The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 +/- 1 subunits of 15254.3 +/- 7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Further-more, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance. (C) 2009 Published by Elsevier Inc.

A presente Dissertação consiste num estudo do comportamento competitivo de entidades, produtoras e comercializadoras de energia eléctrica, num período de tempo antes e após a entrada em vigor do Mercado Ibérico de Electricidade (MIBEL). Esse período de tempo engloba os meses entre Abril de 2007 e Abril de 2008, sendo que o MIBEL teve início a 1 de Julho de 2007. O estudo incide principalmente no grau de competitividade ou ausência da mesma, que as referidas entidades adoptaram após o começo do MIBEL. Para poder efectuar essa avaliação, recorre-se a uma abordagem baseada em variações conjecturais, que constituem um indicador desse grau de competitividade. Ou seja, as variações conjecturais representam a convicção que determinada empresa X tem face ao comportamento que outra empresa Y adoptará após uma acção efectuada pela empresa X. Por acção depreende-se, por exemplo, uma redução ou um aumento da quantidade de produção de uma empresa. Se o valor da variável conjectural for 0, as empresas encontram-se a concorrer à Cournot. Caso seja -1, as empresas encontram-se em concorrência perfeita. Por outro lado, se o valor da variação conjectural for superior a 0 pode existir uma situação de conluio entre as empresas. De modo a obter estes valores das variações conjecturais, utiliza-se e readapta-se um simulador de mercado diário, denominado SiMEC - Simulador do Mercado Eléctrico e do Carbono. Este simulador, que anteriormente simulava apenas períodos temporais precedentes ao MIBEL, foi reajustado de modo a incluir dados pós MIBEL. Finalmente, são então realizados gráficos comparativos e retiradas conclusões acerca do comportamento das entidades face a alterações na estrutura do mercado de electricidade.

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

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Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

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