Roles of Ile209 and Ile210 on the Heme Pocket Structure and Regulation of Histidine Kinase Activity of Oxygen Sensor FixL from Rhizobium meliloti

• Published in: Biochemistry (Easton) 2000-11, Vol.39 (45), p.13810-13816
• Main Authors: Mukai, Masahiro, Nakamura, Kayako, Nakamura, Hiro, Iizuka, Tetsutaro, Shiro, Yoshitsugu
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Carbon Monoxide - chemistry; Enzyme Activation; Ferric Compounds - chemistry; FixL protein; Heme - chemistry; Heme - metabolism; Hemeproteins - genetics; Hemeproteins - metabolism; Histidine - genetics; Histidine Kinase; Isoleucine - chemistry; Isoleucine - genetics; Mutagenesis, Site-Directed; Oxygen - metabolism; Phosphorylation; Protein Binding - genetics; Protein Conformation; Protein Kinases - chemistry; Protein Kinases - metabolism; Rhizobium meliloti; Sinorhizobium meliloti - enzymology; Sinorhizobium meliloti - genetics; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Spectrum Analysis, Raman; Tryptophan - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi001184x

FixL is a sensor histidine kinase having a heme-containing domain as an O2 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 O2 ligand, when it binds to the heme iron. On the basis of the results, we propose an O2 sensing mechanism of RmFixL; the kinase activity is regulated via conformational changes of Ile209 and Ile210 induced by the O2 binding to the sensory center.