PMID:15653746
| Citation |
Zhang, W, Olson, JS and Phillips, GN Jr (2005) Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis. Biophys. J. 88:2801-14 |
|---|---|
| Abstract |
HemAT from Bacillus subtilis is a new type of heme protein responsible for sensing oxygen. The structural and functional properties of the full-length HemAT protein, the sensor domain (1-178), and Tyr-70 mutants have been characterized. Kinetic and equilibrium measurements reveal that both full-length HemAT and the sensor domain show two distinct O(2) binding components. The high-affinity component has a K(dissociation) approximately 1-2 microM and a normal O(2) dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K(dissociation) approximately 50-100 microM and a large O(2) dissociation rate constant equal to approximately 2000 s(-1). The low n-value and biphasic character of the equilibrium curve indicate that O(2) binding to HemAT involves either independent binding to high- and low-affinity subunits in the dimer or negative cooperativity. Replacement of Tyr-70(B10) with Phe, Leu, or Trp in the sensor domain causes dramatic increases in k(O2) for both the high- and low-affinity components. In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr-70 hydroxyl group. These results suggest highly dynamic behavior for the Tyr-70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex. As a result of having both high- and low-affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0-10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system. The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway. |
| Links |
PubMed PMC1305375 Online version:10.1529/biophysj.104.047936 |
| Keywords |
Bacillus subtilis/metabolism; Bacterial Proteins/chemistry; Bacterial Proteins/metabolism; Biophysics/methods; Carbon Monoxide/chemistry; Circular Dichroism; Heme/chemistry; Hydrogen-Ion Concentration; Kinetics; Leucine/chemistry; Ligands; Models, Molecular; Mutation; Oxygen/chemistry; Oxygen/metabolism; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Signal Transduction; Spectrophotometry; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Temperature; Time Factors; Tryptophan/chemistry; Tyrosine/chemistry; Ultracentrifugation; Ultraviolet Rays |
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