Resonance Raman and Ligand Binding Studies of the Oxygen-sensing Signal Transducer Protein HemAT from Bacillus subtilis

Abstract

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 k on ‫؍‬ 32 M ؊1 s ؊1 and k off ‫؍‬ 23 s ؊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°C 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-O 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. CObound HemAT-Bs gave the Fe-CO and C-O bands at 494 and 1964 cm ؊1 , 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 are discussed.Motile bacteria are known to swim toward or away from specific environmental stimuli such as nutrients, oxygen, or light (1, 2). This behavior, termed chemotaxis, is mediated by a signal transduction system consisting of methyl-accepting chemotaxis proteins (MCPs), 1 a histidine kinase CheA, a response regulator CheY, a coupling protein CheW, and the two enzymes that mediate sensory adaptation by covalently modifying the MCPs, CheR and CheB (3-6). Typical MCP is an integral membrane protein with an N-terminal periplasmic substrate binding domain and a C-terminal cytoplasmic signaling domain (7,8). The binding of a substrate, a repellent, or attractant to the periplasmic substrate binding domain is believed to induce a change in the MCP conformation that allows it to activate CheA (7, 8). The activated CheA phosphorylates CheY, and, consequently, the phosphorylated CheY binds to the switch complex at the base of the flagella to control the direction of flagellar rotation (3-6). In this signal transduction system, MCP acts as a sensor for the external signal and as a signal transducer.Hou et al. (9) have recently reported that Bacillus subtilis and Halobacterium salinarum have a signal transducer protein, HemAT-Bs and HemAT-Hs, respectively, for aerotaxis, the migratory response toward or away from oxygen. HemAT-Bs and HemAT-Hs are soluble proteins, and their C-terminal regions, residues 222-489 of HemAT-Hs and 198 -432 of HemAT-Bs, are 30% identical to the cytoplasmic signaling domain of Tsr, an MCP from Escherichia coli (9). Their N-terminal regions, residues 1-184 in HemAT-Hs and 1-175 in HemAT-Bs, show limited homology to myoglobin (9). Recombinant HemAT-Bs and HemAT-Hs are hemoproteins containing a b-type heme as a prost...

Protein Conformation Changes of HemAT-Bs upon Ligand Binding Probed by Ultraviolet Resonance Raman Spectroscopy

“…3 are compatible with the idea that the heme of HemAT-Bs discriminates between O 2 and CO, resulting in different conformational changes in the protein moiety of HemAT-Bs. In addition, binding of O 2 , CO, or NO to a five-coordinate, highspin, ligand-free (reduced) form changes the spin state of the heme iron of HemAT-Bs (15). Thus, the observed structural changes upon O 2 binding are mainly due to conversion of the high-spin to low-spin state and the formation of hydrogen bonds between O 2 and Thr 95 , whereas those observed upon CO or NO binding are probably due to only the change in the spin state of heme iron.…”

“…1A) upon binding of the signaling molecule. The structural changes in the active site upon binding of different ligands have been studied with different techniques (15,16,(22)(23)(24). For instance, by using visible excited RR spectroscopy in combination with site-directed mutagenesis, it is proposed that His 86 forms a hydrogen bond with a heme 6-propionate upon binding of O 2 to the heme (23).…”

Discovery of a reaction intermediate of aliphatic aldoxime dehydratase involving heme as an active center

“…2. The RR spectra of OS-II exhibited no Fe-His band around 226 cm Ϫ1 ; this observation implies the direct binding of the substrate to the heme iron of OxdA, because it is known that the Fe-His line becomes very weak when a ligand is bound to the position trans to the histidine (37,38). The low-frequency heme-ligand vibrational mode can be determined by using an isotope ligand, which causes an isotope shift of the ligand-heme iron vibrational mode due to the mass effect on oscillation (39,40).…”

Binding of Oxygen and Carbon Monoxide to a Heme-regulated Phosphodiesterase from Escherichia coli

“…Ec DOS therefore constitutes a novel class of heme enzymes designated "heme-based sensors" (3)(4)(5). These include proteins such as FixL (6,7), CooA (8,9), sGC (10,11), and Hem-AT (12,13). In these enzymes, association or dissociation of the exogenous axial ligand (O 2 , CO, or NO) from the heme iron leads to protein conformational changes, which in turn transmit signals to other domains to regulate catalysis or binding to DNA.…”