Probing the Effect of Ionic Strength on the Functional Robustness of the Triheme Cytochrome PpcA from Geobacter sulf urreducens:A Contribution for Optimizing Biofuel Cell’s Power Density Joana M. Dantas, † Leonor Morgado, † Ana C. Marques, and Carlos A. Salgueiro* Requimte-CQFB, Departamento de Química, Faculdade de Ciê ncias e Tecnologia, Universidade Nova de Lisboa, Campus Caparica, 2829-516 Caparica, Portugal * S Supporting Information ABSTRACT: The increase of conductivity of electrolytes favors the current production in microbial fuel cells (MFCs). Adaptation of cell cultures to higher ionic strength is a promising strategy to increase electricity production. The bacterium Geobacter sulfurreducens is considered a leading candidate for MFCs. Therefore, it is important to evaluate the impact of the ionic strength on the functional properties of key periplasmic proteins that warrants electron transfer to cell exterior. The effect of the ionic strength on the functional properties of triheme cytochrome PpcA, the most abundant periplasmic cytochrome in G. sulfurreducens, was investigated by NMR and potentiometric methods. The redox properties of heme IV are the most affected ones. Chemical shift perturbation measurements on the backbone NMR signals, at increasing ionic strength, also showed that the region close to heme IV is the most affected due to the large number of positively charged residues, which confer a highly positive electrostatic surface around this heme. The shielding of these positive charges at high ionic strength explain the observed decrease in the reduction potential of heme IV and shows that PpcA was designed to maintain its functional mechanistic features even at high ionic strength. ■ INTRODUCTION In the past decade, several studies have demonstrated that members of the Geobacteraceae family can transfer electrons from the oxidation of organic compounds to carbon dioxide with concomitant extracellular electron transfer toward insoluble acceptors, such as electrode surfaces in microbial fuel cells (MFCs). 1,2 The capability to conduit extracellular electron transfer and the natural abundance of Geobacter species in distinct environments opened routes to the development of Geobacter-based biotechnological applications, which include bioremediation, bioenergy, and biofuel production. 3 MFCs are devices that use bacterial metabolism to produce an electrical current from a wide range of organic substrates. These devices are composed by cathodic and anodic compartments separated by a proton/cation exchange membrane. In the latter compartment, the anode serves as extracellular electron acceptor. 1,4-6 Electrons collected on the anode are transported to the cathode via an external circuit. This direct conversion of organic wastes or renewable biomass to electricity in MFCs constitutes a very promising strategy to produce energy from inexpensive material sources. 1,7-11 Currently, the power output of MFCs is not yet considerable and further research is necessary to optimize energy production. 12 Because one of the key reactions in MFCs is the oxidation of organic matter coupled to electron transfer to the anode, understanding the factors that control the activity of microbes that colonize electrodes and, therefore, the extracellular electron transfer mechanisms is important. Several genetic studies have shown the involvement of different cytochromes in the electron transfer to extracellular acceptors. 13-18 However, these mechanisms are still poorly understood, particularly in the Geobacteraceae family. It is nowadays consensual that these electron routes include multiple redox proteins to ensure an efficient electron transfer from the cytoplasm to the outer cell surface. Among these, the periplasmic cytochromes warrant particular attention as they constitute a crucial interface for electron transfer between inner and outer membrane components. A family of five periplasmic triheme cytochromes (designated PpcA-E) was identified in the bacterium Geobacter sulfurreducens. 19 Genetic studies using G. sulfurreducens cells with genes coding for these cytochromes knocked-out showed that the reduction rates of extracellular acceptors are affected. 20 The redox properties of these cytochromes have been characterized in detail, with the exception of PpcC. 21,22 In the particular case of PpcA, the most abundant periplasmic cytochrome in G. sulfurreducens 23,24 and the most likely electron carrier destined to the outer surface, 25 it was shown that the redox potentials of the three heme groups are modulated by the pH providing the protein the Received: August 5, 2014 Revised: October 2, 2014 Article pubs.acs.org/JPCB © XXXX American Chemical Society A dx.doi.org/10.1021/jp507898x | J. Phys. Chem. B XXXX, XXX, XXX-XXX