Multicopper Enzyme Research Articles

Functional assembly of nitrous oxide reductase provides insights into copper site maturation

The multicopper enzyme nitrous oxide reductase reduces the greenhouse gas N2O to uncritical N2 as the final step of bacterial denitrification. Its two metal centers require an elaborate assembly machinery that so far has precluded heterologous production as a prerequisite for bioremediatory applications in agriculture and wastewater treatment. Here, we report on the production of active holoenzyme in Escherichia coli using a two-plasmid system to produce the entire biosynthetic machinery as well as the structural gene for the enzyme. Using this recombinant system to probe the role of individual maturation factors, we find that the ABC transporter NosFY and the accessory NosD protein are essential for the formation of the [4Cu:2S] site CuZ, but not the electron transfer site CuA Depending on source organism, the heterologous host E. coli can, in some cases, compensate for the lack of the Cu chaperone NosL, while in others this protein is strictly required, underlining the case for designing a recombinant system to be entirely self-contained.

Bifunctional in vivo role of laccase exploited in multiple biotechnological applications.

Laccases are multicopper enzymes present in plants, fungi, bacteria, and insects, which catalyze oxidation reactions together with four electron reduction of oxygen to water. Plant, bacterial, and insect laccases have a polymerizing role in nature, implicated in biosynthesis of lignin, melanin formation, and cuticle hardening, respectively. On the other hand, fungal laccases carry out both polymerizing (melanin synthesis and fruit body formation) as well as depolymerizing roles (lignin degradation). This bifunctionality of fungal laccases can be attributed to the presence of multiple isoforms within the same as well as different genus and species. Interestingly, by manipulating culture conditions, these isoforms with their different induction patterns and unique biochemical characteristics can be expressed or over-expressed for a targeted biotechnological application. Consequently, laccases can be considered as one of the most important biocatalyst which can be exploited for divergent industrial applications viz. paper pulp bleaching, fiber modification, dye decolorization, bioremediation as well as organic synthesis. The present review spotlights the role of fungal laccases in various antagonistic applications, i.e., polymerizing and depolymerizing, and co-relating this dual role with potential industrial significance.

Biodegradation of pyridine raffinate using bacterial laccase isolated from garden soil

Abstract Pyridine raffinate, the residual mixture after the extraction of pyridine, discharges as pollutants from pyridine manufacturing industries. Pyridine and its derivatives are of foremost concern as environmental pollutants. Removal of contaminant or pollutant can be achieved using three kinds of approaches: Physical, Chemical and Biological. Paralleled with other physical and chemical methods such as combustion, photolysis. Biodegradation is a fiscal and environment-friendly alternative. In the present study, bacterial laccase, a multi copper enzyme that uses molecular oxygen to oxidize wide variety of aromatic and non-aromatic compounds, was used for the biodeterioration of pyridine raffinate, sample taken from Jubilant Organosys, Gajraula. Laccase was isolated from the bacterium Pseudomonas fluorescens and found to be an effective method to biodeteriorate pyridine raffinate instead of using harmful chemicals. It was observed that O.D. was reduced (max. 0.2327 at 501 nm) measured by UV/VIS Spectrophotometer taking raffinate as a reference. It signifies the oxidation of compounds present in the effluent that leads to the reduction in C.O.D. and found to be an effective method for the treatment of effluent.

POXC Laccase from Pleurotus ostreatus: A High‐Performance Multicopper Enzyme for Direct Oxygen Reduction Reaction Operating in a Proton‐Exchange Membrane Fuel Cell

AbstractIn this work, POXC, a laccase from Pleurotus ostreatus, was immobilized on carbon nanotubes (CNTs) modified by electrografting anthraquinone and naphthoate diazonium salts. Thanks to a favorable interaction between laccase and covalently modified CNTs, this laccase exhibits high‐potential/high‐current oxygen reduction reaction (ORR) performances, surpassing the performances of the well‐known laccase from Trametes versicolor (TvLAC) immobilized on the same nanostructured electrodes. Furthermore, immobilized POXC demonstrates high ORR activity over a wide range of pH (2 to 8), being also highly active at a gas‐diffusion electrode. Finally, owing to these unique performances, this enzyme was able to operate at the interface of the microporous layer, humidified air and a polymer electrolyte, that is, Nafion®, in a conventional H2/air proton‐exchange membrane fuel.

Walking the seven lines: binuclear copper A in cytochrome c oxidase and nitrous oxide reductase.

The enzymes nitrous oxide reductase (N2OR) and cytochrome c oxidase (COX) are constituents of important biological processes. N2OR is the terminal reductase in a respiratory chain converting N2O to N2 in denitrifying bacteria; COX is the terminal oxidase of the aerobic respiratory chain of certain bacteria and eukaryotic organisms transforming O2 to H2O accompanied by proton pumping. Different spectroscopies including magnetic resonance techniques, were applied to show that N2OR has a mixed-valent Cys-bridged [Cu1.5+(CyS)2Cu1.5+] copper site, and that such a binuclear center, called CuA, does also exist in COX. A sequence motif shared between the CuA center of N2OR and the subunit II of COX raises the issue of a putative evolutionary relationship of the two enzymes. The suggestion of a binuclear CuA in COX, with one unpaired electron delocalized between two equivalent Cu nuclei, was difficult to accept originally, even though regarded as a clever solution to many experimental observations. This minireview in honor of Helmut Sigel traces several of the critical steps forward in understanding the nature of CuA in N2OR and COX, and discusses its unique electronic features to some extent including the contributions made by the development of methodology and the discovery of a novel multi-copper enzyme. Left: X-band (9.130 GHz) and C-band (4.530 GHz, 1st harmonic display of experimental spectrum) EPR spectra of bovine heart cytochrome c oxidase, recorded at 20K. Right: Ribbon presentation of the CuA domain in cytochrome c oxidase and nitrous oxide reductase.

Bacterial laccase: recent update on production, properties and industrial applications.

Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are multi-copper enzymes which catalyze the oxidation of a wide range of phenolic and non-phenolic aromatic compounds in the presence or absence of a mediator. Till date, laccases have mostly been isolated from fungi and plants, whereas laccase from bacteria has not been well studied. Bacterial laccases have several unique properties that are not characteristics of fungal laccases such as stability at high temperature and high pH. Bacteria produce these enzymes either extracellularly or intracellularly and their activity is in a wide range of temperature and pH. It has application in pulp biobleaching, bioremediation, textile dye decolorization, pollutant degradation, biosensors, etc. Hence, comprehensive information including sources, production conditions, characterization, cloning and biotechnological applications is needed for the effective understanding and application of these enzymes at the industrial level. The present review provides exhaustive information of bacterial laccases reported till date.

The catalytic cycle of nitrous oxide reductase — The enzyme that catalyzes the last step of denitrification

The reduction of the potent greenhouse gas nitrous oxide requires a catalyst to overcome the large activation energy barrier of this reaction. Its biological decomposition to the inert dinitrogen can be accomplished by denitrifiers through nitrous oxide reductase, the enzyme that catalyzes the last step of the denitrification, a pathway of the biogeochemical nitrogen cycle. Nitrous oxide reductase is a multicopper enzyme containing a mixed valence CuA center that can accept electrons from small electron shuttle proteins, triggering electron flow to the catalytic sulfide-bridged tetranuclear copper “CuZ center”. This enzyme has been isolated with its catalytic center in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S), proven to be spectroscopic and structurally different. In the last decades, it has been a challenge to characterize the properties of this complex enzyme, due to the different oxidation states observed for each of its centers and the heterogeneity of its preparations. The substrate binding site in those two “CuZ center” forms and which is the active form of the enzyme is still a matter of debate. However, in the last years the application of different spectroscopies, together with theoretical calculations have been useful in answering these questions and in identifying intermediate species of the catalytic cycle. An overview of the spectroscopic, kinetics and structural properties of the two forms of the catalytic “CuZ center” is given here, together with the current knowledge on nitrous oxide reduction mechanism by nitrous oxide reductase and its intermediate species.

Bio-prospecting Bacterial Diversity of Hot Springs in Northern Himalayan Region of India for Laccases.

Bacterial diversity of hot springs of northern Himalayan region of India was studied and explored for laccases, the multicopper enzymes applicable in a large number of industries due to their ability to utilize a wide range of substrates. 220 operational taxonomic units (OTUs) out of 5551 sequence reads for bacterial diversity and 3 OTUs out of 19 sequence reads for Laccase like multicopper oxidases (LMCOs) diversity were generated. Bacteroidetes (74.28%) was the most abundant phylum including genus Paludibacter (66.96%), followed by phylum Proteobacteria (24.53%) including genera Chitinilyticum (7.55%) and Cellvibrio (6.14%). In case of laccase diversity, three LMCO sequences showed affiliation with proteobacteria and one with two domain laccase from uncultivable bacteroidetes. LMCO sequences belonged to H and N families.

Bienzymatic mediatorless sensing of total hydrogen peroxide with catalase and multi-copper enzyme co-adsorbed at carbon nanotube-modified electrodes

The rapid, sensitive, and accurate determination of hydrogen peroxide (H2O2) is of practical importance in physiological, pathological, and environmental fields. In this work, we propose a highly sensitive and selective amperometric biosensor for the detection of hydrogen peroxide. The biosensor consists of a glassy carbon electrode (GCE) covered with multi-walled carbon nanotubes (MWCNTs) and adsorbed enzymes: catalase (Cat) and either laccase (Lac) or bilirubin oxidase (BOX). The stability and durability of the electrode was improved by using glutaraldehyde (GAD). The determination of H2O2 by cyclic voltammetry and chronoamperometry experiments proved the synergy of the laccase and catalase co-adsorbed on the carbon nanotubes. Catalase from bovine liver catalyzed the transformation of H2O2 into water and oxygen, which was further transformed into water by multi-copper enzymes (MCO), either laccase from Trametes versicolor or bilirubin oxidase from Myrothecium verrucaria. The unique property of such a bienzymatic sensing layer is the ability to detect oxygen originating both from catalase activity and from the self-decomposition of H2O2. This makes it possible to evaluate the initial concentration of H2O2 in the analyzed sample.

Osmium(II) Complexes Bearing Chelating N-Heterocyclic Carbene and Pyrene-Modified Ligands: Surface Electrochemistry and Electron Transfer Mediation of Oxygen Reduction by Multicopper Enzymes

We report the synthesis of original osmium(II) complexes bearing chelating N-heterocyclic (NHC) and bipyridine ligands. The pincer ligand 1,1′-dimethyl-3,3′-methylenediimidazole-2,2′-diylidene was used to tune the redox properties of osmium complexes. Bipyridine ligands modified with pyrene groups were chosen to study the electrosynthesis of OsII-NHC-based metallopolymers as well as the noncovalent immobilization of these complexes on carbon-nanotube (CNT) electrodes. Poly-[OsII-NHC] polypyrene polymer was electrogenerated on a GC electrode, whereas the pyrene-modified [OsII-NHC] could interact with the CNTs’ sidewalls through π–π interactions, allowing the immobilization of the NHC complexes at the surface of π-extended nanostructured electrodes. Furthermore, an OsII-NHC complex was studied in water, showing electron transfer mediation with multicopper enzymes. UV–visible and electrochemical experiments demonstrate that redox properties of the OsII-NHC complex provide sufficient driving force for electron transfer with bilirubin oxidase from Myrothecium verrucaria while achieving high potential electroenzymatic oxygen reduction at E = +0.45 V vs Ag/AgCl at pH 6.5.

Macrocyclic metal complexes for metalloenzyme mimicry and sensor development.

Examples of proteins that incorporate one or more metal ions within their structure are found within a broad range of classes, including oxidases, oxidoreductases, reductases, proteases, proton transport proteins, electron transfer/transport proteins, storage proteins, lyases, rusticyanins, metallochaperones, sporulation proteins, hydrolases, endopeptidases, luminescent proteins, iron transport proteins, oxygen storage/transport proteins, calcium binding proteins, and monooxygenases. The metal coordination environment therein is often generated from residues inherent to the protein, small exogenous molecules (e.g., aqua ligands) and/or macrocyclic porphyrin units found, for example, in hemoglobin, myoglobin, cytochrome C, cytochrome C oxidase, and vitamin B12. Thus, there continues to be considerable interest in employing macrocyclic metal complexes to construct low-molecular weight models for metallobiosites that mirror essential features of the coordination environment of a bound metal ion without inclusion of the surrounding protein framework. Herein, we review and appraise our research exploring the application of the metal complexes formed by two macrocyclic ligands, 1,4,7-triazacyclononane (tacn) and 1,4,7,10-tetraazacyclododecane (cyclen), and their derivatives in biological inorganic chemistry. Taking advantage of the kinetic inertness and thermodynamic stability of their metal complexes, these macrocyclic scaffolds have been employed in the development of models that aid the understanding of metal ion-binding natural systems, and complexes with potential applications in biomolecule sensing, diagnosis, and therapy. In particular, the focus has been on "coordinatively unsaturated" metal complexes that incorporate a kinetically inert and stable metal-ligand moiety, but which also contain one or more weakly bound ligands, allowing for the reversible binding of guest molecules via the formation and dissociation of coordinate bonds. With regards to mimicking metallobiosites, examples are presented from our work on tacn-based complexes developed as simplified structural models for multimetallic enzyme sites. In particular, structural comparisons are made between multinuclear copper(II) complexes formed by such ligands and multicopper enzymes featuring type-2 and type-3 copper centers, such as ascorbate oxidase (AO) and laccase (Lc). Likewise, with the aid of relevant examples, we highlight the importance of cooperativity between either multiple metal centers or a metal center and a proximal auxiliary unit appended to the macrocyclic ligand in achieving efficient phosphate ester cleavage. Finally, the critical importance of the Zn(II)-imido and Zn(II)-phosphate interactions in Zn-cyclen-based systems for delivering highly sensitive electrochemical and fluorescent chemosensors is also showcased. The Account additionally highlights some of the factors that limit the performance of these synthetic nucleases and the practical application of the biosensors, and then identifies some avenues for the development of more effective macrocyclic constructs in the future.

Nanolayered Supramolecular Protein Clusters on Electrodes: A Switchable Cascadic Reaction Scheme for Dual-Analyte Detection

Biochemical signal chains and pathways are characterized by an effective coupling of individual reaction steps resulting in a rather high specificity in signal transduction. Additionally such highly complex systems allow a switching between different reaction cascades, depending on the presence of a certain molecule. Apparently, these efficient biological principles have been adopted for the construction of artificial signal chains.[1,2] In such a way protein functionalities can be coupled to electrochemical detection schemes, ensuring signal generation in the presence of individual substances.[3-5] Here we report on a novel system, which allows an activity-switch between two enzymes co-immobilized in a supramolecular network. This supramolecular network is formed by embedding two different biocatalysts - the multi-copper enzyme laccase (Lac), the multi-domain enzyme cellobiose dehydrogenase (CDH) - and the redox protein cytochrome c (cyt c) in an artificial matrix composed of carboxy-modified silica nanoparticles[6] allowing the immobilization of all the components in a layer-by-layer fashion. The formation of the entity with cyt c, Lac, CDH, and SiNPs was confirmed by quartz crystal microbalance experiments, and investigated by cyclic voltammetry.[7] Within this layered architecture two enzymes have been connected to the electrode via cytochrome c. Since the activity of the enzymes is controlled by the delivery or withdrawal of electrons the redox state of cyt c has been used for switching the activity of the biocatalysts on and off. Given that the electron transfer throughout the layered entity is feasible, Lac and CDH in different distances to the electrode can be addressed. The switchable reaction cascades for Lac and CDH are functioning in a non-separated matrix without disturbing the reaction of the other catalyst, allowing lactose and oxygen sensing, respectively.[7] The approach is expected to open the way for the development of multiplex biosensors, and also represents a significant advance in mimicking of biological electron transfer cascades. In addition we also like to show that cyt c has been crystallized on electrodes and that the cyt c molecules in the crystals are in electrical contact with the electrode. Thereby we show for the very first time, that electron transfer within a cyt c-crystal and between the crystal and the electrode is feasible.[8] [1] I. Willner, E. Katz, Angew. Chem. Int. Ed. 2000, 39, 1180. [2] T. Ikeda, K. Kano, J. Biosci. Bioeng. 2001, 92, 9. [3] E. E. Ferapontova, T. Ruzgas, L. Stoica, A. Christenson, J. Tkac, A. I. Yaropolov, L. Gorton, Perspectives in Bioanalysis, Vol. 1, Elsevier, Dordrecht 2005, p. 517. [4] J. F. Rusling, Acc. Chem. Res. 1998, 31, 363. [5] S. C. Feifel, A. Kapp, R. Ludwig, L. Gorton, F. Lisdat, RCS Advances 2013, 3, 3428. [6] S. C. Feifel, F. Lisdat, J. Nanobiotechnol. 2011, 9:59. [7] S. C. Feifel, A. Kapp, R. Ludwig, F. Lisdat, Angew. Chem. Int. E d. 2014, 53, 5676. [8] Roise McGovern, S. C. Feifel, F. Lisdat, P. Crowley, Angew. Chem. Int. Ed. 2015, DOI: 10.1002/anie.201500191.

Mediator-Less Direct Electron Transfer and Oxygen Reduction By Bilirubin Oxidase Adsorbed on to a Bucky Paper Electrode

Direct immobilization of enzyme films on nanostructured electrodes has the advantage of enhancing electrocatalytic currents. Bilirubin oxidase (BOD), a blue multi-copper enzyme, extracted from Myrothecium verrucaria has been recognized as a highly efficient oxygen reduction catalyst at near neutral pH with a minimal overpotential requirement. BOD contains four redox active Cu centers, which are classified based on their spectroscopic characteristics as T1 (one), T2 (one), and T3 (two). T1 Cu-center of BOD has been proposed to be the electron-receiving center from electrode and the catalytic oxygen reduction was suggested to occur at the tri-nuclear Cu clusters of T2/T3.1 Direct electron transfer between BOD and the electrode has been shown in slightly acidic and anaerobic conditions without mediators (E°´ T1 = ~0.5 V, E°′ T2/T3 = ~0.16 V vs Ag/AgCl).2 ,3a Reported studies showed that the oxygen reduction by BOD is highly effective with electrodes modified with a redox polymer hydrogel,3b a poly-lysine composite film,4 gold nanoparticles,5 carbon nanotube-pyrene units,6 and functionalized multiwalled nanotubes (MWNT).7 , 8 Here we will present our findings on the direct electron transfer and electrocatalytic oxygen reduction currents by a BOD film directly adsorbed on to bucky paper electrodes prepared from MWNT. Our preliminary work also suggests the role of bucky paper thickness in controlling the extent of catalytic currents and direct electrochemistry. BOD integrated bucky papers have immense usability in highly efficient biological fuel cells and we envision employing these BOD electrodes as cathodes for enzymatic fuel cell applications.6,9 Acknowledgements. Financial support by the Oklahoma State University is greatly acknowledged. (1) dos Santos, L.; Climent, V.; Blanford, C. F.; Armstrong, F. A. Physical Chemistry Chemical Physics 2010, 12, 13962. (2) Ivnitski, D.; Artyushkova, K.; Atanassov, P. Bioelectrochemistry 2008, 74, 101. (3) (a) Ramírez, P.; Mano, N.; Andreu, R.; Ruzgas, T.; Heller, A.; Gorton, L.; Shleev, S. Biochimica et Biophysica Acta - Bioenergetics 2008, 1777, 1364; (b) Mano, N.; Fernandez, J. L.; Kim, Y.; Shin, W.; Bard, A. J.; Heller, A. Journal of the American Chemical Society 2003, 125, 15290. (4) Tsujimura, S.; Kano, K.; Ikeda, T. Journal of Electroanalytical Chemistry 2005, 576, 113. (5) Murata, K.; Kajiya, K.; Nakamura, N.; Ohno, H. Energy & Environmental Science 2009, 2, 1280. (6) Krishnan, S.; Armstrong, F. A. Chemical Science 2012, 3, 1015. (7) Lopez, R. J.; Babanova, S.; Ulyanova, Y.; Singhal, S.; Atanassov, P. ChemElectroChem 2014, 1, 241. (8) Milton, R. D.; Giroud, F.; Thumser, A. E.; Minteer, S. D.; Slade, R. C. T. Chemical Communications 2014, 50, 94. (9) Bourourou, M.; Elouarzaki, K.; Holzinger, M.; Agnes, C.; Le Goff, A.; Reverdy-Bruas, N.; Chaussy, D.; Party, M.; Maaref, A.; Cosnier, S. Chemical Science 2014, 5, 2885.

Investigation of 3d-transition metal acetates in the oxidation of substituted dioxolene and phenols

Enzymatic reactions have inspired many chemists to design small molecule mimics that would perform the function of the enzymes in aqueous and or non-aqueous medium. Catechol oxidase (CO) and phenoxazinone synthase (PHS) are two multi-copper enzymes in nature, which has led to model complexes of Mn, Fe, Co, Ni, Cu. Based on our earlier work in this area we have probed the commercially available metal acetates of the above metals to establish a trend in reactivity for catalytic conversions similar to those of the two enzymes. The results show that Mn is the best 3d transition metal for similar catalysis. MnII acetate was found to convert 3,5-di-tert-butylcatechol (DTBC) to 3,5-di-tert-butylquinone (DTBQ) with a kcat of 1.3(1)×103h−1and for o-aminophenol (OAP) to 2-aminophenoxazinone (APX) conversion the kcat=111(2)h−1, demonstrating efficient CO and PHS like activity. Kinetic studies show that DTBC oxidation follows a first order kinetics with respect to the substrate for each of those metal(II) acetates with activity order of Mn>>Co>Cu>Fe≥Ni. Through mechanistic investigation we found that the reactive oxygen species detected during the oxidation of DTBC is mostly hydroxyl radical for Mn, Fe and Co whereas Cu and Ni generate H2O2.

Oxygen Reduction Reaction at Multi-Copper Complexes Investigated by Modified Electrode and In Situ XAFS

The oxygen reduction reaction (ORR), which occurs at the cathode in polymer electrolyte fuel cells (PEFCs), requires a large overpotential (more than 200 mV) even at the highly active platinum group metal (PGM) catalysts, since this reaction contains 4-electrons and 4-protons transfer to yield two water molecules from a dioxygen molecule. However, the biocathodes which are constructed by covalently attaching multi-copper enzymes, such as laccase (Lac), at electrode surfaces show nonexsistent overpotential (ca. 20 mV) toward ORR[1-2]. Unfortunately, the pH region, where the Lacs works well as an electrocatalyst for ORR, is limited and the spatial density of reactive sites at such the biocathode seems to be insufficient as compared with the state-of-the-art PGM electrocatalysts. Thus, the extraction of reactive sites in Lac and the effective arrangement on catalyst supports have been desired[3]. Recently, Gewirth and co-workers have achieved a preparation of binuclear copper complex, Cu2(Hdatrz)2, as an electrocatalyst for ORR[4-6]. Although the catalyst shows relatively large overpotential for ORR in acidic solution, the activity in alkaline solution is quite high. The active site for ORR in the catalyst has been proved to be bi-copper centers [6-7] and the investigation of ORR mechanism at the catalyst seems to be valuable.In the present study, we have adopted in situ X-ray absorption fine structure (XAFS) spectroscopy to monitor the electronic structure of Cu centers during ORR and to determine the molecular structure of Cu2(Hdatrz)2 complexes on electrodes. In situ XANES measurement has shown that the ORR at the Cu centers requires the formation of CuI species and the rate-determining step for ORR is dependent on pH.For further development of metal complex catalysts by replacing Cu centers to other metal ions, a easy preparation of model complex electrode should be required. Chemically modified model electrodes are also constructed on gold surface to investigate ORR at multi-metal complex using electrochemistry and spectroscopic methods. This work was supported by New Energy, Industrial Technology Development Organization (NEDO). XAFS experiments were performed under the approval of the Photon Factory Program Advisory Committee (Proposal No. 2010G200 and 2013G173)[1] N. Mano, V. Soukharev, A. Heller, J. Phys. Chem. B, 110, 11180 (2006)[2] M.S. Thorum, C.A. Anderson, J.J. Hatch, A.S. Campbell, N.M. Marshall, S.C. Zimmerman, Y. Liu, A.A. Gewirth, J. Phys. Chem. Lett, 1, 2251 (2010)[3] C.H. Kjargaard, J. Rossmeisi, J.K. Norskov, Inorg. Chem., 49, 3567 (2010)[4] M.S. Thorum, J. Yadav, A.A. Gewirth, Ang. Chem. Int. Ed., 48, 165 (2008)[5] F.R. Brushett, M.S. Thorum, N.S. Lioutas, M.S. Naughton, C. Tornow, H-R. M. Jhong, A.A. Gewirth, P.A. Kenis, J. Am. Chem. Soc., 132, 12185 (2010)[6] M.S. Thorum, J.M. Hankett, A.A. Gewirth, J. Phys. Chem. Lett., 2, 295 (2011)[7] C.C.L. McCrory, A. Devadoss, X. Otterwaelder, R.D. Lowe, T.D.P. Stack, C.E.D. Chidsey, J. Am. Chem. Soc., 133, 3696 (2011)

Inhibition of Phenoloxidase Activity Delays Development inBactrocera dorsalis(Diptera: Tephritidae)

Abstract Phenoloxidase (PO) is a multicopper enzyme, which plays an important role in melanin synthesis. PO is necessary for defense against intruding microorganisms and parasites, and is important in wound healing and cuticle pigmentation. As a specific inhibitor of PO, kojic acid (KA) is commonly used to characterize the functional properties of PO. In this study, we investigated the effect of KA on PO activity during the development of the oriental fruit fly, Bactrocera dorsalis, which is a destructive pest of many horticultural crops. After feeding on a KA-containing artificial diet, the larval and pupal developmental periods were significantly prolonged. In addition, the larvae did not grow to normal size and rates of pupation and emergence were decreased when B. dorsalis larvae had been fed with KA-containing diet for 6 days. Compared to the control, PO activities from whole bodies of B. dorsalis were inhibited, so did in larval cuticles. Consistent with these, kinetic analysis showed that the catal...

Ceruloplasmin-ferroportin system of iron traffic in vertebrates.

Safe trafficking of iron across the cell membrane is a delicate process that requires specific protein carriers. While many proteins involved in iron uptake by cells are known, only one cellular iron export protein has been identified in mammals: ferroportin (SLC40A1). Ceruloplasmin is a multicopper enzyme endowed with ferroxidase activity that is found as a soluble isoform in plasma or as a membrane-associated isoform in specific cell types. According to the currently accepted view, ferrous iron transported out of the cell by ferroportin would be safely oxidized by ceruloplasmin to facilitate loading on transferrin. Therefore, the ceruloplasmin-ferroportin system represents the main pathway for cellular iron egress and it is responsible for physiological regulation of cellular iron levels. The most recent findings regarding the structural and functional features of ceruloplasmin and ferroportin and their relationship will be described in this review.

Layered Protein Assemblies on Electrodes Exploiting Multistep Electron Transfer Chains for Dual Analyte Detection

In recent years there is considerable progress in constructing artificial signal chains which follow natural examples. This can result in sensitive systems which are turned on by the presence of a certain analyte. One approach is based on the construction of protein arrangements using the redox protein cytochrome c (cyt c) [1]. Here electron transfer through several layers of the protein on electrodes has been achieved. The defined arrangement of the biomolecule is an important precondition for a functional system. The layer-by-layer adsorption technique has been shown to be advantageous with this respect. Different building blocks have been applied including polyanilline derivatives, DNA and nanoparticles [2,3]. Furthermore, first examples for the construction of bi-protein assemblies with catalytic properties have been demonstrated, e.g. with sulfite oxidase [4].In this study it is first demonstrated that bi-protein assemblies using cyt c and the enzyme cellobiose dehydrogenase (CDH) can be constructed in a layered design using modified silica nanoparticles as building block [5]. It is found, that glycosylation of the enzyme significantly influences the layered assembly and the electron transfer chain from the substrate to the electrode. Conditions have been found, so that several catalytically active protein layers can be formed on the electrode surface, thus allowing the adjustment of the electrode response to lactose by the numbers of deposited layers.Based on these results a tri-protein architecture has been created on a thiol-modified gold electrode. Again the layer-by-layer deposition method is used, but additionally the multi-copper enzyme Laccase has been immobilised within this structure. The formation of the entity with cyt c, Lac, CDH, and SiNPs is first confirmed by quartz crystal microbalance (QCM) experiments, and then investigated electrochemically by cyclic voltammetry (CV).Within this architecture the two enzymes are connected to the electrode via cyt c. Since the activity of the enzymes is controlled by the delivery or withdrawal of electrons, the redox state of cyt c can be used for switching the activity of the biocatalysts. Since electron transfer throughout the whole layered entity is feasible, Lac and CDH in different distances to the electrode can be addressed. The switchable reaction cascades for Lac and CDH are functioning in an non-separated matrix without disturbing the reaction of the other biocatalyst. Whereas the CDH allows lactose detection, the activity of laccase is influenced by the oxygen content of the solution.The approach represents an advance in mimicking of biological electron transfer cascades by fixing protein-protein reactions to electrodes in the immobilized state without the need of any mediating shuttle molecule..[1 ] F. Lisdat, R. Dronov, H. Möhwald, F. W. Scheller, D. G. Kurth, Chemical Communications3 (2009) 274[2] D. Sarauli; J. Tanne; D. Schäfer; I. W. Schubart; F. Lisdat, Electrochemistry Communications11 (12) (2009) 2288[3] S. C. Feifel, F. Lisdat, Journal of Nanobiotechnology(2011) 9:59[4] R.V. Dronov, D.G. Kurth, H. Möhwald, R. Spricigo, S. Leimkühler, U. Wollenberger, K. V. Rajagopalan, F. W. Scheller, F. Lisdat, JACS130 (4) (2008) 1122[5] S. C. Feifel, A. Kapp, R. Ludwig, L. Gorton, F. Lisdat, RSC Advances 3 (2013) 3428

Laccases from <i>Actinobacteria</i>—What We Have and What to Expect

Laccases are blue multicopper enzymes, capable of oxidizing diverse aromatic and non-aromatic compounds of industrial interest, concomitantly with reduction of molecular oxygen to water. Tolerance to extreme conditions, such as high temperature, salinity or extreme pH, is required for practical industrial applications. Here we focus on bacterial laccases from the phylum Actinobacteria, notably the order Actinomycetales. Currently, less than 10 enzymes have been properly characterized, all belonging to genus Streptomyces, but it is noteworthy that all of them have exhibited industrially important properties. Furthermore, studies with enzymes from this phylum revealed a novel molecular structure of laccases, providing the basis for a distinct family, the two-domain laccases. The relevant traits of actinomycetes laccases emphasize the need for more studies involving the isolation of this bacterial group from lignin-rich environmental samples, detection of their laccase activity and thereafter, characterization of the proteins and related genes. The nonhomogeneous responses of actinomycetes laccases to traditional inhibitors, substrates or metal ions have challenged the currently accepted “laccase concept”. Finally, considering that distinguishing laccase activity in vitro from other ligninolytic enzymes becomes a difficult task due to overlaps in catalytical properties of the enzymes, we proposed a simple flow chart to help experimental assays.

Structural and mechanistic insights into the electron flow through protein for cytochrome c-tethering copper nitrite reductase

Copper-containing nitrite reductases (CuNiRs), which catalyse the reversible one-electron reduction of nitrite to nitric oxide, are members of a large family of multi-copper enzymes that require an interprotein electron transfer (ET) reaction with redox partner proteins. Here, we show that the naturally fused type of CuNiR tethering a cytochrome c (Cyt c) at the C-terminus folds as a unique trimeric domain-swapped structure and has a self-sufficient electron flow system. The C-terminal Cyt c domain is located at the surface of the type 1 copper (T1Cu) site in the N-terminal CuNiR domain from the adjacent subunit, the heme-to-Cu distance (10.6 Å) of which is comparable to the transient ET complex of normal CuNiR with Cyt c. The structural aspects for the domain-domain interface and the ET kinetics indicate that the Cyt c-CuNiR domain interaction should be highly transient. The further electrochemical analysis of the interprotein ET reaction with a cognate redox partner protein suggested that an electron is directly transferred from the partner to the T1Cu. Structural and mechanistic comparisons of Cyt c-CuNiR with another cupredoxin-tethering CuNiR highlight the behaviours of extra domains on the fusion types of CuNiRs required for ET through proteins.