Mechanisms Associated with Superoxide Radical Scavenging Reactions Involving Phenolic Compounds Deduced Based on the Correlation between Oxidation Peak Potentials and Second-Order Rate Constants Determined Using Flow-Injection Spin-Trapping EPR Methods.

Flow-injection spin-trapping electron paramagnetic resonance (FI-EPR) methods that involve the use of 5,5-dimethyl-pyrroline-N-oxide (DMPO) as a spin-trapping reagent have been developed for the kinetic study of the O2•- radical scavenging reactions occurring in the presence of various plant-derived and synthetic phenolic antioxidants (Aox), such as flavonoid, pyrogallol, catechol, hydroquinone, resorcinol, and phenol derivatives in aqueous media (pH 7.4 at 25 °C). The systematically estimated second-order rate constants (ks) of these phenolic compounds span a wide range (from 4.5 × 10 to 1.0 × 106 M-1 s-1). The semilogarithm plots presenting the relationship between ks values and oxidation peak potential (Ep) values of phenolic Aox are divided into three groups (A1, A2, and B). The ks-Ep plots of phenolic Aox bearing two or three OH moieties, such as pyrogallol, catechol, and hydroquinone derivatives, belonged to Groups A1 and A2. These molecules are potent O2•- radical scavengers with ks values above 3.8 × 104 (M-1 s-1). The ks-Ep plots of all phenol and resorcinol derivatives, and a few catechol and hydroquinone derivatives containing carboxyl groups adjacent to the OH groups, were categorized into the group poor scavengers (ks < 1.6 × 103 M-1 s-1). The ks values of each group correlated negatively with Ep values, supporting the hypothesis that the O2•- radical scavenging reaction proceeds via one-electron and two-proton processes. The processes were accompanied by the production of hydrogen peroxide at pH 7.4. Furthermore, the correlation between the plots of ks and the OH proton dissociation constant (pKa•) of the intermediate aroxyl radicals (ks-pKa• plots) revealed that the second proton transfer process could potentially be the rate-determining step of the O2•- radical scavenging reaction of phenolic compounds. The ks-Ep plots provide practical information to predict the O2•- radical scavenging activity of plant-derived phenolic compounds based on those molecular structures.

Revealing the atomic and electronic mechanism of human manganese superoxide dismutase product inhibition.

Human manganese superoxide dismutase (MnSOD) is a crucial oxidoreductase that maintains the vitality of mitochondria by converting superoxide (O2●-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). Human MnSOD has evolved to be highly product inhibited to limit the formation of H2O2, a freely diffusible oxidant and signaling molecule. The product-inhibited complex is thought to be composed of a peroxide (O22-) or hydroperoxide (HO2-) species bound to Mn ion and formed from an unknown PCET mechanism. PCET mechanisms of proteins are typically not known due to difficulties in detecting the protonation states of specific residues that coincide with the electronic state of the redox center. To shed light on the mechanism, we combine neutron diffraction and X-ray absorption spectroscopy of the product-bound, trivalent, and divalent states of the enzyme to reveal the positions of all the atoms, including hydrogen, and the electronic configuration of the metal ion. The data identifies the product-inhibited complex, and a PCET mechanism of inhibition is constructed.

Environmental implications of superoxide radicals: From natural processes to engineering applications.

The roles of superoxide radical (O2•-) in the domains of physiological, physical, and material chemistry are becoming increasingly recognized. Although extensive efforts have been directed to understand O2•- functions in diverse aquatic systems, there is a lack of systematic and in-depth review for its kinetics and mechanisms in various environmental scenarios. This review aims to bridge this gap through discussion of O2•- generation pathways under both natural and controlled conditions. The merits and limitations of the generation and detection methods under various conditions are compared, with emphasis on different approaches for the determination of O2•--triggered reaction kinetics. We summarize the reaction rate constants of O2•- with organic contaminants covering a wide diversity of structures and reactivity. The comparison indicates that O2•- exhibits weak reactivity with most contaminants and lacks selectivity towards compounds with different functional groups, except with quinones which exhibit higher reactivity compared to non-quinones. Further, the reaction mechanisms, namely single electron transfer, nucleophilic substitution, hydrogen atom abstraction, and radical-adduct formation, are critically evaluated. Various environmental implications of O2•- are highlighted including maintenance of biogeochemical iron cycle, synthesis of nanoparticles for antibacterial purposes, desorption of contaminants from heterogeneous interfaces, and synergetic degradation of contaminants.

Moderate elimination of mycotoxins in vegetable oil triggered by superoxide anion and singlet oxygen.

Establishing a moderate elimination strategy for mycotoxins with the maintained food nutrition is significant to food safety. Herein, the Au-NPs decorated defective Bi2WO6 (Au-BWO-OV) with modulated ROS generation was successfully synthesized, integrating the merits of defect-engineering and Au-NPs induced LSPR-effect. The Au-BWO-OV exhibited modified photoelectrochemical property and O2-adsorption capacity, supporting the selective generation of •O2- and 1O2 with moderate oxidizing ability. As a result, >90% of AFB1 and ZEN were eliminated within 100 and 50 min, along with the maintained nutrition in vegetable oil. Moreover, the reasonable degradation mechanism triggered by •O2- and 1O2 was proposed based on the trapping experiments, DFT calculations and LC-MS analysis for intermediate products, including the steps of hydrolysis, oxidative dissociation, cis-trans isomerization, and dehydroxylation. This work not only paved the way for balancing the contradiction between detoxification and nutrient retention, but also casted new insights into the ROS-mediated degradation mechanism.

Development of a nanozyme-based electrochemical catalyst for real-time biomarker sensing of superoxide and nitric oxide anions released from living cells and exogenous donors.

Developing quantitative biosensors of superoxide (O2•-) and nitric oxide (NO) anion is crucial for pathological research. As of today, the main challenge for electrochemical detection is to develop high-selectivity nano-mimetic materials to replace natural enzymes. In this study, the dendritic-like morphological structure of silver organic framework (Ag-MOF) was successfully synthesized via a solvothermal strategy. Owing to the introduction of polymeric composites results in improved electrical conductivity and catalytic activity, which promotes mass transfer and leads to faster electron efficiency. For monitoring the electrochemical signals of O2•- and NO, the Ag-MOF electrode substrate was produced by drop-coating, and composites were designed by cyclic voltammetric potential cycles. The designed electrode substrates demonstrate high sensitivity, wide linear concentrations of 1 nM-1000 µM and 1 nM-850 µM, and low detection limits of 0.27 nM and 0.34 nM (S/N = 3) against O2•- and NO. Aside from that, the sensor successfully monitored the cellular release of O2•-, and NO from HepG2 and RAW 264.7 living cells and has the potential to monitor exogenous NO release from donors of Diethylamine (DEA)-NONOate and sodium nitroprusside (SNP). Additionally, the developed system was applied to the analysis of O2•- and NO in real biological fluid samples, and the results were good satisfactory (94.10-99.57 ± 1.23%). The designed system provides a novel approach to obtaining a good electrochemical biosensor platform that is highly selective, stable, and flexible. Finally, the proposed method provides a quantitative way to follow the dynamic changes in O2•- and NO in biological systems.

An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons.

Enzymes are potent catalysts that increase biochemical reaction rates by several orders of magnitude. Flavoproteins are a class of enzymes whose classification relies on their ability to react with molecular oxygen (O2) during catalysis using ionizable active site residues. Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) is a flavoprotein that oxidizes D-arginine for P. aeruginosa survival and biofilm formation. The crystal structure of PaDADH reveals the interaction of the glutamate 246 (E246) side chain with the substrate and at least three other active site residues, establishing a hydrogen bond network in the active site. Additionally, E246 likely ionizes to facilitate substrate binding during PaDADH catalysis. This study aimed to investigate how replacing the E246 residue with leucine affects PaDADH catalysis and its ability to react with O2 using steady-state kinetics coupled with pH profile studies. The data reveal a gain of O2 reactivity in the E246L variant, resulting in a reduced flavin semiquinone species and superoxide (O2•-) during substrate oxidation. The O2•- reacts with active site protons, resulting in an observed nonstoichiometric slope of 1.5 in the enzyme's log (kcat/Km) pH profile with D-arginine. Adding superoxide dismutase results in an observed correction of the slope to 1.0. This study demonstrates how O2•- can alter the slopes of limbs in the pH profiles of flavin-dependent enzymes and serves as a model for correcting nonstoichiometric slopes in elucidating reaction mechanisms of flavoproteins.

Superoxide dismutase nanozymes: current status and future perspectives on brain disease treatment and diagnosis.

Superoxide dismutase (SOD) is an important metalloenzyme that catalyzes the dismutation of superoxide radicals (O2˙-) into hydrogen peroxide (H2O2) and oxygen (O2). However, the clinical application of SOD is severely limited due to its structural instability and high cost. Compared with natural enzymes, nanomaterials with enzyme-like activity, nanoenzymes, are more stable, economical and easy to modify and their activity can be adjusted. Certain nanozymes that exhibit SOD-like activity have been created and shown to help prevent illnesses brought about by oxidative stress. These SOD-like nanozymes offer an important solution to the problems associated with the clinical application of SOD. In this review, we briefly introduce neurodegenerative diseases, present the research progress of SOD-like nanoenzymes in the diagnosis and treatment of brain diseases, review their mechanism of action in the treatment and diagnosis of brain diseases, and discuss the shortcomings of the current research with a view to providing a reference for future research. We expect more highly active SOD-like nanoenzymes to be developed with a wide range of applications in the diagnosis and treatment of brain diseases.

Role of biochar in superoxide-dominated dye degradation in catalyst-activated peroxymonosulphate process.

In recent times, the application of biochar (BC) as an upcoming catalyst for the elimination of recalcitrant pollutants has been widely explored. Here, an iron loaded bamboo biochar activated peroxymonosulphate (PMS) process was tested for removing Congo red (CR) dye from water medium. The catalyst was synthesized using a green synthesis method using neem extracts and characterized using SEM, FTIR, and XRD. The effects of various operating parameters, including solution pH, catalyst dosage, and pollutant dosage, on dye degradation efficiency were examined. The results showed that at the optimized conditions of 300 mg L-1 PMS concentration, 200 mg L-1 catalyst dosage, and pH 6, about 89.7% of CR dye (initial concentration 10 ppm) was removed at 60 min of operation. Scavenging experiments revealed the significant contribution of O2•-, •OH, and 1O2 for dye degradation, with a major contribution of O2•-. The activation of PMS was mainly done by biochar rather than iron (loaded on biochar). The catalyst was highly active even after four cycles.

Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells.

Detection of superoxide anion (O2·-) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2·-. The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10-9 mol cm-2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+) catalyzes the dismutation of O2·- to O2 and H2O2, and H2O2 is then electrochemically oxidized to generate amperometric signals for determination of O2·-. The sensor demonstrates outstanding detection performance for O2·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM-1.34 µM and 1.34 µM-42.97 µM) with corresponding sensitivity of 0.479 and 0.052 µA µM-1cm-2, respectively. Additionally, the calculated apparent Michaelis constant (Kmapp) of 1.804 µM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.

Enhanced degradation of Direct Red 80 dye via Fenton-like process mediated by cobalt ferrite: generated superoxide radicals and singlet oxygen.

Azo dyes, widely used in the textile industry, contribute to effluents with significant organic content. Therefore, the aim of this work was to synthesize cobalt ferrite (CoFe2O4) using the combustion method and assess its efficacy in degrading the azo dye Direct Red 80 (DR80). TEM showed a spherical structure with an average size of 33 ± 12 nm. Selected area electron diffraction and XRD confirmed the presence of characteristic crystalline planes specific to CoFe2O4. The amount of Co and Fe metals were determined by ICP-OES, indicating an n(Fe)/n(Co) ratio of 2.02. FTIR exhibited distinct bands corresponding to Co-O (455 cm-1) and Fe-O (523 cm-1) bonds. Raman spectroscopy detected peaks associated with octahedral and tetrahedral sites. For the first time, the material was applied to degrade DR80 in an aqueous system, with the addition of persulfate. Consistently, within 60 min, these trials achieved nearly 100% removal of DR80, even after the material had undergone five cycles of reuse. The pseudo-second-order model was found to be the most fitting model for the experimental data (k2 = 0.07007 L mg-1 min-1). The results strongly suggest that degradation primarily occurred via superoxide radicals and singlet oxygen. Furthermore, the presence of UV light considerably accelerated the degradation process (k2 = 1.54093 L mg-1 min-1). The material was applied in a synthetic effluent containing various ions, and its performance consistently approached 100% in the photo-Fenton system. Finally, two degradation byproducts were identified through HPLC-MS/MS analysis.

Class Ib Ribonucleotide Reductases: Activation of a Peroxido-MnIIMnIII to Generate a Reactive Oxo-MnIIIMnIV Oxidant.

In the postulated catalytic cycle of class Ib Mn2 ribonucleotide reductases (RNRs), a MnII2 core is suggested to react with superoxide (O2·-) to generate peroxido-MnIIMnIII and oxo-MnIIIMnIV entities prior to proton-coupled electron transfer (PCET) oxidation of tyrosine. There is limited experimental support for this mechanism. We demonstrate that [MnII2(BPMP)(OAc)2](ClO4) (1, HBPMP = 2,6-bis[(bis(2 pyridylmethyl)amino)methyl]-4-methylphenol) was converted to peroxido-MnIIMnIII (2) in the presence of superoxide anion that converted to (µ-O)(µ-OH)MnIIIMnIV (3) via the addition of an H+-donor (p-TsOH) or (µ-O)2MnIIIMnIV (4) upon warming to room temperature. The physical properties of 3 and 4 were probed using UV-vis, EPR, X-ray absorption, and IR spectroscopies and mass spectrometry. Compounds 3 and 4 were capable of phenol oxidation to yield a phenoxyl radical via a concerted PCET oxidation, supporting the proposed mechanism of tyrosyl radical cofactor generation in RNRs. The synthetic models demonstrate that the postulated O2/Mn2/tyrosine activation mechanism in class Ib Mn2 RNRs is plausible and provides spectral insights into intermediates currently elusive in the native enzyme.

Preparation, characterization, antioxidant and antifungal activities of benzoic acid compounds grafted onto chitosan.

The current study created three novel chitosan derivatives named BACS, PIBACS, and MHBACS by grafting benzoic acid (BA), p-isopropyl benzoic acid (PIBA), and m-hydroxybenzoic acid (MHBA) onto chitosan (CS). The structures of the derivatives were investigated using infrared spectroscopy (FT-IR) and nuclear magnetic resonance (13C NMR). The derivatives were discovered to be 45.06 %-60.49 % substituted using elemental analysis (EA). Based on the findings of in vitro antioxidant experiments (hydroxyl radical scavenging activity, superoxide anion radical scavenging activity, and DPPH radical scavenging activity), all of the derivatives had a higher hydroxyl radical scavenging activity than the chitosan raw material. MHBACS scavenged (31.02 ± 0.90)% of hydroxyl radicals at 0.5 mg/mL, 28.69 % more than chitosan raw. The derivatives scavenged more superoxide anion radicals than the chitosan feedstock at a particular concentration. For instance, at a test dose of 0.2 mg/mL, the scavenging rate of MHBACS on superoxide anion radicals was 7.75 % greater than that of chitosan raw materials. DPPH radical scavenging activity, on the other hand, was not as competent as chitosan feedstock. The growth rate approach was used to assess the potential of the three derivatives to inhibit the development of four phytopathogenic fungi. Chitosan derivatives have better antifungal efficacy than chitosan raw materials. PIBACS, MHBACS, BACS, and Wuyiencin inhibited Phytophthora capsici by (98.03 ± 1.95)%, (81.73 ± 1.63)%, (66.38 ± 1.81)%, and (93.01 ± 2.69)%, respectively, at 1.0 mg/mL. PIBACS had a higher inhibitory impact on Phytophthora capsici than the positive control. Based on the evidence presented above, it is reasonable to conclude that the addition of benzoic acid molecules increased the antioxidant and antifungal capabilities of chitosan.

NIR-II Emissive Superoxide Radical Photogenerator for Photothermal/Photodynamic Therapy against Hypoxic Tumor.

Due to the "Achilles' heels" of hypoxia, complicated location in solid tumor, small molecular photosensitizers with second near-infrared window (NIR-II) fluorescence, type-I photodynamic therapy (PDT), and photothermal therapy (PTT) have attracted great attention. However, these photosensitizers are still few but yet challenging. Herein, an "all in one" NIR-II acceptor-donor-acceptor fused-ring photosensitizer, Y6-Th, is presented for the in-depth diagnosis and efficient treatment of cancer. Benefiting from the strong intramolecular charge transfer, promoted highly efficient intersystem crossing, largely p-conjugated fused-ring structure, and reduced planarity, the fabricated nanoparticles (Y6-Th nanoparticles) can emit NIR-II fluorescence with the peak located at 1020 nm, exclusively generate O2•- for type-I PDT, and display excellent PTT performance under an 808 nm laser stimulation. These characteristics make Y6-Th a distinguished NIR-wavelength-triggered phototheranostic agent, which can effectively therapy the hypoxic tumor using NIR-II-fluorescence-guided type-I PDT/PTT. This work provides a valuable guideline for fabricating high-performing NIR-II emissive superoxide radical photogenerators.

Revealing the generation of reactive oxygen species in hydrochar and pyrochar: Insight into rational regulation of free radicals and catalytic mechanism.

The removal of organic pollutants by biochar has been extensively studied. However, the differences in the removal mechanisms of contaminants by biochar obtained from different preparation techniques have not been thoroughly elucidated. In this study, the catalytic performances of hydrochar (HC) and pyrochar (PC) were compared in the dark and light. Owing to more persistent free radicals (PFRs), greater defects and stronger charge transfer ability on the surface, PC could produce a certain concentration of superoxide radicals (•O2-) even in the dark, making its degradation efficiency for benzoic acid (BA) 11% higher than that of HC. On the contrary, when the light was turned on, HC rather than PC can generate a higher amount of hydroxyl radical (•OH), resulting in an 11% higher degradation efficiency of BA compared to PC. The improvement of catalytic performance in HC originated from its oxygen-containing functional groups (OFGs), which was beneficial for its effective production of singlet oxygen (1O2) and ·OH under light exposure. For PC, its photocatalytic activity depended mainly on the formation of 1O2 induced by the triplet of DOM (dissolved organic matter), but the lack of oxidative ·OH in its system leads to a lower degradation efficiency than that of HC. To prove the universal applicability of this rule for biochar materials, HC and PC materials obtained from soybean residue were also prepared for degrading BA. This work is devoted to an in-depth exploration of the catalytic activation mechanism of biochar obtained by different technological methods, and can create conditions for the generation of more dominant reactive oxygen species (ROS) on biochar, thus providing the guidance for environmental remediation.

Preparation and Antioxidant Activity of New Carboxymethyl Chitosan Derivatives Bearing Quinoline Groups.

A total of 16 novel carboxymethyl chitosan derivatives bearing quinoline groups in four classes were prepared by different synthetic methods. Their chemical structures were confirmed by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and elemental analysis. The antioxidant experiment results in vitro (including DPPH radical scavenging ability, superoxide anion radical scavenging ability, hydroxyl radical scavenging ability, and ferric reducing antioxidant power) demonstrated that adding quinoline groups to chitosan (CS) and carboxymethyl chitosan (CMCS) enhanced the radical scavenging ability of CS and CMCS. Among them, both N, O-CMCS derivatives and N-TM-O-CMCS derivatives showed DPPH radical scavenging over 70%. In addition, their scavenging of superoxide anion radicals reached more than 90% at the maximum tested concentration of 1.6 mg/mL. Moreover, the cytotoxicity assay was carried out on L929 cells by the MTT method, and the results indicated that all derivatives showed no cytotoxicity (cell viability > 75%) except O-CMCS derivative 1a, which showed low cytotoxicity at 1000 µg/mL (cell viability 50.77 ± 4.67%). In conclusion, the carboxymethyl chitosan derivatives bearing quinoline groups showed remarkable antioxidant ability and weak cytotoxicity, highlighting their potential use in food and medical applications.

Separation and antioxidant activities of new acetylated EGCG compounds.

Acetylation could improve the bioavailability of (-)-Epigallocatechin-3-Gallate (EGCG), but the relationship of substitution degree and antioxidant capacity of acetylated EGCG was unclear. The acetylated EGCG products were separated by preparation high performance liquid chromatography (HPLC). Two mono substituted acetylated EGCG, three substituted acetylated EGCG (T-AcE), eight substituted acetylated EGCG (E-AcE) and (-)-Epigallocatechin gallate (EGCG) were isolated. The 7-acetyl-EGCG (S7-ACEGCG) and 7-acetyl-EGCG (T-AcE) were identified for the first time. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of EGCG decreased significantly after acetylation modification. The more EGCG acetylation modification sites, the lower the total antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities. The antioxidant capacity, superoxide anion radical scavenging capacities, and hydroxyl radical scavenging capacities of 5-acetyl-EGCG (S5-ACE) were higher than 7-acetyl-EGCG (S7-AcE). Combining all the results in this and previous studies, acetylation modification is not conducive to the performance of EGCG antioxidant capacity.

New Perylene-Based Chemiluminescent Polymer Nanoparticles for Highly Selective Detection of the Superoxide Anion In Vivo.

The superoxide anion (O2•-) is one of the primary reactive oxygen species in biological systems. Developing a determination system for O2•- in vivo has attracted much attention thanks to its complex biological function. Herein, we proposed a new perylene-based chemiluminescence (CL) probe, the SH-PDI polymer, which was capable of generating strong CL signals with O2•- in comparison with other ROS. The CL mechanism involved was proposed to be a kind of oxidation reaction induced by the breakage of the S-S and S-H bonds into sulfoxide bonds by O2•-. Subsequently, a nanoprecipitation method was introduced, using cumene-terminated poly(styrene-co-maleic anhydride) as the amphiphilic agent, to obtain water-soluble nanoparticles, SPPS NPs, which exhibited not only stronger CL intensity but also higher selectivity toward O2•- than the SH-PDI polymer. Moreover, the CL wavelength of the SPPS-O2•- system was found to be located at 580 and 710 nm, which was conducive to CL imaging. By virtue of these advantages, SPPS NPs were utilized to evaluate the O2•- level in vitro in the range of 0.25-60 µM at pH 7.0, with a detection limit of 8.2 × 10-8 M (S/N = 3). Moreover, SPPS NPs were also capable of imaging O2•- in an LPS-induced acute inflammation mice model and drug-induced acute kidney injury (AKI).

Long-Time Oxygen and Superoxide Localization in Arabidopsis thaliana Cryptochrome.

Cryptochromes are proteins that are highly conserved across species and in many instances bind the flavin adenine dinucleotide (FAD) cofactor within their photolyase-homology region (PHR) domain. The FAD cofactor has multiple redox states that help catalyze reactions, and absorbs photons at about 450 nm, a feature linked to the light-related functions of cryptochrome proteins. Reactive oxygen species (ROS) are produced from redox reactions involving molecular oxygen and are involved in a myriad of biological processes. Superoxide O2•- is an exemplary ROS that may be formed through electron transfer from FAD to O2, generating an electron radical pair. Although the formation of a superoxide-FAD radical pair has been speculated, it is still unclear if the required process steps could be realized in cryptochrome. Here, we present results from molecular dynamics (MD) simulations of oxygen interacting with the PHR domain of Arabidopsis thaliana cryptochrome 1 (AtCRY1). Using MD simulation trajectories, oxygen binding locations are characterized through both the O2-FAD intermolecular distance and the local protein environment. Oxygen unbinding times are characterized through replica simulations of the bound oxygen. Simulations reveal that oxygen molecules can localize at certain sites within the cryptochrome protein for tens of nanoseconds, and superoxide molecules can localize for significantly longer. This relatively long-duration molecule binding suggests the possibility of an electron-transfer reaction leading to superoxide formation. Estimates of electron-transfer rates using the Marcus theory are performed for the identified potential binding sites. Molecular oxygen binding results are compared with recent results demonstrating long-time oxygen binding within the electron-transfer flavoprotein (ETF), another FAD binding protein.

Ligand-Copper(I) Primary O2-Adducts: Design, Characterization, and Biological Significance of Cupric-Superoxides.

In this Account, we overview and highlight synthetic bioinorganic chemistry focused on initial adducts formed from the reaction of reduced ligand-copper(I) coordination complexes with molecular oxygen, reactions that produce ligand-CuII(O2•-) complexes (O2•- ≡ superoxide anion). We provide mostly a historical perspective, starting in the Karlin research group in the 1980s, emphasizing the ligand design and ligand effects, structure, and spectroscopy of these O2 adducts and subsequent further reactivity with substrates, including the interaction with a second ligand-CuI complex to form binuclear species. The Account emphasizes the approach, evolution, and results obtained in the Karlin group, a synthetic bioinorganic research program inspired by the state of knowledge and insights obtained on enzymes possessing copper ion active sites which process molecular oxygen. These constitute an important biochemistry for all levels/types of organisms, bacteria, fungi, insects, and mammals, including humans.Copper is earth abundant, and its redox properties in complexes allow for facile CuII/CuI interconversions. Simple salts or coordination complexes have been well known to serve as oxidants for the stoichiometric or catalytic oxidation or oxygenation (i.e., O-atom insertion) of organic substrates. Thus, copper dioxygen- or peroxide-centered synthetic bioinorganic studies provide strong relevance and potential application to synthesis or even the development of cathodic catalysts for dioxygen reduction to hydrogen peroxide or water, as in fuel cells. The Karlin group's focus however was primarily oriented toward bioinorganic chemistry with the goal to provide fundamental insights into the nature of copper-dioxygen adducts and further reduced and/or protonated derivatives, species likely occurring in enzyme turnover or related in one or more aspects of formation, structure, spectroscopic properties, and scope of reactivity toward organic/biochemical substrates.Prior to this time, the 1980s, O2 adducts of redox-active first-row transition-metal ions focused on iron, such as the porphyrinate-Fe centers occurring in the oxygen carrier proteins myoglobin and hemoglobin and that determined to occur in cytochrome P-450 monooxygenase turnover. Deoxy (i.e., reduced Fe(II)) heme proteins react with O2, giving FeIII-superoxo complexes (preferably referred to by traditional biochemists as ferrous-oxy species). And, it was in the 1970s that great strides were made by synthetic chemists in generating hemes capable of forming O2 adducts, their physiochemical characterization providing critical insights to enzyme (bio)chemistry and providing ideas and important goals leading to countless person years of future research.

Bioinspired Cu-based metal-organic framework mimicking SOD for superoxide anion sensing and scavenging.

Superoxide anion (O2•-) is typically produced in living cells and organisms, while excess O2•- may cause unexpected damage, so monitoring and scavenging the O2•- is of considerable significance to exploring physiological and pathological process. In this study, a Cu-based metal-organic framework (Cu-MOF) which comprise sequential Cu metal ion and conductive organic 2,5-dicarboxylic acid-3,4-ethylene dioxythiophene is synthesized to mimic superoxide dismutase (SOD), in which Cu is the essence of active site. On one hand, the Cu-MOF possesses excellent electrocatalytic activity to detect O2•- at -0.05 V, biased at which potential the electrode showed good linearity toward O2•- with detection limit of 0.283 µM and interference immunity for AA, DA, UA, 5-HT and H2O2. The Cu-MOF modified microelectrode was applied for measuring the O2•- released from living cells real time and monitoring O2•- generation in rat brain. On the other hand, this Cu-MOF has the catalytic activity to mimic the superoxide dismutase for scavenging O2•- in HeLa cells effectively. This work provides a methodology to design metal ion based enzyme mimetic for analyzing and scavenging O2•- in cells and in vivo.