BPS2026 – Biophysical characterization of metal binding in the Ppc family

Dissimilatory metal-reducing bacteria utilize metal ions and metal oxides instead of oxygen in their respiration. Metal-reducing bacterial genomes are very rich (>100) in genes encoding multiheme cytochromes. However, the exact molecular pathways linking cytosolic redox reactions to periplasmic cytochromes and extracellular electron acceptors are not clear. Using UV-visible spectroscopy and isothermal calorimetry (ITC) we observed a 20 μM Cu 2+ binding constant in 20 mM tris buffer—pH 7.5 for PpcA, the most abundant periplasmic cytochrome in Geobacter sulfurreducens. We investigated the surface area responsible for Cu 2+ binding in PpcA. Unlike other Cu 2+ -binding proteins, PpcA does not have surface-exposed histidine and cystine residues available for metal binding. Through a combination of mutagenesis with 1D and 2D NMR analysis, we are pinpointing the surface region involved in Cu 2+ binding. Experimental binding studies and sequence conservation analysis suggest that the other members of the Ppc family are likely to bind Cu 2+ .