154 research outputs found
Substrate binding to a nitrite reductase induces a spin transition
No full textThe multiheme enzyme nitrite reductase catalyzes a 6-electron reduction of nitrite to ammonia. The reaction is initiated by substrate binding to the free axial position of the high spin penta-coordinated heme active site. The spin configuration of the resulting complex is crucial for discrimination between the heterolytic vs homolytic character of the cleavage of the N-O bond and, therefore, subsequent steps of the catalytic cycle. Here, we report the first experimental evidence, based on resonance Raman spectroscopy, that nitrite binding to the enzyme from D. vulgaris induces a transition from the high spin to the low spin configuration in the catalytic heme, thereby favoring the heterolytic route. © 2010 American Chemical Society.Fil: Martins, Gabriel. Universidade Nova de Lisboa; PortugalFil: Rodrigues, Luisa. Universidade Nova de Lisboa; Portugal. Instituto Gulbenkian de Ciencia; PortugalFil: Cunha, Filipa M.. Universidade Nova de Lisboa; PortugalFil: Matos, Daniela. Universidade Nova de Lisboa; PortugalFil: Hildebrandt, Peter. Technishe Universitat Berlin; AlemaniaFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Inorgánica, Analítica y Química Física; ArgentinaFil: Pereira, Inês A. C.. Universidade Nova de Lisboa; PortugalFil: Todorovic, Smilja. Universidade Nova de Lisboa; Portuga
Functional, structural and biophysical view of repair of oxidation damaged DNA in bacteria and Man
No full text"Living organisms have developed multiple DNA repair machinery to maintain
genome integrity. One of the most severe damages that can be inflicted on DNA is
oxidation damage. These damages are mainly caused by Reactive Oxygen Species
(ROS) which are generated by endogenous sources, such as byproducts of cellular
processes (e.g., respiration), or exogenous sources (e.g., environmental stressors and
chemicals). Repairing these damages is crucial for genome maintenance and
preventing the accumulation of mutations that can contribute to various diseases in
humans. The main mechanism responsible for the detection and repair of oxidation
damaged bases in DNA is Base Excision Repair (BER).(...)
Immobilization of metalloproteins for biotechnological applications
No full textHeme peroxidases couple the oxidation of a variety of organic substrates with reduction
of H2O2 to water. They represent an attractive platform for both detection and monitoring
of H2O2 and biocatalysis of organic compounds. DyP-type peroxidases constitute a novel
family of heme peroxidases. They are capable of efficient decolourisation of several dyes,
including anthraquinone-based and azo dyes that are of industrial and environmental
relevance. DyPs are unrelated to other known peroxidases with respect to primary
sequence, catalytic properties and tertiary structure. In this work we establish the first step
towards exploring the potential of DyP-type peroxidases for biotechnological applications.
We present a purification and combined biochemical, spectroscopic and spectroelectrochemical
study of a recombinant DyP-type peroxidase from Pseudomonas putida
MET94 (PpDyP) immobilized on bio-compatibly coated Ag electrodes. The aim of this work
is to provide structural and mechanistic insights into an immobilized DyP-type peroxidase, as
a basis for a rational design of bio-electronic device(s) employing PpDyP.Peroxidases hémicas oxidam uma vasta variedade de substratos orgânicos mediante a
redução de H2O2 a água. Estas enzimas representam uma plataforma atraente para a
detecção e monitorização de H2O2 bem como para a biocatálise de compostos orgânicos.
DyP peroxidases constituem uma nova família de peroxidases hémicas, capazes de
descolorarem eficientemente vários corantes, incluindo corantes derivados da antraquinona
quer corantes azo e que são de elevado interesse industrial e ambiental. Estas peroxidases
não estão relacionadas com outras peroxidases já conhecidas, quer no que diz respeito à sua
sequência primária, quer propriedades catalíticas e até estrutura terciária. Neste trabalho,
pretende-se estabelecer o primeiro passo para explorar o potencial de DyPs para aplicações
biotecnológicas. É apresentada a purificação combinada com um estudo SERR espectroeletroquímico
de uma DyP recombinante, obtida a partir de Pseudomonas putida MET94
(PpDyP), imobilizada num eléctrodo biocompatìvel de prata (Ag). O objectivo deste trabalho
é fornecer informações estruturais e mecanicistas sobre uma DyP imobilizada, como base
para um desenho racional de um dispositivo bioelectrónico (biosensor) baseado nessa
mesma proteína (PpDyP)
Targeting Heme with Single Domain Antibodies
No full textHeme, i.e. iron (Fe) protoporphyrin IX, functions as a prosthetic group in a variety of
hemoproteins that participate in vital biologic functions essential to sustain life.
Heme is a highly reactive molecule, participating in redox reactions, and
presumably for this reason it must be sequestered within the heme pockets of
hemoproteins, controlling its reactivity. However, under biological stress conditions,
hemoproteins can release their prosthetic groups, generating “free heme”, which
binds loosely to proteins or to other molecules and presumably acquires unfettered
redox activity. Moreover, a growing body of evidence supports the notion that “free
heme” can act in a vasoactive, pro-inflammatory and cytotoxic manner when
released from a subset of these hemoproteins, such as extracellular hemoglobin,
generated during hemolytic conditions. (...
Structure, electrocatalysis and dynamics of immobilized cytochrome PccH and its microperoxidase
No full textGeobacter sulfurreducens cells have the ability to exchange electrons with conductive materials, and the periplasmic cytochrome PccH plays an essential role in the direct electrode-to-cell electron transfer in this bacterium. It has atypically low redox potential and unique structural features that differ from those observed in other c-type cytochromes. We report surface enhanced resonance Raman spectroscopic and electrochemical characterization of the immobilized PccH, together with molecular dynamics simulations that allow for the rationalization of experimental observations. Upon attachment to electrodes functionalized with partially or fully hydrophobic self-assembled monolayers, PccH displays a distribution of native and non-native heme spin configurations, similar to those observed in horse heart cytochrome c. The native structural and thermodynamic features of PccH are preserved upon attachment mixed hydrophobic (-CH3/-NH2) surfaces, while pure -OH, -NH2 and -COOH surfaces do not provide suitable platforms for its adsorption, indicating that its still unknown physiological redox partner might be membrane integrated. Neither of the employed immobilization strategies results in electrocatalytically active PccH capable of the reduction of hydrogen peroxide. Pseudoperoxidase activity is observed in immobilized microperoxidase, which is enzymatically produced from PccH and spectroscopically characterized. Further improvement of PccH microperoxidase stability is required for its application in electrochemical biosensing of hydrogen peroxide.Fil: Silveira, Célia M.. Universidade NOVA de Lisboa; PortugalFil: Castro, Maria Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Dantas, Joana M.. Universidade NOVA de Lisboa; PortugalFil: Salgueiro, Carlos. Universidade NOVA de Lisboa; PortugalFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; ArgentinaFil: Todorovic, Smilja. Universidade NOVA de Lisboa; Portuga
Resonance Raman study of the superoxide reductase from Archaeoglobus fulgidus, E12 mutants and a 'natural variant'
No full textThe resonance Raman (RR) spectra of the oxidized wild-type Archaeoglobus fuglidus 1Fe-Superoxide reductase (SOR), E12V and E12Q mutants were studied at different pH conditions upon excitation in resonance with the pH-dependent charge transfer transition to the ferric iron. The wild-type SOR from Nanoarchaeum equitans that lacks the highly conserved glutamate residue was investigated as a ′natural variant′. No substantial differences were observed in the RR spectra of the active sites of the A. fulgidus proteins. Based on the component analysis in the metal-ligand stretching region the modes involving the Fe-S(Cys) stretching coordinates have been identified. The frequencies of these modes reflect the electronic properties of the Fe-S bond which are related to the catalytic activity of SORs, including reduction of superoxide and product dissociation. Moreover, hydroxide binding to the E12 mutant proteins was demonstrated at high pH. It was further observed that the ferric active site of all three SORs from A. fulgidus senses the presence of phosphate, which possibly replaces the hydroxide at high pH.Fil: Todorovic, Smilja. Universidade Nova de Lisboa; PortugalFil: Rodrigues, João V.. Universidade Nova de Lisboa; PortugalFil: Pinto, Ana F.. Universidade Nova de Lisboa; PortugalFil: Thomsen, Christian. Technische Universität Berlin; AlemaniaFil: Hildebrandt, Peter. Technische Universität Berlin; AlemaniaFil: Teixeira, Miguel. Universidade Nova de Lisboa; PortugalFil: Murgida, Daniel Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentin
Health benefits and risks of fermented foods—the PIMENTO initiative
No full textWorldwide, fermented foods (FF) are recognized as healthy and safe. Despite the rapid increase of research papers, there is a lack of systematic evaluation of the health benefits and risks of FF. The COST Action CA20128 “Promoting innovation of fermented foods” (PIMENTO) aims to provide a comprehensive assessment on the available evidence by compiling a set of 16 reviews. Seven reviews will cover clinical and biological endpoints associated with major health indicators across several organ systems, including the cardiovascular, gastrointestinal, neurological, immune, and skeletal systems. Nine reviews will address broader biological questions associated with FF including bioactive compounds and vitamin production, nutrient bioavailability and bioaccessibility, the role of FF in healthy diets and personalized nutrition, food safety, regulatory practices, and finally, the health properties of novel and ethnic FF. For each outcome assessed in the reviews, an innovative approach will be adopted based on EFSA’s published guidance for health claim submissions. In particular, each review will be composed of three parts: (1) a systematic review of available human studies(2) a non-systematic review of the mechanism of action related to the clinical endpoints measured by the human studies identified in part 1and (3) a non-systematic review of the characterization of the FF investigated in the human studies identified in part 1. The evidence and research gaps derived from the reviews will be summarized and published in the form of a strategic road map that will pave the way for future research on FF
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