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Briefly, an approximate final concentration of 1 ��M of each target protein was prepared in a 1 ml cuvette. Dilutions were made from stock solutions of DmsE, MtrD, OTR, ccNiR, and FccA in degassed 20 mM potassium phosphate buffer (pH 7.6) with 100 mM KCl. Each protein was reduced by addition of small amounts of a concentrated solution of sodium dithionite. The absorbance was monitored at 314 nm to avoid excess of reducing agent. Fumarate was added to the reduced protein solutions to a final concentration of ?1 mM. Only when no change was observed in absorbance at 552 nm, the reaction would be initiated by the addition of 1 nM FccA. The spectral changes were monitored over time. Experiments were OTX015 manufacturer performed with constant stirring and the temperature was kept at 25��C using an external thermostatic bath. Protein Electrostatic Surface Potential Calculations The structures of ccNiR (PDB code 3UBR; Youngblut et al., 2012) and the OTR (PDB code 1SP3; Mowat et al., 2004), were used to calculate the electrostatic potential at the surface of both proteins. Both proteins were set in their fully oxidized states, which were the experimental conditions used to study their interactions. The GROMOS 43A1 force field (Scott et al., 1999) was used to set the partial charges of the proteins and co-factors. The MEAD package (Bashford and Karplus, 1990), which solves the Poisson�CBoltzmann equation for a system, was used to calculate the electrostatic potentials. The ionic strength used was 0 mM and the internal and external dielectric constants were set at 2 and 80, respectively. The electrostatic potential was mapped at the surface of the proteins using PyMOL (DeLano, 2003). Results NMR Titrations and Binding Affinities For electron transfer to occur at physiologically relevant rates between two cytochromes, the heme groups of donor and acceptor must be in close proximity (Zhang et al., 2008; Gray and Winkler, 2010). Therefore, when multiheme cytochromes bind in a configuration that is relevant for interprotein electron transfer, NMR spectroscopy can be used to detect this binding through observation of changes in the chemical shifts of signals belonging to the hemes near to the binding sites (Fonseca et al., 2013). This technique is thus highly suited to study interactions between the redox proteins found in the periplasmic space of SOMR-1, revealing the detailed organization of its trans-periplasmic redox network. Figure ?Figure11 illustrates spectral changes for the 181 methyl signal (IUPAC-IUB nomenclature) from heme IV (181CH3IV) of STC in the presence of increasing amounts of OTR. FIGURE 1 1H-1D NMR spectral changes of the signal from methyl 181 belonging to heme IV of small tetraheme cytochrome (STC) in the presence of increasing amounts of octaheme tetrathionate reductase (OTR), illustrating the data used in the chemical shift perturbation ...