Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis

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 O2 binding components. The high-affinity component has a Kdissociation ≈ 1-2 μM and a normal O2 dissociation rate constant, kO2 = 50-80 s -1. The low-affinity component has a Kdissociation ≈ 50-100 μM and a large O2 dissociation rate constant equal to ∼2000 s-1. The low n-value and biphasic character of the equilibrium curve indicate that O2 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, orTrp in the sensor domain causes dramatic increases in kO2 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 μM) and aerobic (50-250 μM) 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. © 2005 by the Biophysical Society.