (Pol III) relies on a transcription factor TFIIF-like Rpc37/53 subcomplex for promoter opening, elongation, termination, and reinitiation. By incorporating the photoreactive amino acid p-benzoyl-L-phenylalanine (BPA) into Rpc37, Rpc53, and the Rpc2 subunit of Pol III, we mapped protein-protein interactions, revealing the position of Rpc37/53 within the Pol III preinitiation complex (PIC). BPA photo-cross-linking was combined with site-directed hydroxyl radical probing to localize the Rpc37/53 dimerization module on the lobe/external 2 domains of Rpc2, in similarity to the binding of TFIIF on Pol II. N terminal to the dimerization domain, Rpc53 binds the Pol III-specific subunits Rpc82 and Rpc34, the Pol III stalk, and the assembly factor TFIIIC, essential for PIC formation. The C-terminal domain of Rpc37 interacts extensively with Rpc2 and Rpc34 and contains binding sites for initiation factor Bdp1. We also located the C-terminal domain of Rpc37 within the Pol III active center in the ternary elongation complex, where it likely functions in accurate termination. Our work explains how the Rpc37/53 dimer is anchored on the Pol III core and acts as a hub to integrate a protein network for initiation and termination.

RNA molecules with conserved catalytic cores but variable peripheries fold along unique energetically optimized pathways

Functional and kinetic constraints must be efficiently balanced during the folding process of all biopolymers. To understand how homologous RNA molecules with different global architectures fold into a common core structure we determined, under identical conditions, the folding mechanisms of three phylogenetically divergent group I intron ribozymes. These ribozymes share a conserved functional core defined by topologically equivalent tertiary motifs but differ in their primary sequence, size, and structural complexity. Time-resolved hydroxyl radical probing of the backbone solvent accessible surface and catalytic activity measurements integrated with structural-kinetic modeling reveal that each ribozyme adopts a unique strategy to attain the conserved functional fold. The folding rates are not dictated by the size or the overall structural complexity, but rather by the strength of the constituent tertiary motifs which, in turn, govern the structure, stability, and lifetime of the folding intermediates. A fundamental general principle of RNA folding emerges from this study: The dominant folding flux always proceeds through an optimally structured kinetic intermediate that has sufficient stability to act as a nucleating scaffold while retaining enough conformational freedom to avoid kinetic trapping. Our results also suggest a potential role of naturally selected peripheral A-minor interactions in balancing RNA structural stability with folding efficiency.

Efficiency and energy requirements for the transformation of organic micropollutants by ozone, O 3/H 2O 2 and UV/H 2O 2

The energy consumptions of conventional ozonation and the AOPs O 3/H 2O 2 and UV/H 2O 2 for transformation of organic micropollutants, namely atrazine (ATR), sulfamethoxazole (SMX) and N-nitrosodimethylamine (NDMA) were compared. Three lake waters and a wastewater were assessed. With p-chlorobenzoic acid (pCBA) as a hydroxyl radical ( •OH) probe compound, we experimentally determined the rate constants of organic matter of the selected waters for their reaction with •OH ( k OH,DOM), which varied from 2.0 × 10 4 to 3.5 × 10 4 L mgC −1 s −1. Based on these data we calculated •OH scavenging rates of the various water matrices, which were in the range 6.1–20 × 10 4 s −1. The varying scavenging rates influenced the required oxidant dose for the same degree of micropollutant transformation. In ozonation, for 90% pCBA transformation in the water with the lowest scavenging rate (lake Zürich water) the required O 3 dose was roughly 2.3 mg/L, and in the water with the highest scavenging rate (Dübendorf wastewater) it was 13.2 mg/L, corresponding to an energy consumption of 0.035 and 0.2 kWh/m 3, respectively. The use of O 3/H 2O 2 increased the rate of micropollutant transformation and reduced bromate formation by 70%, but the H 2O 2 production increased the energy requirements by 20–25%. UV/H 2O 2 efficiently oxidized all examined micropollutants but energy requirements were substantially higher (For 90% pCBA conversion in lake Zürich water, 0.17–0.75 kWh/m 3 were required, depending on the optical path length). Energy requirements between ozonation and UV/H 2O 2 were similar only in the case of NDMA, a compound that reacts slowly with ozone and •OH but is transformed efficiently by direct photolysis.

Hydroxyl radical involvement in the decomposition of hydrogen peroxide by ferrous and ferric-nitrilotriacetate complexes at neutral pH

The relative rates of degradation of three hydroxyl radical probe compounds (atrazine, fenuron and parachlorobenzoic acid (pCBA)) by Fe III/H 2O 2 (pH = 2.85), Fe IIINTA/H 2O 2 (neutral pH), Fe II/O 2, Fe IINTA/O 2, Fe II/H 2O 2 and Fe IINTA/H 2O 2 (neutral pH) have been investigated using the competitive kinetic method. Experiments were carried out in batch and in semi-batch reactors, in the dark, at 25 °C. The data showed that the three probe compounds could be degraded by all the systems studied, and in particular by Fe IINTA/H 2O 2 and Fe IIINTA/H 2O 2 at neutral pH. The relative rate constants of degradation of the three probe compounds obtained for all the systems tested were identical and equal to 1.45 ± 0.03 and 0.47 ± 0.02 for k Atrazine/ k pCBA and k Fenuron/ k pCBA, respectively. These values as well as the decrease of the rates of degradation of the probe compounds upon the addition of hydroxyl radical scavengers ( tert-butanol, bicarbonate ions) suggest that the degradation of atrazine, fenuron and pCBA by Fe IINTA/O 2, Fe IINTA/H 2O 2 and Fe IIINTA/H 2O 2 is initiated by hydroxyl radicals.

Study of electrochemical bleaching of p-nitrosodimethylaniline and its role as hydroxyl radical probe compound

In the present paper, research on the electrochemical bleaching of p-nitrosodimethylaniline (RNO) in different electrolyte systems is presented with special attention to the role of RNO as a selective hydroxyl radical probe compound. At a Ti/Pt90–Ir10 anode, RNO was found to be bleached in 0.050 M sodium sulphate electrolyte due to lattice active oxygen without hydroxyl radicals being intermediately present. In 0.050 M sodium chloride, the bleaching rate was greatly enhanced due to indirect bulk oxidation by active chlorine species, again without the presence of hydroxyl radicals in the oxidation mechanisms. Under galvanostatic electrolysis, a linear relationship was found between the concentration of added chloride to a supporting sodium sulphate electrolyte and the first order rate constant of the bleaching reaction, showing the importance of the indirect bulk chlorine bleaching in chloride electrolyte systems. In this fashion both the chemically bonded active oxygen and the chemical bulk oxidation by active chlorine species proved to be valid bleaching pathways of RNO that according to these findings cannot be regarded as a fully selective hydroxyl radical probe compound. In addition, the difference in the mechanisms of chloride electrolysis at Ti/Pt90–Ir10 and Si–BDD anodes was clearly demonstrated using t-BuOH as hydroxyl radical scavenger.

TRNA/mRNA Mimicry by tmRNA and SmpB inTrans-Translation

Since accurate translation from mRNA to protein is critical to survival, cells have developed translational quality control systems. Bacterial ribosomes stalled on truncated mRNA are rescued by a system involving tmRNA and SmpB referred to as trans-translation. Here, we review current understanding of the mechanism of trans-translation. Based on results obtained by using directed hydroxyl radical probing, we propose a new type of molecular mimicry during trans-translation. Besides such chemical approaches, biochemical and cryo-EM studies have revealed the structural and functional aspects of multiple stages of trans-translation. These intensive works provide a basis for studying the dynamics of tmRNA/SmpB in the ribosome.

Interdependencies govern multidomain architecture in ribosomal small subunit assembly

The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA, thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein (RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the absence of other r-proteins. However, the binding site of S8 at the junction of helices 25-26a, which is transcribed after helix 21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 5' terminus. Moreover, the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex interrelationship is observed between assembly events of independent domains and the incorporation of primary binding proteins during 30S subunit formation.

Feasibility of an electrochemically assisted Fenton method using Fe2 +/HOCl system as an advanced oxidation process

The feasibility of an electrochemically assisted Fenton treatment using a Fenton-type reaction of ferrous iron (Fe(2 + )) and hypochlorous acid (HOCl) is discussed in this research. The reactor used was composed of an undivided single cell with a ruthenium dioxide-coated titanium anode and a stainless steel cathode, in which Fe(2 + ) and HOCl were catalytically regenerated from ferric iron at the cathode and chloride ion at the anode, respectively. Although the reactor functioned well, the degradation rate of 1,4-dioxane as a hydroxyl radical probe decreased at the current density more than 6.92 mA cm(-2). The decrease in degradation rate was inferred to be caused by the vain consumption of hydroxyl radicals by excess HOCl and the deposition of ferric hydroxide on the cathode at relatively high current density. The current efficiency of 1,4-dioxane removal remained more than 90% at the current density less than 6.92 mA cm(-2) and the iron concentration not less than 1.0 mmol L(-1). Consequently, this technique is thought to be applicable to the treatment of wastewater containing high concentration of chloride ion such as landfill leachate, scrubber wastewater from incineration plants, etc.

The Osmolyte TMAO Stabilizes Native RNA Tertiary Structures in the Absence of Mg2+: Evidence for a Large Barrier to Folding from Phosphate Dehydration

The stabilization of RNA tertiary structures by ions is well known, but the neutral osmolyte trimethylamine oxide (TMAO) can also effectively stabilize RNA tertiary structure. To begin to understand the physical basis for the effects of TMAO on RNA, we have quantitated the TMAO-induced stabilization of five RNAs with known structures. So-called m values, the increment in unfolding free energy per molal of osmolyte at constant KCl activity, are ∼0 for a hairpin secondary structure and between 0.70 and 1.85 kcal mol−1m−1 for four RNA tertiary structures (30–86 nt). Further analysis of two RNAs by small-angle X-ray scattering and hydroxyl radical probing shows that TMAO reduces the radius of gyration of the unfolded ensemble to the same endpoint as seen in titration with Mg2+ and that the structures stabilized by TMAO and Mg2+ are indistinguishable. Remarkably, TMAO induces the native conformation of a Mg2+ ion chelation site formed in part by a buried phosphate, even though Mg2+ is absent. TMAO interacts weakly, if at all, with KCl, ruling out the possibility that TMAO stabilizes RNA indirectly by increasing salt activity. TMAO is, however, strongly excluded from the vicinity of dimethylphosphate (unfavorable interaction free energy, +211 cal mol−1m−1 for the potassium salt), an ion that mimics the RNA backbone phosphate. We suggest that formation of RNA tertiary structure is accompanied by substantial phosphate dehydration (loss of 66–173 water molecules in the RNA structures studied) and that TMAO works principally by reducing the energetic penalty associated with this dehydration. The strong parallels we find between the effects of TMAO and Mg2+ suggest that RNA sequence is more important than specific ion interactions in specifying the native structure.

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding

Tethered hydroxyl-radical probing has been used to determine the orientation of binding of polypyrimidine tract-binding protein (PTB) to the poliovirus type 1 (Mahoney) (PV-1(M)) internal ribosome entry site/segment (IRES)-the question of which RNA-binding domain (RBD) binds to which sites on the IRES. The results show that under conditions in which PTB strongly stimulates IRES activity, a single PTB is binding to the IRES, a finding which was confirmed by mass spectrometry of PTB/IRES complexes. RBDs1 and 2 interact with the basal part of the Domain V irregular stem loop, very close to the binding site of eIF4G, and RBDs3 and 4 interact with the single-stranded regions flanking Domain V. The binding of PTB is subtly altered in the presence of the central domain (p50) of eIF4G, and p50 binding is likewise modified if PTB is present. This suggests that PTB stimulates PV-1(M) IRES activity by inducing eIF4G to bind in the optimal position and orientation to promote internal ribosome entry, which, in PV-1(M), is at an AUG triplet 30 nt downstream of the base of Domain V.

Molecular mimicry of tRNA / mRNA during trans‐translation

In trans-translation, transfer-messanger RNA (tmRNA) rescues the stalled ribosome, which is caused by truncated or other problemtic mRNAs. When tmRNA enters the stalled ribosome, there is no codon-anticodon interaction, but with a protein factor SmpB. Here, we studied the binding site of SmpB on the ribosome by directed hydroxyl radical probing. It revealed that there are two SmpB binding sites on a ribosome, the A-site and P-site, suggesting that these binding sites reflect pre- and post-translocation states of SmpB during trans-translation. It is also suggested that the N-terminal region (1–133) and the C-terminal tail (134–160) of SmpB mimic the lower half of tRNA and mRNA, respectively. We also examined the role of the C-terminal tail of SmpB using an in vitro trans-translation system. Our data indicate that the residues of the C-terminal tail of SmpB are involved in the early stage of trans-translation. This work was supported by a grant-in-aid for Young Scientists (Start-up) from Japan Society for the Promotion of Science (JSPS) to D.K. (No. 21880009) and a Research Fellowship of Hirosaki University for D.K.

Role of the C-terminal tail of SmpB in the early stage of trans-translation

Trans-translation relieves a stalled translation on the bacterial ribosome by transfer-messenger RNA (tmRNA) with the help of SmpB, an essential cofactor of tmRNA. Here, we examined the role of the unstructured C-terminal tail of SmpB using an in vitro trans-translation system. It was found that truncation of the C-terminal tail or substitution of tryptophan residue at 147 in the middle of the C-terminal tail affected the activity in the early stage of trans-translation. Our investigations also revealed that the C-terminal tail is not required for the events until GTP is hydrolyzed by EF-Tu in complex with tmRNA-SmpB. A synthetic peptide corresponding to the C-terminal tail of SmpB inhibited peptidyl-transfer of alanyl-tmRNA and A-site binding of SmpB, but not GTP hydrolysis. These results suggest that the C-terminal tail has a role in the step of accommodation of alanyl-tmRNA-SmpB into the A-site. Directed hydroxyl radical probing indicated that tryptophan residue at 147 is located just downstream of the decoding center in the mRNA path when SmpB is in the A-site.

Persistent organic pollutants (POPs) degradation in natural waters using a V-UV/UV/TiO2 reactor

This paper presents the use of a V-UV/UV/TiO2 reactor (M300 water purifier®) for the removal of sulfamethoxazole and atrazine from natural water. The efficiency of the different processes (photolysis, Vacuum UV (V-UV) and photocatalysis) within the photoreactor was investigated using para-chlorobenzoic acid (pCBA) which is a good probe for hydroxyl radicals. The effect of pH, dissolved organic carbon (DOC) and total inorganic carbon (TIC) was also studied. V-UV was found to be the most efficient process for pCBA degradation within this unit. No photocatalytic degradation was observed. The water quality largely affected the efficiency of the system. Indeed, both DOC and TIC lowered the hydroxyl radical concentration in the system, but DOC did it to a larger extent. Atrazine and sulfamethoxazole, were successfully degraded, and as for pCBA, V-UV was the most efficient process. Sulfamethoxazole displayed a better removal by photocatalysis than atrazine and pCBA. In addition, there was a larger contribution of photolysis during the degradation of sulfamethoxazole than of atrazine. The UV254 nm absorbance of the water affected the photolysis of sulfamethoxazole which has a high molar extinction coefficient and quantum yield at 254 nm, while the DOC is mainly competing with the pollutants for the hydroxyl radicals.

Trans-translation by tmRNA and SmpB

tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNA's tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the interaction of SmpB with ribosome and tmRNA, we have performed directed hydroxyl radical probing. It revealed that there are two SmpB binding sites at the A-site and P-site, and their C-terminal regions reside along the mRNA path in the 30S subunit. From these results, we propose a new type of molecular mimicry, in which both tmRNA and SmpB mimic the structures and functions of tRNA and mRNA during trans-translation, addressing how tmRNA preferentially recognizes the stalled ribosome, and what substitutes for a codon-anticodon interaction.

Position of eukaryotic translation initiation factor eIF1A on the 40S ribosomal subunit mapped by directed hydroxyl radical probing

The universally conserved eukaryotic initiation factor (eIF), eIF1A, plays multiple roles throughout initiation: it stimulates eIF2/GTP/Met-tRNAiMet attachment to 40S ribosomal subunits, scanning, start codon selection and subunit joining. Its bacterial ortholog IF1 consists of an oligonucleotide/oligosaccharide-binding (OB) domain, whereas eIF1A additionally contains a helical subdomain, N-terminal tail (NTT) and C-terminal tail (CTT). The NTT and CTT both enhance ribosomal recruitment of eIF2/GTP/Met-tRNAiMet, but have opposite effects on the stringency of start codon selection: the CTT increases, whereas the NTT decreases it. Here, we determined the position of eIF1A on the 40S subunit by directed hydroxyl radical cleavage. eIF1A's OB domain binds in the A site, similar to IF1, whereas the helical subdomain contacts the head, forming a bridge over the mRNA channel. The NTT and CTT both thread under Met-tRNAiMet reaching into the P-site. The NTT threads closer to the mRNA channel. In the proposed model, the NTT does not clash with either mRNA or Met-tRNAiMet, consistent with its suggested role in promoting the ‘closed’ conformation of ribosomal complexes upon start codon recognition. In contrast, eIF1A-CTT appears to interfere with the P-site tRNA-head interaction in the ‘closed’ complex and is likely ejected from the P-site upon start codon recognition.

Polypyrimidine Tract Binding Protein Stabilizes the Encephalomyocarditis Virus IRES Structure via Binding Multiple Sites in a Unique Orientation

Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3' end, and RBD3 contacting the 5' end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES.

Gemin5-snRNA interaction reveals an RNA binding function for WD repeat domains

Gemin5 binds specifically to the small nuclear RNA (snRNA)-defining small nuclear ribonucleoprotein (snRNP) code sequence and is essential, together with other components of the survival of motor neurons (SMN) complex, for the biogenesis of snRNPs, the major constituents of spliceosomes. We show that this binding is mediated by Gemin5's WD repeat domain, a common domain not previously known to bind RNA independently. The entire WD repeat domain, comprising 13 WD motifs, is both necessary and sufficient for sequence-specific, high-affinity binding of Gemin5 to its RNA targets. Using an RNA-mediated hydroxyl radical probing method and mass spectrometry, we mapped a discrete region of the WD repeat domain that contacts snRNAs and demonstrated by mutagenesis that specific amino acids in this region are crucial for Gemin5-snRNA binding. The WD repeat domain is thus a previously undescribed RNA binding domain, and we suggest that the presence of WD repeats should be considered as predictive of potential function in RNA binding.