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* Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005; and Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
Correspondence: Address reprint requests to George N. Phillips Jr., Dept. of Biochemistry, University of Wisconsin, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-263-6142; Fax: 608-262-3453; E-mail: phillips{at}biochem.wisc.edu.
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, or Trp 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.
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