Raman Spectroscopy of Metalloproteins
Research in the Laboratory for Raman spectroscopy of metalloproteins is focused on structural and functional characterization of redox proteins that perform diverse functions in cells, including electron transport, detoxification and enzymatic catalysis.
Phone (+351) 214469717
We are interested in the biophysico-chemical properties of several metalloproteins with the specific goal of understanding how different proteins fine-tune the reactivity of their metal cofactors and how such metal centers help to tailor the dynamics and stability of protein molecules. We employ Raman, resonance Raman (RR) and surface enhanced resonance Raman (SERR) spectroscopies in both stationary and time resolved modes, complemented with MD calculations and electrochemical and other spectroscopic (EPR, IR, SEIRA) methods, to reveal the structural and mechanistic features of various soluble and membrane metalloproteins. Special emphasis is given to investigations of the redox processes of membrane proteins under conditions that reproduce basic features of natural membrane. At the membrane, the mobility of the reaction partners is restricted and electron transfer (ET) takes place under the influence of strong electric fields and along pathways with different dielectric constants. These conditions are mimicked by immobilizing proteins onto biocompatible metal electrodes, which are then probed by surface enhanced vibrational spectroscopy to provide information about the structure of the redox sites, the thermodynamics and kinetics of ET and also protein dynamics. We study inter- and intra-protein ET in heme-copper oxygen reductases, employing systems with different levels of complexity, including domains of subunits, individual subunits and integral proteins. Using these approaches, we have established the molecular basis for the functioning of several oxygen reductases.
We are also interested in utilizing immobilized proteins as biosensors. In such systems the structural integrity of the immobilized enzyme, its catalytic activity and the efficiency of electronic communication between the enzyme and the metal support are crucial determinants for the performance of the enzyme-based device. To this end, we have contributed to the characterization of these parameters in immobilized nitrite reductases, peroxidases and cytochromes P450, by employing SERR spectroelectrochemistry.
We employ highly sensitive RR spectroscopy to provide insights into physiological processes that involve heme proteins. These include: i) the sensing of external stimuli by chemotaxis proteins; ii) the interaction of antibiotics with proteins that help pathogens to evade the hosts´ immune system, and iii) the fate of the heme group upon release from hemoglobin under inflammatory conditions of mammalian tissues.
- Murat Sezer, post doc fellow
- Célia Silveira, post doc fellow
- Zélia Gouveia, PhD student
- Daniela Presa, research student
- Patrycja Kielb, Erasmus student
- Todorovic, S., Pereira M., Bandeiras, T., Teixeira, M., Hildebrandt, P., Murgida, D. H. (2005) `Midpoint potentials of Hemes a and a3 in the Quinol Oxidase from Acidianus ambivalens are Inverted` J. Am. Chem. Soc. 127 13561-13566
- Todorovic, S. Veríssimo, A., Pereira, M., Teixeira, M., Hildebrandt, P., Zebger, I., Wisitruangsakul, N., Murgida, D. (2008) ´SERR-spectroelectrochemical study of a cbb3 oxygen reductase in a biomimetic construct´ J. Phys. Chem. B, 112 16952-16959
- Sezer, M., Genebra, T., Mendes, S., Martins, L. O., Todorovic, S. (2012) ´A DyP-type peroxidase at a biocompatible interface: structural and mechanistic insights´ Soft Matter, DOI: 10.1039/c2sm26310f
For further information visit the laboratory's website
A espectroscopia de ressonância Raman (RR) permite a caracterização estrutural e funcional de proteinas contendo centros metálicos, tais como ferro hémico ou não hémico, cobre, cobalto ou ferro-enxofre. Com a RR também podemos investigar diferentes processos, incluindo: a libertação de hemo da hemoglobina em mamíferos com doenças inflamatórias; a detecção de NO ou CO por proteínas sensoras que bactérias utilizam para reagir a condições externas; ou ainda a acção de antibióticos inibitórios de bactérias patogénicas.
A RR aumentada de superfície (SERR) é utilizada em estudos de transferência electrónica de proteínas membranares. Esta abordagem permite-nos compreender processos enzimáticos visando o desenvolvimento de dispositivos bioelectrónicos, nomeadamente biosensores. A reductase de nitrito, peroxidases ou citocromo P450, os quais detectam nitrito e peróxido de hidrogénio ou outros substratos com grande sensibilidade e selectividade, são exemplos de componentes enzimaticas de biosensores.