Heme proteins have evolved numerous functions, many of which involve allosteric regulation, including O2 transport by hemoglobin and substrate-dependent conformational changes in P450s. Globin-coupled sensor (GCS) proteins are a class of signaling proteins that have been implicated in O₂ dependent regulation of pathogenic phenotypes, including biofilm formation, motility, and virulence in several pathogens through allosteric regulation. While a handful of GCS proteins have been characterized to date, the diversity of the protein family has precluded a detailed mechanistic understanding of O₂ dependent signaling. Characterization of GCS protein from plant pathogen, Pectobacterium carotovorum (PccGCS) and the mechanism of O2 binding has been investigated using a combination of resonance Raman spectroscopy, structural methods, and enzymatic assays. These studies have identified positive cooperativity in ligand binding by bacterial globins outside of the hemoglobin family and identified residues essential for allosteric communication between heme moieties. In addition, new insights have been gained into the mechanism by which ligand binding is transmitted throughout the multi-domain protein. Taken together, these findings offer a novel perspective on O2 binding cooperativity within heme proteins and clarify how changes in the ligation/redox state of the sensor globin heme are communicated through full-length GCS proteins to govern cellular behaviors.