Small electron transfer proteins as mediators in enzymatic electrochemical biosensors

The final publication is available at Springer."Electrochemical mediators transfer redox equivalents between the active sites of enzymes and electrodes and, in this way, trigger bioelectrocatalytic redox processes. This has been very useful in the development of the so-called second generation biosensors, where they are able to transduce the catalytic event into an electrical signal. Among other pre-requisites, redox mediators must be readily oxidized/reduced at the electrode surface and easily interact with the biorecognition component. Small chemical compounds (e.g. ferrocene derivatives, ruthenium or osmium complexes and viologens) are frequently used for this purpose, but lately, small redox proteins (e.g. horse heart cytochrome c) have also played the role of redox partners in biosensing applications. In general, the docking between two complementary proteins introduces a second level of selectivity to the biosensor and enlarges the list of compounds targeted for analysis. Moreover, electrochemical interferences are frequently minimized owing to the small overpotentials achieved. This paper aims to provide an overview of enzyme biosensors that are mediated by electron transfer proteins. The article begins with a few considerations on mediated electrochemistry in biosensing 2 systems and proceeds with a detailed description of relevant works concerning the cooperative use of redox enzymes and biological electron donors/acceptors.

A comprehensive view of nitrite reductases

The authors acknowledge the support of the research centers Applied Molecular Biosciences Unit-UCIBIO, which is co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). CMS acknowledges support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI). Publisher Copyright: © 2022 Elsevier B.V.The last years have witnessed a steady increase of social and political awareness for the need of studying, monitoring, and controlling several anthropological activities that are dramatically impacting the environment and human health. The increasing turnover rates of the nitrogen cycle across the Planet are of major concern, so the understanding of the biological, chemical, and physical processes associated with the biogeochemical nitrogen cycle has been attracting the attention of several scientific disciplines. For many years, the primary focus has been the so-called “dissimilatory reduction of nitrate”, which refers to the stepwise conversion of nitrate into molecular nitrogen, closely followed by the assimilatory nitrate reduction pathway, which allow nitrogen incorporation into biomolecules. The contribution of bioinorganic chemists to better understand the enzymology underlying these two branches of the N-cycle has been remarkable. The constant development of mechanistic, structural, and biological tools has been keeping this bioinorganic chemistry field very active, making it a highly relevant research area still today. In this paper, we review the state-of-the-art in both dissimilatory and assimilatory nitrite reducing enzymes, highlighting the structural peculiarities of the different metalloenzymes involved in this step.publishersversionpublishe

Electrocatalysis by heme enzymes—applications in biosensing

Funding Information: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Funding Information: Acknowledgments: We acknowledge the support from Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE 2020-Programa Operacional Competitividade e Internacionalização (POCI); from FCT—Fundação para a Ciência e a Tecnologia (PTDC/BIA-BFS/31026/2017 and 2020.05017.BD) and from the European Union's Horizon 2020 Research and Innovation Program, through TIMB3 and B-LigZymes projects (grant agreements No 810856 and 824017, respectively). We thank Edilson Galdino for critical reading of the manuscript and helpful discussions. Funding Information: Funding: The APC was funded by TIMB3 project, European Union's Horizon 2020 Research and Innovation Program grant agreement No 810856. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Heme proteins take part in a number of fundamental biological processes, including oxygen transport and storage, electron transfer, catalysis and signal transduction. The redox chemistry of the heme iron and the biochemical diversity of heme proteins have led to the development of a plethora of biotechnological applications. This work focuses on biosensing devices based on heme proteins, in which they are electronically coupled to an electrode and their activity is determined through the measurement of catalytic currents in the presence of substrate, i.e., the target analyte of the biosensor. After an overview of the main concepts of amperometric biosensors, we address transduction schemes, protein immobilization strategies, and the performance of devices that explore reactions of heme biocatalysts, including peroxidase, cytochrome P450, catalase, nitrite reductase, cytochrome c oxidase, cytochrome c and derived microperoxidases, hemoglobin, and myoglobin. We further discuss how structural information about immobilized heme proteins can lead to rational design of biosensing devices, ensuring insights into their efficiency and long-term stability.publishersversionpublishe

Development of new analytical tools for monitoring of cardiovascular disease markers – towards the detection of homocysteine-thiolactone

Poster presented at the 4th International Conference on Bio-Sensing Technology, 10-13 May 2015, Lisbon, Portuga

New PON1-based biosensor for the detection of homocysteine-thiolactone in human plasma

Poster presented at the XXIII International Symposium on Bioelectrochemistry and Bioenergetics, 14-18 June 2015, Malmo, Sweden

Respiratory versatility in Desulfovibrio desulfuricans ATCC 27774 – a proteomic approach

Poster presented at the Bacterial Electron Transfer Processes and their Regulation Meeting, European Federation of Biotechnology Microbial Physiology Section, 15-18 March 2015, Vimeiro, Portugal

Measuring the cytochrome c nitrite reductase activity—practical considerations on the enzyme assays

Hindawi Publishing Corporation Bioinorganic Chemistry and Applications Volume 2010, Article ID 634597, 8 pagesThe cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force

Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile Deinococcus radiodurans

Funding Information: This research was funded by FCT, Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit (UIDB/04612/2020, UIDP/04612/2020) and LS4FUTURE Associated Laboratory (LA/P/0087/2020), with national funds through FCT, Fundação para a Ciência e a Tecnologia (PTDC/BBBEBB/0122/2014, IF/00710/2014, PTDC/BII-BBF/29564/2017 and PTDC/BIA-BFS/31026/2017), post doc fellowship SFRH/BPD/97493/2013 (EM), and PhD fellowship COVID/BD/152598/2022 (BS). Funding is also acknowledged for the TIMB3, European Union’s Horizon 2020 research and innovation program, under grant agreement No. 810856. Publisher Copyright: © 2024 by the authors.Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium Deinococcus radiodurans is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H2O2 detected at lower pH. A sequence alignment with other DyPs reveals that DrDyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting DrDyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of DrDyP and DrDyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of DrDyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H2O2 substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to DrDyP.publishersversionpublishe

Measuring the Cytochrome c

The cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC 27774 is able to reduce nitrite to ammonia in a six-electron transfer reaction. Although extensively characterized from the spectroscopic and structural points-of-view, some of its kinetic aspects are still under explored. In this work the kinetic behaviour of ccNiR has been evaluated in a systematic manner using two different spectrophotometric assays carried out in the presence of different redox mediators and a direct electrochemical approach. Solution assays have proved that the specific activity of ccNiR decreases with the reduction potential of the electronic carriers and ammonium is always the main product of nitrite reduction. The catalytic parameters were discussed on the basis of the mediator reducing power and also taking into account the location of their putative docking sites with ccNiR. Due to the fast kinetics of ccNiR, electron delivering from reduced electron donors is rate-limiting in all spectrophotometric assays, so the estimated kinetic constants are apparent only. Nevertheless, this limitation could be overcome by using a direct electrochemical approach which shows that the binding affinity for nitrite decreases whilst turnover increases with the reductive driving force

Star-Shaped Gold Nanoparticles as Friendly Interfaces for Protein Electrochemistry: the Case Study of Cytochrome c

UID/QUI/50006/2019 POCI‐01‐0145‐FEDER‐007265 UID/Multi/04378/2019 POCI‐01‐0145‐FEDER‐007728 PTDC/NAN‐MAT/30589/2017 grant NORTE‐01‐0145‐FEDER‐000011 SFRH/BD/132057/2017Gold nanostars with an average tip-to-tip length of 52±6 nm were functionalized with different capping agents and used as electrode modification materials for protein electrochemistry. Direct electron transfer between cytochrome c and nanostar-coated pyrolytic graphite electrodes was observed with the protein in solution. The electrochemical response was improved at nanostars functionalized with a 1 : 1 mixture of 11-mercaptoundecanoic acid and 4-mercaptobenzoic acid in comparison with gold nanospheres coated with a similar functionalization. Further immobilization of cytochrome c on pyrolytic graphite while conjugated with the same nanostars guaranteed the maintenance of the protein's native properties, whereas direct adsorption on the bare or nanostar-modified electrodes resulted in an altered conformational state. The pseudo-peroxidase activity of the altered cytochrome c was enhanced in the presence of the nanostars.publishe