Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies.
FixL of Rhizobium meliloti (RmFixL) is a sensor histidine kinase of the two-component system, which regulates the expression of the genes related to nitrogen fixation in the root nodule in response to the O(2) levels. The crystal structure of the sensor domain of FixL (RmFixLH), which contains a heme (Fe-porphyrin) as a sensing site, was determined at 1.4 A resolution. Based on the structural and spectroscopic analyses, we propose the O(2) sensing mechanism that differs from the case proposed in BjFixLH as follows; conformational changes in the F/G loop, which are induced by steric repulsion between the bent-bound O(2) and the Ile209 side-chain, would be transmitted to the histidine kinase domain. Interaction between the iron-bound O(2) and Ile209 was also observed in the resonance Raman spectra of RmFixLH as evidenced by the fact that the Fe-O(2) and Fe-CN stretching frequencies were shifted from 575 to 570 cm(-1) (Fe-O(2)), and 504 to 499 cm(-1), respectively, as the result of the replacement of Ile209 with an Ala residue. In the I209A mutant of RmFixL, the O(2) sensing activity was destroyed, thus confirming our proposed mechanism.
Miyatake
H
H
RIKEN Harima Institute/SPring-8, 1-1-1 Koto, Hyogo, Mikazuki-cho, 679-5148, Japan. miyatake@postman.riken.go.jp
Mukai
M
M
Park
S Y
SY
Adachi
S
S
Tamura
K
K
Nakamura
H
H
Nakamura
K
K
Tsuchiya
T
T
Iizuka
T
T
Shiro
Y
Y
D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000302:isolation & purification; D000071677:Histidine Kinase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification; D017434:Protein Structure, Tertiary; D016962:Sinorhizobium meliloti / Q000737:chemistry*; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D006420:Hemeproteins; D006418:Heme; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen