Resonance Raman and ligand binding studies of the oxygen-sensing signal transducer protein HemAT from Bacillus subtilis.
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 microm(-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 degrees 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 nu(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 nu(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 nu(Fe-CO) and nu(C-O) bands at 494 and 1964 cm(-1), respectively, which fall on the same nu(C-O) versus nu(Fe-CO) correlation line as that of Mb. Based on these results, the structural and functional properties of HemAT-Bs are discussed.
Aono
Shigetoshi
S
School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, Japan. aono@jaist.ac.jp
Kato
Toshiyuki
T
Matsuki
Mayumi
M
Nakajima
Hiroshi
H
Ohta
Takehiro
T
Uchida
Takeshi
T
Kitagawa
Teizo
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007554:Isotopes; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D013059:Spectrum Analysis, Raman; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D007554:Isotopes; D008024:Ligands; D011994:Recombinant Proteins; C404449:heme protein, bacteria; D006418:Heme; D006859:Hydrogen; D010100:Oxygen