Role of Metals in Biology

Bioinorganic, Biophysics, Biocatalysis
Electron Transfer, Biocycles, Bioconversion of Energy
Metal Transport and Homeostasis
Inorganic Systems as Models for Metalloenzymes
Spectroscopy (NMR, EPR and Mössbauer)
(Bio)Electrochemistry and Biosensing
Computational Chemistry

Study the role of metal ions in Biology has been a long-standing objective. The focus has been maintained in the structural and functional characterisation of metalloenzymes, metal ions metabolism (tolerance and toxicity), metallodrugs and reactive oxygen and nitrogen species metabolism (formation, consumption and tolerance). For these purposes, the group has gathered competencies in Biochemistry and Biophysics and in other scientific areas such as (but not restricted to) Molecular Bbiology, Microbiology, Proteomics, Bioinorganic Chemistry, Computational Chemistry, Crystallography, Spectroscopy and Electrochemistry.

The main goal of the group is to understand electron transfer chains involved in important bacterial processes relevant to biocatalysis, bioenergy and ecology, namely denitrification and sulfate reduction. The metal centres studied include haems, iron-sulfur centres, as well as centres of molybdenum, tungsten, copper, cobalt, zinc and nickel. Presently, one of the mostly targeted pathway is denitrification, in particular the least characterised enzymes, nitric oxide reductase and nitrous oxide reductase. The research is primarily directed toward the 3D structure and the study of the catalytic mechanism, by identification and characterisation of catalytic intermediate species.

The objectives were designed to promote intervention in interface areas in order to solve complex problems using knowledge and know-how from Chemistry and Biology, with large implications on areas such as biomolecular structure, molecular recognition, environment (biocycles), (bio)energy, bioremediation, biosensing. The activities contribute (in the past and we hope in the future) to an increase of the critical mass and competences, and develop synergies resulting in the strengthening of the areas of research in an international way (areas of Life Sciences).

DENITRIFICATION AND THE NITROGEN BIOCYCLE
Denitrification is a stepwise sequential pathway that transforms nitrate in dinitrogen, having nitrite, NO and N₂O as intermediates. Nitrate, nitrite, NO and N₂O reductases and their mechanism of reduction are highlighted below.

Nitrate Reductases
The active sites of nitrate reductases have been studied in detail and mechanisms proposed by spectroscopy, enzymatic kinetics, crystallography and DFT calculations. The active sites of nitrate reductases and formate dehydrogenases are very close and crucial changes have been identified. The molybdenum (or tungsten) reactivity and specificity toward a substrate is determined by the polypeptide chain of the enzyme, which tunes the chemical properties of the metal ion.
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Nitrite Reductases
Nitrite reduction can be accomplished by copper (CuNiR) and cytochrome c and d₁ enzymes (cd₁NiR) to NO or to ammonia by multiheme cytochrome c type enzymes (CcNiR).
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Nitric Oxide Reductase
A nitric oxide reductase (NOR) was purified from Pseudomonas nautica. It is a membrane bound enzyme that consists of two subunits: NOR B (heme c, a low spin heme b and a non-heme iron) and NOR C (one c type heme)...
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Nitrous Oxide Reductase
The final step of bacterial denitrification, the two electron reduction of N2O to N2 is catalyzed by a multicopper enzyme named nitrous oxide reductase (N2OR). The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The structure of CuZ centre opened a novel area of research in metallobiochemistry. In the last decade, there has been progress in defining the structure of the CuZ centre, characterizing the mechanism of nitrous oxide reduction, and identifying intermediates of this reaction.
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MOLYBDENUM AND TUNGSTEN ENZYMES
Reactivity, Structures and Mechanisms
Molybdenum and tungsten are heavy metallic elements, belonging to the 6^th group of the ''d-block'' of the Periodic Table. They are trace elements, either on the Universe or on Earth crustal rocks and oceans. In spite of that scarcity, molybdenum is essential to most organisms, from archaea and bacteria to higher plants and mammals, being found in the active site of enzymes that catalyse redox reactions involving carbon, nitrogen and sulfur atoms of key metabolites. Some of the molybdenum-dependent reactions constitute key steps in the global biogeochemical cycles of carbon, nitrogen, sulfur and oxygen. Presently, more than 50 molybdenum-containing enzymes are known, with the great majority being prokaryotic, and eukaryotes holding only a restricted number of molybdoenzymes (humans have 4 molybdoenzymes). Tungsten, probably because of its limited bioavailability, is, by far, less used. It is present in anaerobic prokaryotes, most of them termophilics (but not exclusively, as first though), and also in some strictly aerobic bacteria (e.g., in some methylotrophs).
The group has been extensively involved in the study of several molybdo- and tungstoenzymes. Namely:
(i) molybdenum-containing aldehyde oxidoreductase (bacterial), xanthine dehydrogenase, xanthine oxidase and aldehyde oxidase (mammals), from the xanthine oxidase family;
(ii) molybdenum- and tungsten-containing formate dehydrogenases (bacterial), from the DMSOR family;
(iii) the molybdenum-containing nitrate reductases, respiratory and periplasmatic enzymes (bacterial), also from the DMSOR family.
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NOVEL MOLYBDENUM CENTERS
Proteins Containing Novel Molybdenum Heterometallic Clusters
The orange protein (ORP), first isolated from the sulfate-reducing organism Desulfovibrio gigas, is found in several anaerobic bacteria. It harbors a unique molybdenum/copper heterometallic cluster, [S₂Mo^VIS₂Cu^IS₂Mo^VIS₂]³⁻ (Mo/Cu), revealed by EXAFS...
The biological function of ORP is still unknown; however, it is tempting to suggest that it may be related to its ability to chelate copper... Yet, to our knowledge, in spite of this well-known chemistry, the Mo/Cu complex is unique to ORP, and its identification in the D. gigas ORP in 20001 was surprising.
In this project, we aim to understand the mechanism of the in vivo formation of the ORP Mo/Cu cluster, as well as, its physiological role.
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NEW PATHWAYS FOR NITRIC OXIDE FORMATION
Generation of NO by molybdoenzymes
Nitric oxide radical (NO) is a signalling molecule that plays important regulatory roles in several physiological processes, in all forms of life. In mammals, NO is predominantly catalysed by NO synthases ... Recent studies have shown that NO can also be formed by nitrite reduction through several pathways, including by the molybdenum-containing enzyme xanthine oxidoreductase... To contribute to a better characterisation of this new NO production pathway, we are investigating if the nitrate/nitrite reductase activities are a common feature of other molyboenzymes (prokaryotic and eukaryotic enzymes) and we are studying the molecular mechanism of these reductions and its physiological implications.
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ATMOSPHERIC CARBON DIOXIDE UTILIZATION
Using bacterial formate dehydrogenases to develop new (bio)catalysts for carbon dioxide reduction
The global energy demand and the present high dependence on fossil fuels have caused an unprecedented increase in the Earth’s atmosphere carbon dioxide concentration... the carbon dioxide abundance and low cost make it an interesting source for the production of chemical feedstocks and fuels...
In this project, we propose to use the bacterial molybdenum and tungsten-containing formate dehydrogenase enzymes as a model to understand the mechanistic strategies and key chemical features needed to efficiently and specifically reduce carbon dioxide to formate. We want to understand the structure-activity relationships of the enzymes active site and the possible reaction mechanisms at atomic level. The information gathered will guide the development of new efficient (bio)catalysts for the atmospheric carbon dioxide utilization, to produce energy and chemical feedstocks, while reducing an important environmental pollutant.
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DEVELOPMENT OF ELECTROCHEMICAL BIOSENSORS
In recent years, there has been a budding increase in the social and political awareness of the need for monitoring and controlling environmental and industrial processes. As a consequence, there is a strong need to develop improved analytical tools which allow the reliable inspection of the chemical or biological composition of every material which interacts with consumers or nature. In this context, biosensors technology constitutes a very important area of R&D. The electrochemical enzyme based devices are of particular interest due to their operational simplicity, low cost fabrication, portability and real-time monitoring ability. Several electrode configurations have been tested, using a variety of immobilization materials, such as non-conducting polymers, electropolymerized films functionalized with redox groups, sol-gel glasses and modified clays. The direct electron transfer between nitrite reductase and a pyrolytic graphite electrode has been also explored. As expected, the analytical performance (detection limit, linear range, sensitivity and operational stability) of each system is strongly dependent on the electrode design.
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BIOCORROSION
BIOCOR is a Marie Curie Initial Training Network (EU FP7) focused on research and training in the area of biocorrosion.
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