Gene knock-out studies on Geobacter sulfurreducens (Gs) cells showed that the periplasmic triheme cytochrome PpcD is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. More recently, it was also shown that the gene encoding for PpcD has higher transcript abundance when Gs cells utilize graphite electrodes as sole electron donors to reduce fumarate. This sets PpcD as the first multiheme cytochrome to be involved in Gs respiratory pathways that bridge the electron transfer between the cytoplasm and cell exterior in both directions. Nowadays, extracellular electron transfer (EET) processes are explored for several biotechnological applications, which include bioremediation, bioenergy and biofuel production. Therefore, the structural characterization of PpcD is a fundamental step to understand the mechanisms underlying EET. However, compared to non-heme proteins, the presence of numerous proton-containing groups in the redox centers presents additional challenges for protein signal assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with 1H, 13C and 15N backbone and side chain assignments of the reduced form of PpcD.

Gene knock-out studies on Geobacter sulfurreducens cells showed that the periplasmic triheme cytochrome PpcA is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI) oxides. The crucial role of this protein in bridging the electron transfer between the cytoplasm and cell exterior was further supported by proteomics studies. In comparison with non-heme proteins, the presence of numerous proton-containing groups in the heme groups causes additional challenges to the full protein assignment and structure calculation. Here, we report the complete assignment of the heme proton signals together with the 1H and 15N backbone and side chain assignments of the reduced form of PpcA.

Gene knockout studies on Geobacter sulfurreducens (Gs) cells showed that the outer membrane cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) citrate and U(VI) oxide. In addition, microarray analysis of OmcF-deficient mutant versus the wild-type strain revealed that many of the genes with decreased transcript level were those whose expression is upregulated in cells grown with a graphite electrode as electron acceptor. This suggests that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electrical current production by Gs in microbial fuel cells. Extracellular electron transfer processes (EET) constitute nowadays the foundations to develop biotechnological applications in biofuel production, bioremediation and bioenergy. Therefore, the structural characterization of OmcF is a fundamental step to understand the mechanisms underlying EET. Here, we report the complete assignment of the heme proton signals together with (1)H, (13)C and (15)N backbone and side chain assignments of the OmcF, excluding the hydrophobic residues of the N-terminal predicted lipid anchor.

The bacterium Geobacter metallireducens is capable of transferring electrons to the cell exterior, a process designated extracellular electron transfer. This mechanism allows the microorganism to reduce extracellular acceptors such as Fe(III) (hydr)oxides and water toxic and/or radioactive contaminants including Cr(VI) and U(VI). It is also capable of oxidizing waste water aromatic organic compounds being an important microorganism for bioremediation of polluted waters. Extracellular electron transfer also allows electricity harvesting from microbial fuel cells, a promising sustainable form of energy production. However, extracellular electron transfer processes in this microorganism are still poorly characterized. The triheme c-type cytochrome PpcA from G. metallireducens is abundant in the periplasm and is crucial for electron transfer between the cytoplasm and the cell’s exterior. In this work, we report near complete assignment of backbone, side chain and heme resonances for PpcA in the oxidized state that will permit its structure determination and identification of interactions with physiological redox partners.

Microbial electrosynthesis is an emerging green technology that explores the capability of a particular group of microorganisms to drive their metabolism toward the production of hydrogen or value-added chemicals from electrons supplied by electrode surfaces. The cytochrome PccH showed the largest increase in transcription when electrons are supplied to Geobacter sulfurreducens biofilms. Gene knock-out experiments have shown that the electron transfer toward G. sulfurreducens cells was completely inhibited by the deletion of the gene encoding for cytochrome PccH. This identifies a crucial role for this protein in G. sulfurreducens microbial electrosynthesis mechanisms, which are currently unknown. In this work, we present the backbone (1H, 13C and 15N) and heme assignment for PccH in the oxidized state. The data obtained paves the way to identify and structurally map the molecular interaction regions between the cytochrome PccH and its physiological redox partners.

Using 2D-NMR the four haems of Desulfovibrio vulgaris (Hildenborough) cytochromes, within the X-ray structure were fully cross-assigned according to their redox potential. The strategy used was based on a complete network of chemical exchange connectivities between the NMR signals obtained for all oxidation levels to the corresponding ones in the fully reduced spectrum [1992, Eur. J. Biochem., in press]. This unequivocal cross-assignment disagrees within earlier results obtained for the similar protein from Desulfovibrio vulgaris (Miyazaki F.) [1991, FEBS Lett. 285, 149–151]

The discovery of microorganisms with the ability of Extracellular Electron Transfer (EET), nearly three decades ago, sparked interest due to their ability to be used in diverse applications that can range from bioremediation to electricity production in Microbial Fuel Cells (MFC). Microbial respiration is based on electron transfer from a donor to an electron acceptor, through a series of stepwise electron transfer events that generate the necessary metabolic energy. Some microorganisms, such as Pseudomonas species, Shewanella putrefaciens or Geothrix fermentans are able to produce electrochemical mediators to increase the EET. The mechanical stability of the biofilm is provided by the biofilm matrix, a hydrated extracellular polymeric matrix that encases the biofilm cells. The biofilm matrix could potentially offer a resistance pathway to EET unless bacteria develop strategies to increase its conductivity. MFC devices currently being used and studied do not generate sufficient power to support widespread and cost-effective applications.

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely 31P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8α-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.