Estudo de novos centros contendo ferro Isolamento e caracterizacao de proteinas de desulfovibrio desulfuricans ATCC 27774

Abstract

Although neglected for a long time, non-heme iron proteins are now acknowledge to participate on important metabolic systems as the Krebs cycle, photosynthesis, respiratory chain and DNA synthesis and maintenance. For many years, sulfate reducing bacteria have been a major source of this type of proteins. Enzymes such as APS reductases, hydrogenases, sulfite reductases or ferredoxins have been isolated and characterized, revealing unique biochemical and spectroscopic properties. In this thesis is described the isolation and characterization of several proteins purified from Desulfovibrio desulfuricans ATCC 27774, possessing new types of non heme iron centers. Rubrerithryn is a dimeric protein (2 x 24 kDa). Each monomer contains two different types of centers: i) a monomeric rubredoxin type center, where the iron atom is tetrahedrically coordinated to cysteinyl residues and ii) a binuclear iron center with a #mu#-oxo bridge, similar to the one found in ribonucleotide reductase B2 subunit. This unusual association of centers result in unique spectroscopic properties. The UV-visible spectrum of rubrerithryn has maxima at 370 and 493 nm. Upon subtraction of Desulfovibrio desulfuricans ATCC 27774 rubredoxin spectrum a new maximum at 362 nm is obtained, typical for the binuclear center. The EPR spectrum shows resonances at g=9.6 and 4.3, typical of rubredoxin centers, and a high field rhombic signal (g=1.98, 1.76 and 1.56) that can be ascribed to the binuclear center half reduced form. Analysis of Mossbauer data shows the presence of these two types of centers in all the possible oxidation states. Desulfoferrodoxin is another protein that possesses a new association of iron centers. A single polipeptide chain (13.9 kDa) provides the ligands for the two monometric centers. One of the centers is similar to the iron center of Desulfovibrio gigas desulforedoxin. In this center the iron atom is coordinated by four cysteinyl residues in distorted tetrahedral geometry. Spectroscopically this center gives rise to UV-visible maxima at 495 and 368 nm and EPR resonances at g=7.7, 5.7, 4.1 and 1.8. The Mossbauer spectrum of the oxidized state is correctly simulated using the..Available from Fundacao para a Ciencia e a Tecnologia, Servico de Informacao e Documentacao, Av. D. Carlos I, 126, 1200 Lisboa / FCT - Fundação para o Ciência e a TecnologiaSIGLEPTPortuga