title: Regulatory Implications of Structural Changes in Tyr201 of the Oxygen Sensor Protein FixL.
abstract: FixL is a heme-based oxygen-sensing histidine kinase that induces the expression of nitrogen fixation genes under hypoxic conditions. Oxygen dissociation from heme iron in the sensor domain of FixL initiates protein conformational changes that are transmitted to the histidine kinase domain, activating autophosphorylation activity. Conversely, oxygen binding inhibits FixL kinase activity. It is essential to elucidate the changes that occur in the protein structure upon this oxygen dissociation for understanding of the allosteric transduction mechanism. We measured ultraviolet resonance Raman spectra of FixL and its mutants for deoxy, oxy, and carbonmonoxy forms to examine the changes in protein structure upon oxygen dissociation. The observed spectral changes indicated that Tyr201 and its neighboring residues undergo structural changes upon oxygen dissociation. Kinase assays showed that substitution of Tyr201 significantly decreased the inhibition of kinase activity upon oxygen binding. These data mean that weakening of the hydrogen bond of Tyr201 that is induced by oxygen dissociation is essential for inhibition of kinase activity. We also observed spectral changes in Tyr residues in the kinase domain upon oxygen dissociation from FixL, which is the first observation of oxygen-dependent structural changes in the kinase domain of FixL. The observed structural changes support the allosteric transduction pathway of FixL which we proposed previously [ Yano, S., Ishikawa, H., Mizuno, M., Nakamura, H., Shiro, Y., and Mizutani, Y. ( 2013 ) J. Phys. Chem. B 117 , 15786 - 15791 ].
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University , 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Takeo
authors.initials: T
authors.lastname: Yamawaki
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University , 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Haruto
authors.initials: H
authors.lastname: Ishikawa
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University , 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Misao
authors.initials: M
authors.lastname: Mizuno
authors.affiliation: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Hiro
authors.initials: H
authors.lastname: Nakamura
authors.affiliation: RIKEN SPring-8 Center , 1-1-1 Kouto, Sayo, Hyogo 679-5148, Japan.
authors.forename: Yoshitsugu
authors.initials: Y
authors.lastname: Shiro
authors.affiliation: Department of Chemistry, Graduate School of Science, Osaka University , 1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan.
authors.forename: Yasuhisa
authors.initials: Y
authors.lastname: Mizutani
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020369:Bradyrhizobium / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D006420:Hemeproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman
chemical_list: D001426:Bacterial Proteins; D006420:Hemeproteins; D011994:Recombinant Proteins; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen