Research Lab

Biochemistry and Bioenergetics of Heme Proteins

Biochemistry and Bioenergetics of Heme Proteins
29673

Lab Members

Lab leader

Postdoc

PhD student

Other members

Master Students

Research Interests

The main research lines of our group aim to contribute to the optimization of extracellular electron transfer processes in exoelectrogens using the bacteria Geobacter as a model. The rationale for this choice is based on the ubiquitous presence of these bacteria in the environment and their high current densities output in microbial fuel devices. We are developing new methodologies to assist the structural and functional characterization of electron transfer components of Geobacter cells. This information will contribute to the rational engineering of exoelectrogens electron transfer components and to optimize the Geobacter-based biotechnological applications (bioremediation, microbial electrosynthesis and bioenergy).

Research Highlights
Molecular interactions between Geobacter sulfurreducens triheme cytochromes, redox active analogue for humic substances and iron citrate

The bacterium Geobacter sulfurreducens can transfer electrons to quinone moieties of humic substances or to anthraquinone-2,6-disulfonate (AQDS), a model for the humic acids. The reduced form of AQDS (AH2 QDS) can also be used as energy source by G. sulfurreducens. Such bidirectional utilization of humic substances confers competitive advantages to these bacteria in Fe(III) enriched environments. Among others, Fe(III)- citrate can also be utilized as terminal acceptor by G. sulfurreducens. Stopped-flow kinetic measurements and biomolecular NMR interaction studies showed that the triheme periplasmic cytochromes have a bifunctional behavior toward the humic substance analogue. They can reduce AQDS, but the proteins can also be reduced by AH2 QDS. The extent of the electron transfer is thermodynamically controlled favoring the reduction of the cytochromes. The chemical shift perturbations on cytochromes backbone NH and 1 H heme methyl signals were used to map their interaction regions with AQDS, showing that each protein forms a low-affinity binding complex through well-defined positive surface regions in the vicinity of the heme groups. Overall, the results obtained provided important structural-functional relationships to rationalize the microbial respiration of humic substances in G. sulfurreducens. NMR chemical shift perturbation studies were also explored to evaluate the molecular interactions between the periplasmic triheme cytochromes and Fe(III) citrate. Also in this case, the complex interfaces encompassed positively charged patches in the cytochromes (BBA - Bioenergetics, Vol. 1859,619, 2018; Dalton Trans, Vol. 46, 2350, 2017).

fig1Molecular interactions between periplasmic triheme cytochrome PpcB and Fe(III)-citrate, mapped by NMR

 

Structural and functional characterization of the outer membrane cytochrome OmcF from G. sulfurreducens

Gene knockout studies on G. sulfurreducens cells showed that the outer membrane-associated monoheme cytochrome OmcF is involved in respiratory pathways leading to the extracellular reduction of Fe(III) and U(VI). In addition, microarray analysis of an OmcF-deficient mutant revealed that many of the genes with decreased transcript level were those whose expression is up-regulated in cells grown with a graphite electrode as electron acceptor, suggesting that OmcF also regulates the electron transfer to electrode surfaces and the concomitant electricity production by G. sulfurreducens in microbial fuel cells. 15N,13C-labeled OmcF was produced and NMR spectroscopy was used to determine the solution structure of the protein in the fully reduced state and the pH-dependent conformational changes. In addition, 15N relaxation NMR experiments were used to characterize the overall and internal backbone dynamics of OmcF. Complementary, the detailed electrochemical characterization of this cytochrome showed that OmcF displays high electron transfer efficiency compared to other monoheme cytochromes. The pH dependence of the redox potential indicates that the protein has an important redox-Bohr effect in the physiological pH range for G. sulfurreducens growth. The redox-Bohr centre was assigned to the side chain of His47 residue and its pKa values in the reduced and oxidized states were determined. The enthalpy, entropy and Gibbs free energy associated with the redox transaction indicated that the reduced form of OmcF is the most favourable. Overall the data showed that G. sulfurreducens cells evolved to warrant a down-hill electron transfer from the periplasm to the outer-membrane associated cytochrome OmcF (BBA - Bioenergetics, Vol. 1858,733, 2017; BBA - Bioenergetics, Vol. 2728, 30218, 2018).

 

 

fig2Structural map of pH-linked conformational changes in OmcF

 

Representative Projects

  • “Look out over the wall: rational powering of microbial nanowires for sustainable bioenergy-based applications”, FCT-MCTES, Total and Unit funding: €220,459, Carlos Salgueiro (PI).
  • “The way forward: optimization of respiratory electron transfer chains toward sustainable microbial electricity production”, FCT-MCTES, Total and Unit funding: €166,862, Carlos Salgueiro (PI).
  • “The difference a cell wall makes: optimization of bioelectrochemical systems by exploring the paradigm of extracellular electron transfer in Gram positive bacteria”, FCT-MCTES, Total funding: €196,926, Unit funding: €24,000, Carlos Salgueiro (Collaborator).

Selected Publications

Fernandes, TM; Portela, PC; Dantas, JM; Salgueiro, CA. 2018. Microbial electricity production: Biomolecular elucidation of extracellular electron transfer mechanisms of Geobacter metallireducens. FREE RADICAL BIOLOGY AND MEDICINE, 120, DOI: 10.1016/j.freeradbiomed.2018.04.287
Fernandes, AP; Nunes, TC; Paquete, CM; Salgueiro, CA. 2017. Interaction studies between periplasmic cytochromes provide insights into extracellular electron transfer pathways of Geobacter sulfurreducens. BIOCHEMICAL JOURNAL, 474, DOI: 10.1042/BCJ20161022
Dantas, JM; Brausemann, A; Einsle, O; Salgueiro, CA. 2017. NMR studies of the interaction between inner membrane-associated and periplasmic cytochromes from Geobacter sulfurreducens. FEBS LETTERS, 591, DOI: 10.1002/1873-3468.12695
Morgado, L; Bruix, M; Pokkuluri, PR; Salgueiro, CA; Turner, DL. 2017. Redox- and pH-linked conformational changes in triheme cytochrome PpcA from Geobacter sulfurreducens. BIOCHEMICAL JOURNAL, 474, DOI: 10.1042/BCJ20160932
Silveira, CM; Castro, MA; Dantas, JM; Salgueiro, C; Murgida, DH; Todorovic, S. 2017. Structure, electrocatalysis and dynamics of immobilized cytochrome PccH and its microperoxidase. PHYSICAL CHEMISTRY CHEMICAL PHYSICS, 19, DOI: 10.1039/c6cp08361g