RESUMEN

An artificial hydrogenase is constructed when the natively noncatalytic α-domain of the Cys-rich protein metallothionein (MT) is assembled with NiII. αMT binds four eq. of NiII in a non-cooperative manner where the addition of the 1st NiII eq. affords the most catalytically active species with little effect on photocatalytic H2 production during subsequent metal addition. The critical role of protonated Cys residue(s) in H-H bond formation is demonstrated.

Asunto(s)

Hidrogenasas , Hidrogenasas/química , Cisteína/química

RESUMEN

We present a Nip site model of acetyl coenzyme-A synthase (ACS) within a de novo-designed trimer peptide that self-assembles to produce a homoleptic Ni(Cys)3 binding motif. Spectroscopic and kinetic studies of ligand binding demonstrate that Ni binding stabilizes the peptide assembly and produces a terminal NiI-CO complex. When the CO-bound state is reacted with a methyl donor, a new species is quickly produced with new spectral features. While the metal-bound CO is albeit unactivated, the presence of the methyl donor produces an activated metal-CO complex. Selective outer sphere steric modifications demonstrate that the physical properties of the ligand-bound states are altered differently depending on the location of the steric modification above or below the Ni site.

Asunto(s)

Complejos de Coordinación , Metaloproteínas , Metaloproteínas/metabolismo , Acetilcoenzima A/metabolismo , Cinética , Ligandos , Níquel/química , Óxido Nítrico Sintasa/metabolismo , Aldehído Oxidorreductasas/metabolismo

RESUMEN

De novo metalloprotein design involves the construction of proteins guided by specific repeat patterns of polar and apolar residues, which, upon self-assembly, provide a suitable environment to bind metals and produce artificial metalloenzymes. While a wide range of functionalities have been realized in de novo designed metalloproteins, the functional repertoire of such constructs towards alternative energy-relevant catalysis is currently limited. Here we show the application of de novo approach to design a functional H2 evolving protein. The design involved the assembly of an amphiphilic peptide featuring cysteines at tandem a/d sites of each helix. Intriguingly, upon NiII addition, the oligomers shift from a major trimeric assembly to a mix of dimers and trimers. The metalloprotein produced H2 photocatalytically with a bell-shape pH dependence, having a maximum activity at pH 5.5. Transient absorption spectroscopy is used to determine the timescales of electron transfer as a function of pH. Selective outer sphere mutations are made to probe how the local environment tunes activity. A preferential enhancement of activity is observed via steric modulation above the NiII site, towards the N-termini, compared to below the NiII site towards the C-termini.

Asunto(s)

Metaloproteínas , Metaloproteínas/química , Hidrógeno , Metales , Cisteína/química , Péptidos/química

RESUMEN

Alzheimer's disease severely perturbs transition metal homeostasis in the brain leading to the accumulation of excess metals in extracellular and intraneuronal locations. The amyloid beta protein binds these transition metals, ultimately causing severe oxidative stress in the brain. Metal chelation therapy is an approach to sequester metals from amyloid beta and relieve the oxidative stress. Here we have designed a mixed N/O donor Cu chelator inspired by the proposed ligand set of Cu in amyloid beta. We demonstrate that the chelator effectively removes Cu from amyloid beta and suppresses reactive oxygen species (ROS) production by redox silencing and radical scavenging both in vitro and in cellulo. The impact of ROS on the extent of oxidation of the different aggregated forms of the peptide is studied by mass spectrometry, which, along with other ROS assays, shows that the oligomers are pro-oxidants in nature. The aliphatic Leu34, which was previously unobserved, has been identified as a new oxidation site.

Asunto(s)

Péptidos beta-Amiloides/antagonistas & inhibidores , Quelantes/farmacología , Cobre/farmacología , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Quelantes/síntesis química , Quelantes/química , Cobre/química , Humanos , Ligandos , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

Hydrogenase enzymes produce H2 gas, which can be a potential source of alternative energy. Inspired by the [NiFe] hydrogenases, we report the construction of a de novo-designed artificial hydrogenase (ArH). The ArH is a dimeric coiled coil where two cysteine (Cys) residues are introduced at tandem a/d positions of a heptad to create a tetrathiolato Ni binding site. Spectroscopic studies show that Ni binding significantly stabilizes the peptide producing electronic transitions characteristic of Ni-thiolate proteins. The ArH produces H2 photocatalytically, demonstrating a bell-shaped pH-dependence on activity. Fluorescence lifetimes and transient absorption spectroscopic studies are undertaken to elucidate the nature of pH-dependence, and to monitor the reaction kinetics of the photochemical processes. pH titrations are employed to determine the role of protonated Cys on reactivity. Through combining these results, a fine balance is found between solution acidity and the electron transfer steps. This balance is critical to maximize the production of NiI -peptide and protonation of the NiII -H- intermediate (Ni-R) by a Cys (pKa ≈6.4) to produce H2 .

Asunto(s)

Materiales Biomiméticos/química , Cisteína/química , Diseño de Fármacos , Hidrogenasas/metabolismo , Procesos Fotoquímicos , Protones

RESUMEN

Copper-containing metalloenzymes constitute a major class of proteins which catalyze a myriad of reactions in nature. Inspired by the structural and functional characteristics of this unique class of metalloenzymes, we report the conception, design, characterization, and functional studies of a de novo artificial copper peptide (ArCuP) within a trimeric self-assembled polypeptide scaffold that activates and reduces peroxide. Using a first principles approach, the ArCuP was designed to coordinate one Cu via three His residues introduced at an a site of the peptide scaffold. X-ray crystallographic, UV-vis and EPR data demonstrate that Cu binds via the Nε atoms of His forming a T2Cu environment. When reacted with hydrogen peroxide, the putative copper-hydroperoxo species is formed where a reductive priming step accelerates the rate of its formation and reduction. Mass spectrometry was used to identify specific residues undergoing oxidative modification, which showed His oxidation only in the reduced state. The redox behavior of the ArCuP was elucidated by protein film voltammetry. Detailed characterization of the electrocatalytic behavior of the ArCuP led us to determine the catalytic parameters (KM, kcat), which established the peroxidase activity of the ArCuP. Combined spectroscopic and electrochemical data showed a pH-dependence on the reactivity, which was optimum at pH 7.5.

RESUMEN

Hydrogen is a clean and sustainable form of fuel that can minimize our heavy dependence on fossil fuels as the primary energy source. The need of finding greener ways to generate H2 gas has ignited interest in the research community to synthesize catalysts that can produce H2 by the reduction of H+ . The natural H2 producing enzymes hydrogenases have served as an inspiration to produce catalytic metal centers akin to these native enzymes. In this article we describe recent advances in the design of a unique class of artificial hydrogen evolving catalysts that combine the features of the active site metal(s) surrounded by a polypeptide component. The examples of these biosynthetic catalysts discussed here include i) assemblies of synthetic cofactors with native proteins; ii) peptide-appended synthetic complexes; iii) substitution of native cofactors with non-native cofactors; iv) metal substitution from rubredoxin; and v) a reengineered Cu storage protein into a Ni binding protein. Aspects of key design considerations in the construction of these artificial biocatalysts and insights gained into their chemical reactivity are discussed.

Asunto(s)

Hidrógeno , Hidrogenasas , Compuestos Orgánicos/química , Catálisis , Dominio Catalítico , Hidrógeno/química , Hidrogenasas/química , Hidrogenasas/metabolismo

RESUMEN

The O2 reactivity of an artificial biomolecular hydrogenase, the nickel binding protein (NBP) is investigated. Kinetic analyses revealed a complete 4e- reduction of O2 to H2O under catalytic conditions with associated k0 for ET in the order of 10-6 cm s-1. Protein destabilization and S oxygenation are contributing factors to the deactivation of NBP under oxic conditions. Computational studies provided insight into the S oxygenation and the reaction intermediates of a proposed mechanistic pathway for O2 activation by NBP.

Asunto(s)

Hidrogenasas/química , Níquel/química , Oxígeno/química , Catálisis , Electrólisis , Cinética , Modelos Moleculares , Oxidación-Reducción , Agua/química

RESUMEN

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H+ reduction to H2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.

RESUMEN

Creating new environmentally friendly and non-toxic biomaterials with novel properties is required for numerous applications in healthcare and sensing. Protein bound gold nanoclusters constitute one such class of materials that offer promise in fluorescence imaging and sensing applications. However, unlike alkane thiol-protected gold nanoclusters, the number of protein-templated gold nanoclusters with such properties is limited and there is a need to expand the repertoire of such attractive hybrid quantum clusters. Herein, we report the synthesis, characterization, and applications of new fluorescent gold nanoclusters with tunable emission properties including blue, orange, and red, within a four-helix bundle copper storage protein (Csp1). The template protein consists of 13 cysteines along the length of the helix, which are suitable ligands to template Au and stabilize the resulting 14-19 atom clusters within the protein. The resulting clusters were extensively characterized by employing spectroscopic, microscopic and other analytical methods. The optical emission, relative quantum yields, and the excited state lifetime of the clusters are shown to depend on synthetic conditions. The clusters were found to be sensitive to the ppm level of transition metal ions with the quenching capabilities following the Irving-Williams series of metals (Co2+ < Ni2+ < Cu2+), which is rationalized based on the relative affinities of transition metals for a given set of ligands. The clusters were also found to be stable across the pH range 4-8.5 which, along with tunable emission properties paves the path for live bio-imaging and bio-sensing applications under physiological conditions.

RESUMEN

Metal nanoclusters containing a few to several hundred atoms with sizes ranging from sub-nanometer to ∼2 nm occupy an intermediate size regime that bridges larger plasmonic nanoparticles and smaller metal complexes. With strong quantum confinement, metal nanoclusters exhibit molecule-like properties. This Account focuses on noble metal nanoclusters that are synthesized within a single stranded DNA template. Compared to other ligand protected metal nanoclusters, DNA-templated metal nanoclusters manifest intriguing physical and chemical properties that are heavily influenced by the design of DNA templates. For example, DNA-templated silver nanoclusters can show bright fluorescence, tunable emission colors, and enhanced stability by tuning the sequence of the encapsulating DNA template. DNA-templated gold nanoclusters can also serve as excellent cocatalysts, which are integratable with other biocatalysts such as enzymes. In this Account, DNA-templated silver and gold nanoclusters are selected as paradigm systems to showcase their emergent properties and unique applications. We first discuss the DNA-templated silver nanoclusters with a focus on the creation of a complementary palette of emission colors, which has potential applications for multiplex assays. The importance of the DNA template toward enhanced stability of silver nanoclusters is also demonstrated. We then introduce a special class of activable fluorescence probes that are based on the fluorescence turn-on phenomena of DNA-templated silver nanoclusters, which are named nanocluster beacons (NCBs). NCBs have distinct advantages over molecular beacons for nucleic acid detection, and their emission mechanisms are also discussed in detail. We then discuss a universal method of creating novel DNA-silver nanocluster aptamers for protein detection with high specificity. The remainder of the Account is devoted to the DNA-templated gold nanoclusters. We demonstrate that DNA-gold nanoclusters can serve as enhancers for enzymatic reduction of oxygen, which is one of the most important reactions in biofuel cells. Although DNA-templated metal nanoclusters are still in their infancy, we anticipate they will emerge as a new type of functional nanomaterial with wide applications in biology and energy science. Future research will focus on the synthesis of size selected DNA-metal nanoclusters with atomic monodispersity, structural determination of different sized DNA-metal nanoclusters, and establishment of structure-property correlations. Some long-standing mysteries, such as the origin of fluorescence and mechanism for emission color tunability, constitute the central questions regarding the photophysical properties of DNA-metal nanoclusters. On the application side, more studies are required to understand the interaction between nanocluster and biological systems. In the foreseeable future, one can expect that new biosensors, catalysts, and functional devices will be invented based on the intriguing properties of well-designed DNA-metal nanoclusters and their composites. Overall, DNA-metal nanoclusters can add additional spotlights into the highly vibrant field of ligand protected, quantum sized metal nanoclusters.

Asunto(s)

ADN/química , Nanopartículas del Metal/química , Aptámeros de Nucleótidos/química , Fuentes de Energía Bioeléctrica , Técnicas Biosensibles/métodos , Oro/química , Ácidos Nucleicos/análisis , Oxidación-Reducción , Oxígeno/química , Polimorfismo de Nucleótido Simple , Proteínas/análisis , Plata/química

RESUMEN

Transition-metal-catalyzed oxidative stress is a widespread concern in the pathogenesis of Alzheimer's disease. However, the exact role of amyloid beta oligomers towards oxidative stress is widely debated. Assessing the oxidative nature of the oligomers in vitro is complicated by the different experimental conditions under which they are prepared. We have investigated Cu2+ -catalyzed reactive oxygen species (ROS) generation by using oligomers prepared in phosphate-buffered saline (AßO-PBS ) and in cell culture medium (AßO-CCM ), and compared their activities with respect to the monomers and fibrils prepared at neutral and acidic pH. Although both are deca- to dodecamers, the AßO-PBS oligomers have a spherical morphology and are smaller than the AßO-CCM . The AßO-PBS behaved as pro-oxidants; in contrast, AßO-CCM quench OH. generation attributed to CCM itself. Although the pro-oxidant oligomers showed oxidation, they also partially protect themselves from radical damage and maintain their overall spherical arrangement. The monomers and fibrils manifested antioxidant properties: radical scavenging as opposed to redox silencing. A dual role of Aß species depending on the stage of the disease is proposed. In the earlier stages, the monomers can act as antioxidants, whereas at the later stages, the oligomers take on a pro-oxidant role. Kaempferol, a natural flavonoid, bound Cu2+ in 2:1 ratio and abolished ROS production in all Aß species. It also distinctly modified the folding landscape of Aß species into new or altered morphologies.

Asunto(s)

Enfermedad de Alzheimer , Péptidos beta-Amiloides/metabolismo , Antioxidantes/metabolismo , Cobre/metabolismo , Agregación Patológica de Proteínas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Biocatálisis , Humanos , Técnicas In Vitro , Quempferoles/metabolismo

RESUMEN

The presence of a nonheme metal, such as copper and iron, in the heme-copper oxidase (HCO) superfamily is critical to the enzymatic activity of reducing O2 to H2O, but the exact mechanism the nonheme metal ion uses to confer and fine-tune the activity remains to be understood. We herein report that manganese and cobalt can bind to the same nonheme site and confer HCO activity in a heme-nonheme biosynthetic model in myoglobin. While the initial rates of O2 reduction by the Mn, Fe, and Co derivatives are similar, the percentages of reactive oxygen species (ROS) formation are 7%, 4%, and 1% and the total turnovers are 5.1 ± 1.1, 13.4 ± 0.7, and 82.5 ± 2.5, respectively. These results correlate with the trends of nonheme-metal-binding dissociation constants (35, 22, and 9 µM) closely, suggesting that tighter metal binding can prevent ROS release from the active site, lessen damage to the protein, and produce higher total turnover numbers. Detailed spectroscopic, electrochemical, and computational studies found no evidence of redox cycling of manganese or cobalt in the enzymatic reactions and suggest that structural and electronic effects related to the presence of different nonheme metals lead to the observed differences in reactivity. This study of the roles of nonheme metal ions beyond the Cu and Fe found in native enzymes has provided deeper insights into nature's choice of metal ion and reaction mechanism and allows for finer control of the enzymatic activity, which is a basis for the design of efficient catalysts for the oxygen reduction reaction in fuel cells.

Asunto(s)

Cobalto/química , Hemo/química , Manganeso/química , Modelos Moleculares , Oxidorreductasas/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Oxidación-Reducción , Espectroscopía de Absorción de Rayos X

RESUMEN

Metalloproteins are essential to numerous reactions in nature, and constitute approximately one-third of all known proteins. Molecular dynamics of proteins has been elucidated with great success both by experimental and theoretical methods, revealing atomic level details of function involving the organic constituents on a broad spectrum of time scales. However, the characterization of dynamics at biomolecular metal sites on nanosecond time scales is scarce in the literature. The aqua ions of many biologically relevant metal ions exhibit exchange of water molecules on the nanosecond time scale or faster, often defining their reactivity in aqueous solution, and this is presumably also a relevant time scale for the making and breaking of coordination bonds between metal ions and ligands at protein metal sites. Ligand exchange dynamics is critical for a variety of elementary steps of reactions in metallobiochemistry, for example, association and dissociation of metal bound water, association of substrate and dissociation of product in the catalytic cycle of metalloenzymes, at regulatory metal sites which require binding and dissociation of metal ions, as well as in the transport of metal ions across cell membranes or between proteins involved in metal ion homeostasis. In Perturbed Angular Correlation of γ-rays (PAC) spectroscopy, the correlation in time and space of two γ-rays emitted successively in a nuclear decay is recorded, reflecting the hyperfine interactions of the PAC probe nucleus with the surroundings. This allows for characterization of molecular and electronic structure as well as nanosecond dynamics at the PAC probe binding site. Herein, selected examples describing the application of PAC spectroscopy in probing the dynamics at protein metal sites are presented, including (1) exchange of Cd2+ bound water in de novo designed synthetic proteins, and the effect of remote mutations on metal site dynamics; (2) dynamics at the ß-lactamase active site, where the metal ion appears to jump between the two adjacent sites; (3) structural relaxation in small blue copper proteins upon 111Ag+ to 111Cd2+ transformation in radioactive nuclear decay; (4) metal ion transfer between two HAH1 proteins with change in coordination number; and (5) metal ion sensor proteins with two coexisting metal site structures. With this Account, we hope to make our modest contribution to the field and perhaps spur additional interest in dynamics at protein metal sites, which we consider to be severely underexplored. Relatively little is known about detailed atomic motions at metal sites, for example, how ligand exchange processes affect protein function, and how the amino acid composition of the protein may control this facet of metal site characteristics. We also aim to provide the reader with a qualitative impression of the possibilities offered by PAC spectroscopy in bioinorganic chemistry, especially when elucidating dynamics at protein metal sites, and finally present data that may serve as benchmarks on a relevant time scale for development and tests of theoretical molecular dynamics methods applied to biomolecular metal sites.

Asunto(s)

Rayos gamma , Metales/química , Proteínas/química , Análisis Espectral/métodos , Sitios de Unión

RESUMEN

Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

Asunto(s)

Cobre/química , Hierro/química , Oxidorreductasas/química , Oxígeno/química , Biocatálisis , Cobre/metabolismo , Técnicas Electroquímicas , Hierro/metabolismo , Cinética , Modelos Teóricos , Oxidación-Reducción , Oxidorreductasas/metabolismo , Espectrofotometría Infrarroja , Zinc/química

RESUMEN

Nanosecond ligand exchange dynamics at metal sites within proteins is essential in catalysis, metal ion transport, and regulatory metallobiochemistry. Herein we present direct observation of the exchange dynamics of water at a Cd2+ binding site within two de novo designed metalloprotein constructs using 111mCd perturbed angular correlation (PAC) of γ-rays and 113Cd NMR spectroscopy. The residence time of the Cd2+-bound water molecule is tens of nanoseconds at 20 °C in both proteins. This constitutes the first direct experimental observation of the residence time of Cd2+ coordinated water in any system, including the simple aqua ion. A Leu to Ala amino acid substitution ∼10 Å from the Cd2+ site affects both the equilibrium constant and the residence time of water, while, surprisingly, the metal site structure, as probed by PAC spectroscopy, remains essentially unaltered. This implies that remote mutations may affect metal site dynamics, even when structure is conserved.

Asunto(s)

Cadmio/química , Proteínas/química , Agua/química , Ligandos , Modelos Moleculares , Factores de Tiempo

RESUMEN

Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.

Asunto(s)

Proteínas Bacterianas/metabolismo , Hemo/metabolismo , Modelos Moleculares , Mioglobina/metabolismo , Oxidorreductasas/metabolismo , Ingeniería de Proteínas , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Dimerización , Hemo/química , Interacciones Hidrofóbicas e Hidrofílicas , Mutación , Mioglobina/química , Mioglobina/genética , Proteínas de Hierro no Heme/química , Proteínas de Hierro no Heme/genética , Proteínas de Hierro no Heme/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Conformación Proteica , Subunidades de Proteína , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Espectrometría Raman , Cachalote

RESUMEN

Cu(A) is a binuclear electron transfer (ET) center found in cytochrome c oxidases (CcOs), nitrous oxide reductases (N2ORs), and nitric oxide reductase (NOR). In these proteins, the Cu(A) centers facilitate efficient ET (kET > 104s⁻¹) under low thermodynamic driving forces (10-90 mV). While the structure and functional properties of Cu(A) are well understood, a detailed mechanism of the incorporation of copper into the protein and the identity of the intermediates formed during the Cu(A) maturation process are still lacking. Previous studies of the Cu(A) assembly mechanism in vitro using a biosynthetic model Cu(A) center in azurin (Cu(A)Az) identified a novel intermediate X (Ix) during reconstitution of the binuclear site. However, because of the instability of Ix and the coexistence of other Cu centers, such as Cu(A)' and type 1 copper centers, the identity of this intermediate could not be established. Here, we report the mechanism of Cu(A) assembly using variants of Glu114XCuAAz (X = Gly, Ala, Leu, or Gln), the backbone carbonyl of which acts as a ligand to the Cu(A) site, with a major focus on characterization of the novel intermediate Ix. We show that Cu(A) assembly in these variants proceeds through several types of Cu centers, such as mononuclear red type 2 Cu, the novel intermediate Ix, and blue type 1 Cu. Our results show that the backbone flexibility of the Glu114 residue is an important factor in determining the rates of T2Cu → Ix formation, suggesting that Cu(A) formation is facilitated by swinging of the ligand loop, which internalizes the T2Cu capture complex to the protein interior. The kinetic data further suggest that the nature of the Glu114 side chain influences the time scales on which these intermediates are formed, the wavelengths of the absorption peaks, and how cleanly one intermediate is converted to another. Through careful understanding of these mechanisms and optimization of the conditions, we have obtained Ix in ∼80-85% population in these variants, which allowed us to employ ultraviolet-visible, electron paramagnetic resonance, and extended X-ray absorption fine structure spectroscopic techniques to identify the Ix as a mononuclear Cu(Cys)(2)(His) complex. Because some of the intermediates have been proposed to be involved in the assembly of native Cu(A), these results shed light on the structural features of the important intermediates and mechanism of Cu(A) formation.

Asunto(s)

Azurina/química , Cobre/química , Thermus thermophilus/química , Dominio Catalítico , Cisteína/química , Espectroscopía de Absorción de Rayos X

RESUMEN

We report the synthesis and characterization of a new DNA-templated gold nanocluster (AuNC) of ∼1 nm in diameter and possessing ∼7 Au atoms. When integrated with bilirubin oxidase (BOD) and single walled carbon nanotubes (SWNTs), the AuNC acts as an enhancer of electron transfer (ET) and lowers the overpotential of electrocatalytic oxygen reduction reaction (ORR) by ∼15 mV as compared to the enzyme alone. In addition, the presence of AuNC causes significant enhancements in the electrocatalytic current densities at the electrode. Control experiments show that such enhancement of ORR by the AuNC is specific to nanoclusters and not to plasmonic gold particles. Rotating ring disk electrode (RRDE) measurements confirm 4e(-) reduction of O2 to H2O with minimal production of H2O2, suggesting that the presence of AuNC does not perturb the mechanism of ORR catalyzed by the enzyme. This unique role of the AuNC as enhancer of ET at the enzyme-electrode interface makes it a potential candidate for the development of cathodes in enzymatic fuel cells, which often suffer from poor electronic communication between the electrode surface and the enzyme active site. Finally, the AuNC displays phosphorescence with large Stokes shift and microsecond lifetime.

Asunto(s)

ADN/química , Enzimas/química , Oro/química , Nanopartículas del Metal/química , Oxígeno/química , Microscopía Electrónica de Transmisión

RESUMEN

This Forum Article focuses on recent advances in structural and spectroscopic studies of biosynthetic models of nitric oxide reductases (NORs). NORs are complex metalloenzymes found in the denitrification pathway of Earth's nitrogen cycle where they catalyze the proton-dependent two-electron reduction of nitric oxide (NO) to nitrous oxide (N2O). While much progress has been made in biochemical and biophysical studies of native NORs and their variants, a clear mechanistic understanding of this important metalloenzyme related to its function is still elusive. We report herein UV-vis and nuclear resonance vibrational spectroscopy (NRVS) studies of mononitrosylated intermediates of the NOR reaction of a biosynthetic model. The ability to selectively substitute metals at either heme or nonheme metal sites allows the introduction of independent (57)Fe probe atoms at either site, as well as allowing the preparation of analogues of stable reaction intermediates by replacing either metal with a redox inactive metal. Together with previous structural and spectroscopic results, we summarize insights gained from studying these biosynthetic models toward understanding structural features responsible for the NOR activity and its mechanism. The outlook on NOR modeling is also discussed, with an emphasis on the design of models capable of catalytic turnovers designed based on close mimics of the secondary coordination sphere of native NORs.

Asunto(s)

Resonancia Magnética Nuclear Biomolecular , Oxidorreductasas/química , Modelos Moleculares , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Óxido Nitroso/química , Óxido Nitroso/metabolismo , Oxidorreductasas/metabolismo , Teoría Cuántica , Espectrofotometría Ultravioleta