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 approximate to 1-2 mu M and a normal O-2 dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K-dissociation approximate to 50-100 mu M and a large O-2 dissociation rate constant equal to; 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 mu M) and aerobic (50-250 mu M) 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.