O2-specific regulation of the ferrous heme-based sensor kinase FixL from Sinorhizobium meliloti and its aberrant inactivation in the ferric form.
FixL, a rhizobial heme-based O2-sensing histidine kinase, catalyzes autophosphorylation in the deoxy form at low O2 tension, while the kinase activity is inhibited in the case of the O2-bound form. The present study unambiguously shows that the binding of CO and NO does not significantly inhibit the kinase activity of dithiothreitol (DTT)-reduced ferrous FixL from Sinorhizobium meliloti, which is inconsistent with the spin state mechanism previously reported. Kinase inactivation is caused by aberrant disulfide (S-S) bond formation at Cys301 in the ferric homodimer, which explains these contradictory observations. The addition of DTT cleaved the S-S bond, leading to restoration of kinase activity in the ferric form as well as heme reduction, but, sodium hydrosulfite treatment produced the kinase-inactive deoxy form without S-S cleavage. On the basis of these experimental results, it can be concluded that ferrous FixL discriminates O2 from CO and NO, and signals the O2-bound state by downregulating the phosphoryl transfer reaction.
Akimoto
Satoru
S
RIKEN Harima Institute/SPring-8, Mikazuki, 679-5148, Hyogo, Japan.
Tanaka
Atsunari
A
Nakamura
Kayako
K
Shiro
Yoshitsugu
Y
Nakamura
Hiro
H
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000494:pharmacology; D015536:Down-Regulation; D005290:Ferric Compounds / Q000378:metabolism; D005296:Ferrous Compounds / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry / Q000378:metabolism*; D000071677:Histidine Kinase; D008024:Ligands; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000502:physiology*; D010766:Phosphorylation; D016962:Sinorhizobium meliloti / Q000201:enzymology*; D013447:Sulfites / Q000494:pharmacology
D001426:Bacterial Proteins; D005290:Ferric Compounds; D005296:Ferrous Compounds; D006420:Hemeproteins; D008024:Ligands; D013447:Sulfites; D009569:Nitric Oxide; D002248:Carbon Monoxide; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D003545:Cysteine; C042345:hydrogen sulfite; D010100:Oxygen; D004229:Dithiothreitol