HemAT from Bacillus subtilis (HemAT-Bs) is a heme-based O2 sensor protein that acts as a signal transducer responsible for aerotaxis. HemAT-Bs discriminates its physiological effector (O2) from other gas molecules (CO and NO), although all of them bind to a heme. To monitor the conformational changes in the protein moiety upon binding of different ligands we have investigated ultraviolet resonance Raman (UVRR) spectra of the ligand-free, and O2-, CO- and NO-bound forms of full-length HemAT-Bs and several mutants (Tyr70Phe, His86Ala, Thr95Ala, and, Tyr133Phe), and found that Tyr70 in the heme distal side, and Tyr133 and Trp132 from G-helix in the heme proximal side undergo environmental changes upon ligand binding. In addition, the UVRR results confirmed our previous model, which suggested that Thr95 forms a hydrogen bond with heme-bound O2 but Tyr70 does not. It is deduced from this study that hydrogen bonds between Thr95 and heme bound-O2 and between His86 and heme 6-propionate communicate the heme structural changes to the protein moiety upon O2-binding but not upon CO- and NO binding. Accordingly, the present UVRR results suggest that O2 binding to heme causes displacement of G-helix, which would be important for transduction of the conformational changes from the sensor domain to the signaling domain.