Roles of Ile209 and Ile210 on the heme pocket structure and regulation of histidine kinase activity of oxygen sensor FixL from Rhizobium meliloti.
FixL is a sensor histidine kinase having a heme-containing domain as an O(2) sensing site. In the study presented here, Ile209 and Ile210 located near the heme iron of the heme domain of Rhizobium meliloti FixL (RmFixL) were mutated, and the mutational effects on the regulation of the kinase activity and the heme pocket structure were examined by the autophosphorylation assay and UV-visible absorption and resonance Raman (RR) spectroscopies. The mutation of these residues disrupted the regulation of the kinase activity by the sensor (heme) domain, indicating that Ile209 and Ile210 play important roles in the signal transduction between the heme and the kinase domains. By measurement of the resonance Raman and optical absorption spectra of Ile209 and Ile210 mutants in several oxidation, spin, and ligation states, it was found that both residues are highly flexible, and their side chains sterically interact with the O(2) ligand, when it binds to the heme iron. On the basis of the results, we propose an O(2) sensing mechanism of RmFixL; the kinase activity is regulated via conformational changes of Ile209 and Ile210 induced by the O(2) binding to the sensory center.
Mukai
M
M
RIKEN Harima Institute/Spring-8, Mikazuki-cho, Hyogo 679-5148, Japan.
Nakamura
K
K
Nakamura
H
H
Iizuka
T
T
Shiro
Y
Y
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002248:Carbon Monoxide / Q000737:chemistry; D004789:Enzyme Activation; D005290:Ferric Compounds / Q000737:chemistry; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D000071677:Histidine Kinase; D007532:Isoleucine / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000235:genetics
D001426:Bacterial Proteins; D005290:Ferric Compounds; D006420:Hemeproteins; D007532:Isoleucine; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D014364:Tryptophan; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen