Structural and functional studies of PpcA: a key protein in the electron transfer pathways of Geobacter sulfurreducens

Dissertação para obtenção do Grau de Doutor em Bioquímica, ramo de BiotecnologiaGeobacter species show an impressive respiratory versatility and can sustain their growth by using insoluble extracellular compounds as terminal electron acceptors. The genome of Geobacter sulfurreducens has been completely sequenced and it revealed an unprecedented number of putative c-type cytochromes, 73 of which with more than one heme binding site. Although numerous electron transfer proteins have been identified, the electron transfer pathways that allow G. sulfurreducens to obtain energy are still far from being understood. Five homologous triheme cytochromes (PpcA-E) were identified in G. sulfurreducens periplasm and gene knockout studies revealed their involvement in Fe(III) and U(VI) extracellular reduction. This thesis focuses on the characterization of these proteins, with special emphasis on PpcA, the most abundant in G. sulfurreducens’ periplasm. PpcA, PpcB, PpcD and PpcE were thermodynamically characterized in detail using Nuclear Magnetic Resonance and ultraviolet-visible spectroscopy. The results obtained showed that PpcA and PpcD were able to perform e-/H+ energy transduction in addition to their role in the electron transfer pathways. No evidence for coupling of e-/H+ transfer was observed for PpcB and PpcE. The functional implications of these results are discussed. PpcA solution structure in the fully reduced state was determined using NMR spectroscopy and the redox-Bohr center responsible for controlling the e-/H+ transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. In order to elucidate the physiologic function of PpcA individual key residues and understand its functional mechanism, a family of mutants covering the entire protein was prepared using site-directed mutagenesis. The results obtained revealed how proper tuning of the reduction potentials of the heme groups is fundamental to achieve concerted e-/H+ transfer.Fundação para a Ciência e Tecnologia - Projecto de Re-Equipamento Científico (REDE/1517/RMN/2005); Bolsa de Doutoramento SFRH/BD/37415/2007; Projecto PTDC/QUI/70182/2006 atribuído ao Prof. Carlos Salgueiro, e à Fundação das Universidades Portuguesas através da Acção Integrada E-69/0