Catalytic Cofactor Research Articles

Regulation of Atp7a RNA contributes to differentiation-dependent Cu redistribution in skeletal muscle cells.

Cu (Cu) is essential for several biochemical pathways due to its role as a catalytic cofactor or allosteric regulator of enzymes. Its import and distribution are tightly controlled by transporters and metallochaperones and Cu homeostasis is maintained by balancing Cu uptake and export. Genetic diseases are caused by impaired Cu transporters CTR1, ATP7A, or ATP7B but little is known about the regulatory mechanisms by which these proteins meet the fluctuating demands of Cu in specific tissues. Cu is required for differentiation of skeletal myoblasts to myotubes. Here, we demonstrate that ATP7A is needed for myotube formation and that its increased abundance during differentiation is mediated by stabilization of Atp7a mRNA via the 3'untranslated region. Increased ATP7A levels during differentiation resulted in increased Cu delivery to lysyl oxidase, a secreted cuproenzyme that needed for myotube formation. These studies identify a previously unknown role for Cu in regulating muscle differentiation and have broad implications for understanding Cu-dependent differentiation in other tissues.

Expression and copper binding studies of a Plasmodium falciparum protein with Cox19 copper binding motifs

Copper can exist in an oxidized and a reduced form, which enables the metal to play essential roles as a catalytic co-factor in redox reactions in many organisms. Copper confers redox activity to the terminal electron transport chain cytochrome c oxidase protein. Cytochrome c oxidase in yeast obtains copper for the CuB site in the Cox1 subunit from Cox11 in association with Cox19. When copper is chelated in growth medium, Plasmodium falciparum parasite development in infected red blood cells is inhibited and excess copper is toxic for the parasite. The gene of a 26 kDa Plasmodium falciparum PfCox19 protein with two Cx9C Cox19 copper binding motifs, was cloned and expressed as a 66 kDa fusion protein with maltose binding protein and affinity purified (rMBP-PfCox19). rMBP-PfCox19 bound copper measured by: a bicinchoninic acid release assay; an in vivo bacterial host growth inhibition assay; ascorbate oxidation inhibition and differential scanning fluorimetry. The native protein was detected by antibodies raised against rMBP-PfCox19. PfCox19 binds copper and is predicted to associate with PfCox11 in the insertion of copper into the CuB site of Plasmodium cytochrome c oxidase. Characterisation of the proteins involved in Plasmodium spp. copper metabolism will help us understand the role of cytochrome c oxidase and this essential metal in Plasmodium homeostasis.

A ribozyme that uses lanthanides as cofactor.

To explore how an early, RNA-based life form could have functioned, in vitro selection experiments have been used to develop catalytic RNAs (ribozymes) with relevant functions. We previously identified ribozymes that use the prebiotically plausible energy source cyclic trimetaphosphate (cTmp) to convert their 5'-hydroxyl group to a 5'-triphosphate. While these ribozymes were developed in the presence of Mg2+, we tested here whether lanthanides could also serve as catalytic cofactors because lanthanides are ideal catalytic cations for this reaction. After an in vitro selection in the presence of Yb3+, several active sequences were isolated, and the most active RNA was analyzed in more detail. This ribozyme required lanthanides for activity, with highest activity at a 10:1 molar ratio of cTmp : Yb3+. Only the four heaviest lanthanides gave detectable signals, indicating a high sensitivity of ribozyme catalysis to the lanthanide ion radius. Potassium and Magnesium did not facilitate catalysis alone but they increased the lanthanide-mediated kOBS by at least 100-fold, with both K+ and Mg2+ modulating the ribozyme's secondary structure. Together, these findings show that RNA is able to use the unique properties of lanthanides as catalytic cofactor. The results are discussed in the context of early life forms.

Smartphone-Based Chemiluminescence Glucose Biosensor Employing a Peroxidase-Mimicking, Guanosine-Based Self-Assembled Hydrogel

Chemiluminescence is widely used for hydrogen peroxide detection, mainly exploiting the highly sensitive peroxidase-luminol-H2O2 system. Hydrogen peroxide plays an important role in several physiological and pathological processes and is produced by oxidases, thus providing a straightforward way to quantify these enzymes and their substrates. Recently, biomolecular self-assembled materials obtained by guanosine and its derivatives and displaying peroxidase enzyme-like catalytic activity have received great interest for hydrogen peroxide biosensing. These soft materials are highly biocompatible and can incorporate foreign substances while preserving a benign environment for biosensing events. In this work, a self-assembled guanosine-derived hydrogel containing a chemiluminescent reagent (luminol) and a catalytic cofactor (hemin) was used as a H2O2-responsive material displaying peroxidase-like activity. Once loaded with glucose oxidase, the hydrogel provided increased enzyme stability and catalytic activity even in alkaline and oxidizing conditions. By exploiting 3D printing technology, a smartphone-based portable chemiluminescence biosensor for glucose was developed. The biosensor allowed the accurate measurement of glucose in serum, including both hypo- and hyperglycemic samples, with a limit of detection of 120 µmol L−1. This approach could be applied for other oxidases, thus enabling the development of bioassays to quantify biomarkers of clinical interest at the point of care.

Protection of catalytic cofactors by polypeptides as a driver for the emergence of primordial enzymes.

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects protection of bound heme from common degradation processes and a resulting longer life time and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations.

Abstract 6255: Anti-proliferative effect of two copper complexes against medulloblastoma cells

Abstract Background and Purpose: Copper is a crucial structural component for many significant enzymes, as well as a key catalytic co-factor in redox processes. The flexible Cu(I/II) redox behavior facilitates the formation of copper complexes that are more potent and less toxic. The anticancer activity of several agents can be enhanced by forming copper complexes. The purpose of this study is to evaluate the anti-proliferative effects of two copper (II) complexes, copper-tolfenamic acid (Cu-TA) and copper thiosemicarbazone (Cu-acetylethTSC or CuTSC) against medulloblastoma (MB). MB is a cancer of the cerebellum which is associated with frequent relapse and drug resistance with the current treatments. It necessitates the development of alternative strategies. The anti-cancer activity of Cu-TA was evaluated in laboratory testing in some cancer models, but it has not been studied in MB. CuTSC against colorectal cancer, leukemia and breast cancer has been studied but it has not in MB. Methods: TA and TSC were complexed with Cu and Cu-TA and CuTSC complexes were characterized through colorimetric/mass spectrometric analyses. Cancer cells and cardiomyocytes were cultured using standard protocols and cell viability was measured using Cell Titer-Glo kit (Promega). Protein expression was determined by Western blot analysis. Experiments/Results: CuTA and CuTSC complexes showed proper characterization and stability during the testing period. Cardiomyocytes (H9C2) and human cancer cell lines representing breast, Ewing sarcoma and MB were screened for anti-proliferative activity of these two Cu complexes. While, cell viability was inhibited in all cancer cell lines, MB cell lines (DAOY and D283) showed higher efficacy while H9C2 cell viability was not affected. To understand the underlying mechanism of action, the effect of Cu-TA was assessed on the expression of c-PARP, Specificity protein 1 (Sp1) and an antiapoptotic protein, survivin. Western blot results revealed a clear decrease in the expression of both Sp1 and survivin and upregulation of c-PARP. Discussion/Conclusion: Both Cu-TA and CuTSC treatments result in a dose-time dependent anti-proliferative activity against cancer cells but not affecting non-malignant cardiomyocyte cells. Cu-TA can inhibit Sp1 and survivin and increase PARP cleavage suggesting the effect on Sp1-regulated oncogenes and an induction of apoptotic pathways. The studies to establish crossing of Blood Brain Barrier and further understand the underling mechanisms of these two Cu complexes in single and combination treatment alongside chemotherapeutic agents are currently under investigation. Citation Format: Hope Korshie Fiadjoe, Christoffer Lambring, Umesh T. Sankpal, Duaa Alajroush, Alvin A. Holder, Riyaz Basha. Anti-proliferative effect of two copper complexes against medulloblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6255.

Connecting the geometric and electronic structures of the nitrogenase iron-molybdenum cofactor through site-selective 57Fe labelling.

Understanding the chemical bonding in the catalytic cofactor of the Mo nitrogenase (FeMo-co) is foundational for building a mechanistic picture of biological nitrogen fixation. A persistent obstacle towards this goal has been that the 57Fe-based spectroscopic data-although rich with information-combines responses from all seven Fe sites, and it has therefore not been possible to map individual spectroscopic responses to specific sites in the three-dimensional structure. Here we have addressed this challenge by incorporating 57Fe into a single site of FeMo-co. Spectroscopic analysis of the resting state informed on the local electronic structure of the terminal Fe1 site, including its oxidation state and spin orientation, and, in turn, on the spin-coupling scheme for the entire cluster. The oxidized resting state and the first intermediate in nitrogen fixation were also characterized, and comparisons with the resting state provided molecular-level insights into the redox chemistry of FeMo-co.

The molecular origin of various DNA-repair quantum yield in photolyases.

Photolyases is a blue-light-activated flavoenzyme that use FADH− as the catalytic cofactor to repair UV-induced DNA lesions including cyclobutene pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4 PP). Different classes of CPD photolyases show diverse genetic sequences but have similar folding structure. Class I CPD photolyases from bacteria have much higher repair quantum yields than class II CPD photolyases from plants. The difference mainly comes from a bifurcation in initial electron transfer: class I CPD photolyases mainly use a tunnelling pathway (electron from the isoalloxazine ring directly tunnels to the CPD substrate), while class II CPD photolyases mainly use a two-step hopping pathway (electron first jumps to the adenine and then jumps to the CPD substrate). In this study, we switched two key residues in the active sites of class I (N341, R342 in EcPL from Escherichia coli) and class II photolyases (G381, F382 in AtPL from Arabidopsis thaliana). Steady-state repair quantum yield measurements show dramatic lower repair quantum yields in EcPL mutants compared to EcPL while a different trend is observed in AtPL and its mutants. To reveal how these residues affect the repair reaction, ultrafast laser spectroscopy was used to determine the reaction rates of seven electron-transfer reactions in 10 elementary steps. We found that the repair quantum yields can be tuned by favoring either electron tunnelling or hopping channel, and thus adjusting the quantum yield of electron injection to the CPD substrate. Photolyases evolved to have high affinity and specificity towards DNA lesion with reasonably repair quantum yield. Solely increasing repair quantum yield by single mutation may disrupt the delicate balance between substrate binding and CPD repair.

Reduced Flavin in Aqueous Solution Is Nonfluorescent.

Flavins are blue-light-absorbing chromophores with rich redox activity. Biologically, the most important are riboflavin (vitamin B2), flavin mononucleotide, and flavin adenine dinucleotide, the latter two of which are catalytic cofactors in enzymes. Flavins pivot between oxidized, one electron-, and two electron-reduced forms in different protonation states, depending on enzymatic requirements. Some flavoenzymes use light as a reagent for chemical bond formation, photoinduced electron transfer, or conformational changes required for light-sensitive signaling. Therefore, the photochemistry and photophysics of flavins have received wide attention. Fluorescence from oxidized flavin is often used to detect and track changes in flavin oxidation states. However, there have been conflicting reports over the past 45 years as to whether reduced flavin in solution has detectable fluorescence. Here, using single photon counting emission spectroscopy with rigorous sample preparation, we show definitively that reduced flavins are essentially nonfluorescent, having a quantum yield more than three orders of magnitude lower than oxidized flavin. This result will force a re-evaluation of experiments and models that assumed otherwise.

Conformational and mechanical stability of the isolated large subunit of membrane-bound [NiFe]-hydrogenase from Cupriavidus necator.

Comprising at least a bipartite architecture, the large subunit of [NiFe]-hydrogenase harbors the catalytic nickel-iron site while the small subunit houses an array of electron-transferring Fe-S clusters. Recently, some [NiFe]-hydrogenase large subunits have been isolated showing an intact and redox active catalytic cofactor. In this computational study we have investigated one of these metalloproteins, namely the large subunit HoxG of the membrane-bound hydrogenase from Cupriavidus necator (CnMBH), targeting its conformational and mechanical stability using molecular modelling and long all-atom Gaussian accelerated molecular dynamics (GaMD). Our simulations predict that isolated HoxG is stable in aqueous solution and preserves a large portion of its mechanical properties, but loses rigidity in regions around the active site, in contrast to the MBH heterodimer. Inspired by biochemical data showing dimerization of the HoxG protein and IR measurements revealing an increased stability of the [NiFe] cofactor in protein preparations with higher dimer content, corresponding simulations of homodimeric forms were also undertaken. While the monomeric subunit contains several flexible regions, our data predicts a regained rigidity in homodimer models. Furthermore, we computed the electrostatic properties of models obtained by enhanced sampling with GaMD, which displays a significant amount of positive charge at the protein surface, especially in solvent-exposed former dimer interfaces. These data offer novel insights on the way the [NiFe] core is protected from de-assembly and provide hints for enzyme anchoring to surfaces, which is essential information for further investigations on these minimal enzymes.

Cyanide Binding to [FeFe]-Hydrogenase Stabilizes the Alternative Configuration of the Proton Transfer Pathway.

Hydrogenases are H2 converting enzymes that harbor catalytic cofactors in which iron (Fe) ions are coordinated by biologically unusual carbon monoxide (CO) and cyanide (CN- ) ligands. Extrinsic CO and CN- , however, inhibit hydrogenases. The mechanism by which CN- binds to [FeFe]-hydrogenases is not known. Here, we obtained crystal structures of the CN- -treated [FeFe]-hydrogenase CpI from Clostridium pasteurianum. The high resolution of 1.39 Å allowed us to distinguish intrinsic CN- and CO ligands and to show that extrinsic CN- binds to the open coordination site of the cofactor where CO is known to bind. In contrast to other inhibitors, CN- treated crystals show conformational changes of conserved residues within the proton transfer pathway which could allow a direct proton transfer between E279 and S319. This configuration has been proposed to be vital for efficient proton transfer, but has never been observed structurally.

Ultrahigh-Throughput Directed Evolution of a Metal-Free α/β-Hydrolase with a Cys-His-Asp Triad into an Efficient Phosphotriesterase.

Finding new mechanistic solutions for biocatalytic challenges is key in the evolutionary adaptation of enzymes, as well as in devising new catalysts. The recent release of man-made substances into the environment provides a dynamic testing ground for observing biocatalytic innovation at play. Phosphate triesters, used as pesticides, have only recently been introduced into the environment, where they have no natural counterpart. Enzymes have rapidly evolved to hydrolyze phosphate triesters in response to this challenge, converging onto the same mechanistic solution, which requires bivalent cations as a cofactor for catalysis. In contrast, the previously identified metagenomic promiscuous hydrolase P91, a homologue of acetylcholinesterase, achieves slow phosphotriester hydrolysis mediated by a metal-independent Cys-His-Asp triad. Here, we probe the evolvability of this new catalytic motif by subjecting P91 to directed evolution. By combining a focused library approach with the ultrahigh throughput of droplet microfluidics, we increase P91's activity by a factor of ≈360 (to a kcat/KM of ≈7 × 105 M-1 s-1) in only two rounds of evolution, rivaling the catalytic efficiencies of naturally evolved, metal-dependent phosphotriesterases. Unlike its homologue acetylcholinesterase, P91 does not suffer suicide inhibition; instead, fast dephosphorylation rates make the formation of the covalent adduct rather than its hydrolysis rate-limiting. This step is improved by directed evolution, with intermediate formation accelerated by 2 orders of magnitude. Combining focused, combinatorial libraries with the ultrahigh throughput of droplet microfluidics can be leveraged to identify and enhance mechanistic strategies that have not reached high efficiency in nature, resulting in alternative reagents with novel catalytic machineries.

Peroxynitrite Electrochemical Signature on Selenium and Selenium-Decorated Graphene Depositions

Reactive oxygen species (ROS) such as peroxynitrite have been linked to oxidative stress-induced diseases such as cardiovascular failure. Determining the cause and progression of such disease, as well as testing the efficacy of potential treatments all hinge on the ability to accurately detect these species in biological systems. Antioxidant uses of selenium have garnered the attention of scientists in recent years. Latest investigations have revealed that selenium is essential for redox regulation as a ROS modulator and as a catalytic cofactor of endogenous antioxidative mechanisms.The advantages of preparing selenium electrochemical depositions as sensitive surfaces for peroxynitrite detection and quantification were revealed in our research. Electrochemical sensors with high sensitivity and selectivity were developed by dissolving selenium dioxide in an acidic medium. Graphene nanostructures loaded with selenium were used to modify one set of glassy carbon electrodes (GCEs), while selenium nanoparticles were used to modify another set of GCEs for comparison purposes. The shape and surface chemistry of selenium and selenium-decorated graphene nanostructures were investigated using scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). The electrochemical catalytic activity of these systems was determined using cyclic voltammetry and chronoamperometry. The thickness of the deposited selenium interfaces was measured using Quartz Crystal Microbalance (QCM) analysis.Elemental selenium nanoparticles showed an increase in the sensitivity toward peroxynitrite when embedded into graphene sheets. In addition, it allowed for the use of low positive potential, leading to improved segregation of peroxynitrite from other analytes that are sensitive to higher potentials.

The Contribution of Proton-Donor pKa on Reactivity Profiles of [FeFe]-hydrogenases.

The [FeFe]-hydrogenases are enzymes that catalyze the reversible activation of H2 coupled to the reduction–oxidation of electron carriers. Members of the different taxonomic groups of [FeFe]-hydrogenases display a wide range of preference, or bias, for H2 oxidation or H2 production reactions, despite sharing a common catalytic cofactor, or H-cluster. Identifying the properties that control reactivity remains an active area of investigation, and models have emerged that include diversity in the catalytic site coordination environments and compositions of electron transfer chains. The kinetics of proton-coupled electron transfer at the H-cluster might be expected to be a point of control of reactivity. To test this hypothesis, systematic changes were made to the conserved cysteine residue that functions in proton exchange with the H-cluster in the three model enzymes: CaI, CpII, and CrHydA1. CaI and CpII both employ electron transfer accessory clusters but differ in bias, whereas CrHydA1 lacks accessory clusters having only the H-cluster. Changing from cysteine to either serine (more basic) or aspartate (more acidic) modifies the sidechain pKa and thus the barrier for the proton exchange step. The reaction rates for H2 oxidation or H2 evolution were surveyed and measured for model [FeFe]-hydrogenases, and the results show that the initial proton-transfer step in [FeFe]-hydrogenase is tightly coupled to the control of reactivity; a change from cysteine to more basic serine favored H2 oxidation in all enzymes, whereas a change to more acidic aspartate caused a shift in preference toward H2 evolution. Overall, the changes in reactivity profiles were profound, spanning 105 in ratio of the H2 oxidation-to-H2 evolution rates. The fact that the change in reactivity follows a common trend implies that the effect of changing the proton-transfer residue pKa may also be framed as an effect on the scaling relationship between the H-cluster di(thiolmethyl)amine (DTMA) ligand pKa and Em values of the H-cluster. Experimental observations that support this relationship, and how it relates to catalytic function in [FeFe]-hydrogenases, are discussed.

13C ENDOR Characterization of the Central Carbon within the Nitrogenase Catalytic Cofactor Indicates That the CFe6 Core Is a Stabilizing "Heart of Steel".

Substrates and inhibitors of Mo-dependent nitrogenase bind and react at Fe ions of the active-site FeMo-cofactor [7Fe-9S-C-Mo-homocitrate] contained within the MoFe protein α-subunit. The cofactor contains a CFe6 core, a carbon centered within a trigonal prism of six Fe, whose role in catalysis is unknown. Targeted 13C labeling of the carbon enables electron-nuclear double resonance (ENDOR) spectroscopy to sensitively monitor the electronic properties of the Fe-C bonds and the spin-coupling scheme adopted by the FeMo-cofactor metal ions. This report compares 13CFe6 ENDOR measurements for (i) the wild-type protein resting state (E0; α-Val70) to those of (ii) α-Ile70, (iii) α-Ala70-substituted proteins; (iv) crystallographically characterized CO-inhibited "hi-CO" state; (v) E4(4H) Janus intermediate, activated for N2 binding/reduction by accumulation of 4[e-/H+]; (vi) E4(2H)* state containing a doubly reduced FeMo-cofactor without Fe-bound substrates; and (vii) propargyl alcohol reduction intermediate having allyl alcohol bound as a ferracycle to FeMo-cofactor Fe6. All states examined, both S = 1/2 and 3/2 exhibited near-zero 13C isotropic hyperfine coupling constants, Ca = [-1.3 ↔ +2.7] MHz. Density functional theory computations and natural bond orbital analysis of the Fe-C bonds show that this occurs because a (3 spin-up/3 spin-down) spin-exchange configuration of CFe6 Fe-ion spins produces cancellation of large spin-transfers to carbon in each Fe-C bond. Previous X-ray diffraction and DFT both indicate that trigonal-prismatic geometry around carbon is maintained with high precision in all these states. The persistent structure and Fe-C bonding of the CFe6 core indicate that it does not provide a functionally dynamic (hemilabile) "beating heart"─instead it acts as "a heart of steel", stabilizing the structure of the FeMo-cofactor-active site during nitrogenase catalysis.

Epigallocatechin gallate is a potent inhibitor of cystathionine beta-synthase: Structure-activity relationship and mechanism of action

Epigallocatechin gallate (EGCG) is the main bioactive component of green tea. Through screening of a small library of natural compounds, we discovered that EGCG inhibits cystathionine β-synthase (CBS), a major H2S-generating enzyme. Here we characterize EGCG's mechanism of action in the context of CBS-derived H2S production. In the current project, biochemical, pharmacological and cell biology approaches were used to characterize the effect of EGCG on CBS in cellular models of cancer and Down syndrome (DS). The results show that EGCG binds to CBS and inhibits H2S-producing CBS activity almost 30-times more efficiently than the canonical cystathionine formation (IC50 0.12 versus 3.3 μM). Through screening structural analogs and building blocks, we identified that gallate moiety of EGCG represents the pharmacophore responsible for CBS inhibition. EGCG is a mixed-mode, CBS-specific inhibitor with no effect on the other two major enzymatic sources of H2S, CSE and 3-MST. Unlike the prototypical CBS inhibitor aminooxyacetate, EGCG does not bind the catalytic cofactor of CBS pyridoxal-5′-phosphate. Molecular modeling suggests that EGCG blocks a substrate access channel to pyridoxal-5′-phosphate. EGCG inhibits cellular H2S production in HCT-116 colon cancer cells and in DS fibroblasts. It also exerts effects that are consistent with the functional role of CBS in these cells: in HCT-116 cells it decreases, while in DS cells it improves viability and proliferation. In conclusion, EGCG is a potent inhibitor of CBS-derived H2S production. This effect may contribute to its pharmacological effects in various pathophysiological conditions.

Structures of the nitrogenase complex prepared under catalytic turnover conditions.

The enzyme nitrogenase couples adenosine triphosphate (ATP) hydrolysis to the multielectron reduction of atmospheric dinitrogen into ammonia. Despite extensive research, the mechanistic details of ATP-dependent energy transduction and dinitrogen reduction by nitrogenase are not well understood, requiring new strategies to monitor its structural dynamics during catalytic action. Here, we report cryo-electron microscopy structures of the nitrogenase complex prepared under enzymatic turnover conditions. We observe that asymmetry governs all aspects of the nitrogenase mechanism, including ATP hydrolysis, protein-protein interactions, and catalysis. Conformational changes near the catalytic iron-molybdenum cofactor are correlated with the nucleotide-hydrolysis state of the enzyme.

Electrochemical Detection of Peroxynitrite Using Selenium-Decorated Graphene

Peroxynitrite and other reactive oxygen species (ROS) have shown contribution to oxidative stress-driven diseases including cardiovascular dysfunction. A precise detection of these species in biological systems is not only crucial for understanding the genesis and progression of diseases, but also for developing and evaluating effective therapies. In the past decade, selenium attracted the attention of many researchers for its antioxidative applications. Research studies have found that selenium plays a key role in redox regulation as a ROS modulator and as a catalytic cofactor of endogenous antioxidative systems.Our research study revealed the advantages of preparing electrochemical depositions of selenium as sensitive interfaces for peroxynitrite detection and quantification. In this regard, we fabricated highly sensitive and selective electrochemical sensors from a solution contains selenium dioxide in acidic medium. A series of glassy carbon electrodes (GCEs) have been modified with selenium nanoparticles, while another series of GCEs were modified with selenium-decorated graphene nanostructures for comparison purposes. The morphology and surface chemistry of the selenium and selenium-decorated graphene nanostructures were characterized using Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray analysis (EDX). The electrochemical catalytic activities of these platforms were measured using cyclic voltammetry and chronoamperometry. The particle size of the prepared interfaces was determined by microscopic imaging particle analysis.It was shown that elemental selenium nanoparticles incorporated into graphene sheets enhanced the sensitivity toward peroxynitrite. Selenium tends to stimulate the current response to aliquots of peroxynitrite in solution. Additionally, it enabled the use of fewer positive potentials, resulting in better differentiation between peroxynitrite and other analytes that are sensitive to more positive potentials.

Mineral Element Insiders and Outliers Play Crucial Roles in Biological Evolution.

The geosphere of primitive Earth was the source of life’s essential building blocks, and the geochemical interactions among chemical elements can inform the origins of biological roles of each element. Minerals provide a record of the fundamental properties that each chemical element contributes to crustal composition, evolution, and subsequent biological utilization. In this study, we investigate correlations between the mineral species and bulk crustal composition of each chemical element. There are statistically significant correlations between the number of elements that each element forms minerals with (#-mineral-elements) and the log of the number of mineral species that each element occurs in, and between #-mineral-elements and the log of the number of mineral localities of that element. There is a lesser correlation between the log of the crustal percentage of each element and #-mineral-elements. In the crustal percentage vs. #-mineral-elements plot, positive outliers have either important biological roles (S, Cu) or toxic biological impacts (Pb, As), while negative outliers have no biological importance (Sc, Ga, Br, Yb). In particular, S is an important bridge element between organic (e.g., amino acids) and inorganic (metal cofactors) biological components. While C and N rarely form minerals together, the two elements commonly form minerals with H, which coincides with the role of H as an electron donor/carrier in biological nitrogen and carbon fixation. Both abundant crustal percentage vs. #-mineral-elements insiders (elements that follow the correlation) and less abundant outsiders (positive outliers from the correlation) have important biological functions as essential structural elements and catalytic cofactors.

Trapping an Oxidized and Protonated Intermediate of the [FeFe]-Hydrogenase Cofactor under Mildly Reducing Conditions

The H-cluster is the catalytic cofactor of [FeFe]-hydrogenase, a metalloenzyme that catalyzes the formation of dihydrogen (H2). The catalytic diiron site of the H-cluster carries two cyanide and three carbon monoxide ligands, making it an excellent target for IR spectroscopy. In previous work, we identified an oxidized and protonated H-cluster species, whose IR signature differs from that of the oxidized resting state (Hox) by a small but distinct shift to higher frequencies. This "blue shift" was explained by a protonation at the [4Fe-4S] subcomplex of the H-cluster. The novel species, denoted HoxH, was preferentially accumulated at low pH and in the presence of the exogenous reductant sodium dithionite (NaDT). When HoxH was reacted with H2, the hydride state (Hhyd) was formed, a key intermediate of [FeFe]-hydrogenase turnover. A recent publication revisited our protocol for the accumulation of HoxH in wild-type [FeFe]-hydrogenase, concluding that inhibition by NaDT decay products rather than cofactor protonation causes the spectroscopic "blue shift". Here, we demonstrate that HoxH formation does not require the presence of NaDT (or its decay products), but accumulates also with the milder reductants tris(2-carboxyethyl)phosphine, dithiothreitol, or ascorbic acid, in particular at low pH. Our data consistently suggest that HoxH is accumulated when deprotonation of the H-cluster is impaired, thereby preventing the regain of the oxidized resting state Hox in the catalytic cycle.