title: A memory of oxygen binding explains the dose response of the heme-based sensor FixL.
abstract: Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxia by coupling the status of a heme-binding domain to a histidine-protein kinase activity. The oxygen-bound form is the "off-state". The unliganded form is the "on-state" active kinase that phosphorylates a transcription factor, FixJ. We have developed methods to optimize the kinase reactions of FixL and measure the turnover rates (kcat) for reactions catalyzed by highly inhibited states of this sensor at constant, precisely known oxygen saturations. The resulting oxygen dose-response curve shows that an in vitro system with FixL and the response regulator FixJ as its only proteins manifests such a sharp ligand response that, when the proportion of deoxy-FixL decreases less than 3-fold, the kinase activity drops over 50-fold, and by the time the deoxy-FixL declines just 8-fold, the activity is inhibited over 1100-fold. This response is entirely reversible and similar to that reported for the in vivo hypoxic induction of FixLJ-regulated genes. FixL binds oxygen noncooperatively. When complexed with FixJ, FixL is dimeric in both oxy and deoxy states. Therefore traditional models involving cooperative binding of ligand or robust allosteric regulation cannot account for the extremely nonlinear kinase response to the heme saturation. This response, however, can be explained by a form of enzyme hysteresis with the simple assumptions that (i) on association of oxygen with the heme, the kinase is rapidly switched off; (ii) after dissociation of oxygen, the kinase remains inhibited longer than the average time that it takes a deoxy-heme to encounter an oxygen molecule at most oxygen saturations.
authors.affiliation: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038, USA.
authors.forename: Eduardo Henrique Silva
authors.initials: EH
authors.lastname: Sousa
authors.forename: Jason Robert
authors.initials: JR
authors.lastname: Tuckerman
authors.forename: Gonzalo
authors.initials: G
authors.lastname: Gonzalez
authors.forename: Marie-Alda
authors.initials: MA
authors.lastname: Gilles-Gonzalez
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005786:Gene Expression Regulation; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D000071677:Histidine Kinase; D000860:Hypoxia / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D010100:Oxygen / Q000378:metabolism*; D011494:Protein Kinases
chemical_list: D001426:Bacterial Proteins; D006420:Hemeproteins; C069289:FixJ protein, Bacteria; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen