A Memory of Oxygen Binding Explains the Dose Response of the Heme-Based Sensor FixL
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- 作者：Eduardo Henrique Silva Sousa ; Jason Robert Tuckerman ; Gonzalo Gonzalez ; Marie-Alda Gilles-Gonzalez
- 刊名：Biochemistry
- 出版年：2007
- 出版时间：May 29, 2007
- 年：2007
- 卷：46
- 期：21
- 页码：6249 - 6257
- 全文大小：363K
- 年卷期：v.46,no.21(May 29, 2007)
- ISSN：1520-4995
文摘
Bradyrhizobium japonicum FixL is a modular oxygen sensor that directs adaptations to hypoxiaby 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 atranscription factor, FixJ. We have developed methods to optimize the kinase reactions of FixL and measurethe turnover rates (kcat) for reactions catalyzed by highly inhibited states of this sensor at constant, preciselyknown oxygen saturations. The resulting oxygen dose-response curve shows that an in vitro system withFixL 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, andby the time the deoxy-FixL declines just 8-fold, the activity is inhibited over 1100-fold. This response isentirely 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 deoxystates. Therefore traditional models involving cooperative binding of ligand or robust allosteric regulationcannot 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 ofoxygen with the heme, the kinase is rapidly switched off; (ii) after dissociation of oxygen, the kinaseremains inhibited longer than the average time that it takes a deoxy-heme to encounter an oxygen moleculeat most oxygen saturations.