Biochemistry / EPR, UV-visible and Mössbauer Spectroscopies / Fast kinetics / Molecular Biology / Iron containing enzymes / Oxidative stress / Denitrification / Membrane-bound enzymes / Iron storage proteins
Research work focused in oxidative stress and detoxification mechanisms.
Enzymes isolated from denitrifying bacteria that catalyze elimination of oxidizing species derived from molecular oxygen, namely Pseudomonas stutzeri cytochrome c peroxidase and Pseudomonas nautica nitric oxide reductase (an integral membrane enzyme). Establishment of the expression conditions in the original host, purification, equilibrium and fast kinetic, EPR, UV-visible and NMR spectroscopies and molecular biology techniques.
Catalytical mechanism of bacterial iron storage enzymes, such as Ferritins , baterioferritins and the oxidative stress DNA protection enzyme DPS. A membrane-bound, iron containing, plant desaturase, an enzyme involved in resistance to environmental conditions, was also studied.
Study of a bacterial oxidative stress sensor – PerR – in a strict anaerobic bacterium, Desulfovibrio vulgaris. This bacterial repressor, contains a structural Zinc metallic centre and an active Iron centre. In the presence of both Iron and hydrogen peroxide, it adopts an open conformation, unable to bind DNA, allowing for oxidative stress protection enzymes, such as catalases and bacterioferritins to be expressed.
More recently, study of an haemic protein, isolated from the bacterium Shewanella baltica, able to transiently bind molecular oxygen and which possesses a highly unusual axial coordination.
Genes coding for the proteins were cloned and overexpressed in Escherichia coli. Spectroscopic techniques were used in the detection and identification of reaction intermediates, with special emphasis on Mössbauer spectroscopy. A general approach is to use stopped-flow absorption measurements for initial identification of the number of colored accumulating states in a specific reaction, and the formation and decay timescale of the transient species. Based on these information rapid-freeze quench EPR and Mössbauer experiments are designed for further analysis
1. Cristina G. Timóteo, Pedro Tavares, Graham W. Pettigrew and Isabel Moura, “Calcium in Bacterial Peroxidases - Pseudomonas stutzeri cytochrome c peroxidase“ (2001) J Inorg Biochem, 86, 456.
2. Cristina G. Timóteo, Pedro Tavares, Celia F. Goodhew, Luís C. Duarte, Kornelia Jumel, Francisco M.F. Gírio, Steve Harding, Graham W. Pettigrew and Isabel Moura, “Calcium and Bacterial Peroxidases - The cytochrome c peroxidase from Pseudomonas stutzeri “ (2003) J Biol Inorg Chem, 8, 29-37.
3. Cecília Bonifácio, João M. Dias, Carlos Cunha, Axel Muller, Cristina G. Timóteo, Isabel Moura and Maria J. Romão, “Crystallization and Preliminary X-Ray Diffraction Analysis of the Di-heme Cytochrome c Peroxidase from Pseudomonas stutzeri” (2003) Acta Cryst, D59, 345-347.
4. Carlos E. Martins, Cristina Cordas, Cristina G. Timóteo, Pedro Tavares, Alice S. Pereira, José J.G. Moura and Isabel Moura. “Nitric oxide reductase from Pseudomonas nautica” (2005) Eur. Biophys. J., 34, 663.
5. Cristina M. Cordas, Alice S. Pereira, Carlos E. Martins, Cristina G. Timóteo, Isabel Moura, José J. G. Moura and Pedro Tavares, “Nitric Oxide Reductase: Direct Electrochemistry and Electrocatalytic Activity” (2006) ChemBioChem, 7 (12), 1878-81.
6. Katja Conrath, Alice S. Pereira, Carlos E. Martins, Cristina G. Timóteo, Pedro Tavares, Silvia Spinelli, Joerg Kinne, Christophe Flaudrops, Christian Cambillau, Serge Muyldermans, Isabel Moura, Jose J. G. Moura, Mariella Tegoni and Aline Desmyter, “Camelid Nanobodies Raised against an Integral Membrane Enzyme,Nitric Oxide Reductase”, (2009) Protein Science, 18, 619-628.
7. Cristina G. Timóteo, Alice S. Pereira, Carlos E. Martins, Sunil G. Naik, Américo G. Duarte, José J. G. Moura, Pedro Tavares, Boi Hanh Huynh and Isabel Moura, “Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas náutica”(2011) Biochemistry, 50, 4251-4262.
8. Patrícia M. Paes de Sousa, David Rodrigues, Cristina G. Timóteo, Maria L. Simões Gonçalves, Graham W. Pettigrew, Isabel Moura, José J. G. Moura and Maria M. Correia dos Santos, “Analysis of the activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase through an electron transfer chain”, (2011) J Biol Inorg Chem, 16, 881-888.