Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.
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.
Zhang
Wei
W
Department of Biochemistry and Cell Biology, W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005, USA.
Olson
John S
JS
Phillips
George N
GN
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001703:Biophysics / Q000379:methods*; D002248:Carbon Monoxide / Q000737:chemistry; D002942:Circular Dichroism; D006418:Heme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013053:Spectrophotometry; D013055:Spectrophotometry, Infrared; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014461:Ultracentrifugation; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D008024:Ligands; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D002248:Carbon Monoxide; D014364:Tryptophan; D007930:Leucine; D010100:Oxygen