Ligand and oxidation-state specific regulation of the heme-based oxygen sensor FixL from Sinorhizobium meliloti.
Phosphorylation of the transcription factor RmFixJ is the key step in the hypoxic induction of Sinorhizobium meliloti nitrogen fixation genes. Oxygen regulates this process by binding reversibly to RmFixL, a heme protein kinase whose deoxy form catalyzes the phosphoryl transfer from ATP to RmFixJ. Here we present the first quantitative measure of the extent by which various heme ligands inhibit the turnover of RmFixJ to phospho-RmFixJ. We also quantitate the inhibition by ligands of the reaction of RmFixL with ATP, in the absence of RmFixJ, to form phospho-RmFixL, i.e., the "autophosphorylation". Phospho-RmFixL formed from autophosphorylation will transfer its phosphoryl group to RmFixJ in an oxygen-independent "phosphotransfer." Here we show that the mode of substrate presentation, i.e., simultaneous versus sequential, influences the regulation of phosphoryl transfer by heme status. Inhibition factors for O(2), CO, NO, CN(-), and imidazole in the presence of RmFixJ are drastically different from the inhibition of autophosphorylation by the same ligands. Oxidation of the heme iron in unliganded RmFixL is known to have no effect on either of the sequential reactions; yet oxidation causes a 100-fold decrease in RmFixJ turnover when ATP and RmFixJ are presented simultaneously. The profound difference between the regulation of isolated RmFixL versus the complex of RmFixL with RmFixJ shows that interaction of a response regulator with its histidine-kinase partner need not be limited to the enzymatic regions of the histidine kinase, but can extend also to its sensory domain.
Tuckerman
Jason R
JR
Department of Biochemistry, The Ohio State University, 1060 Carmack Road, Columbus, Ohio 43210-1002, USA.
Gonzalez
Gonzalo
G
Dioum
Elhadji M
EM
Gilles-Gonzalez
Marie-Alda
MA
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D006418:Heme / Q000737:chemistry; D006420:Hemeproteins / Q000737:chemistry* / Q000378:metabolism*; D000071677:Histidine Kinase; D007700:Kinetics; D008024:Ligands; D008954:Models, Biological; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000378:metabolism
D001426:Bacterial Proteins; D006420:Hemeproteins; D008024:Ligands; D011994:Recombinant Proteins; D006418:Heme; D000255:Adenosine Triphosphate; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen