Roles of the heme distal residues of FixL in O2 sensing: a single convergent structure of the heme moiety is relevant to the downregulation of kinase activity.
FixL is a heme-based O(2) sensor, in which the autophosphorylation is regulated by the binding of exogenous ligands such as O(2) and CN(-). In this study, mutants of the heme distal Arg200, Arg208, Ile209, Ile210, and Arg214 residues of SmFixL were characterized biochemically and physicochemically, because it has been suggested that they are significant residues in ligand-linked kinase regulation. Measurements of the autoxidation rate, affinities, and kinetics of ligand binding revealed that all of the above residues are involved in stabilization of the O(2)-heme complex of FixL. However, Arg214 was found to be the only residue that is directly relevant to the ligand-dependent regulation of kinase activity. Although the wild type and R214K and R214Q mutants exhibited normal kinase regulation, R214A, R214M, R214H, and R214Y did not. (13)C and (15)N NMR analyses for (13)C(15)N(-) bound to the truncated heme domains of the Arg214 mutants indicated that, in the wild type and the foregoing two mutants, the heme moiety is present in a single conformation, but in the latter four, the conformations fluctuate possibly because of the lack of an interaction between the iron-bound ligand and residue 214. It is likely that such a rigid conformation of the ligand-bound form is important for the downregulation of histidine kinase activity. Furthermore, a comparison of the NMR data between the wild type and R214K and R214Q mutants suggests that a strong electrostatic interaction between residue 214 and the iron-bound ligand is not necessarily required for the single convergent structure and eventually for the downregulation of FixL.
Tanaka
Atsunari
A
Yokohama City University International Graduate School of Arts and Sciences, Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045, Japan.
Nakamura
Hiro
H
Shiro
Yoshitsugu
Y
Fujii
Hiroshi
H
D001120:Arginine / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003486:Cyanides / Q000378:metabolism; D015536:Down-Regulation / Q000502:physiology*; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism; D000071677:Histidine Kinase; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D016962:Sinorhizobium meliloti / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D001426:Bacterial Proteins; D003486:Cyanides; D006420:Hemeproteins; D007093:Imidazoles; D008024:Ligands; D007532:Isoleucine; D006418:Heme; C029899:imidazole; D001120:Arginine; D007501:Iron; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen