Structure and function of the heme-based gas sensor proteins

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HemAT-Bs is a heme-containing signal transducer protein responsible for aerotaxis of Bacillus subtilis. The recombinant HemAT-Bs expressed in Escherichia coli was purified as the oxy form in which oxygen was bound to the ferrous heme. Oxygen binding and dissociation rate constants were determined to be kon = 32 μM^<-1> s^<-1> and koff = 23^<-1>, respectively, revealing that HemAT-Bs has a moderate oxygen affinity similar to that of sperm whale myoglobin (Mb). The rate constant for autoxidation at 37 ℃ was 0.06 h^<-1>, which is also close to that of Mb. Although the electronic absorption spectra of HemAT-Bs were similar to those of Mb, HemAT-Bs showed some unique characteristics in its resonance Raman spectra. Oxygen-bound HemAT-Bs gave the Fe-0_2 band at a noticeably low frequency (560 cm^<-1>), which suggests a unique hydrogen bonding between a distal amino acid residue and the proximal atom of the bound oxygen molecule. Deoxy HemAT-Bs gave the Fe-His band at a higher frequency (225 cm<-1>) than those of ordinary His-coordinated deoxy heme proteins. CO-bound HemAT-Bs gave the Fe-CO and C-O bands at 494 and 1964 cm1, respectively, which fall on the same C-O versus Fe-CO correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs has been elucidated.In this work, a new CooA homologue from C.hydrogenoformans (Ch-CooA) was also studied. Ch-CooA also contains a protoheme as the active site for sensing CO. The heme in Ch-CooA is six-coordinate, low spin state in the ferric, ferrous and CO-bound forms. The heme environmental structure of Ch-CooA was elucidated by mutagenesis and spectroscopic studies.