A repressor composed of homodimeric subunits, as is often found in bacteria, possesses two effector-binding sites per molecule, enabling sophisticated regulation by the cooperative binding of two effector molecules. Positive cooperativity generates a narrower region of effector concentration for switching, but little is known about the role of negative cooperativity. d-camphor, an inducer for Pseudomonas putida cytochrome P450cam hydroxylase operon (camDCAB), binds to the homodimeric cam repressor (CamR). Here, we report solid evidence that the complex of CamR and an operator DNA is not dissociated by the first binding of d-camphor but, at a higher concentration, is dissociated by the second binding. d-camphor thus binds to the CamR in two steps with negative cooperativity, yielding two distinct dissociation constants of K(d1 ) =( ) 0.064 ± 0.030 and K(d2 ) =( ) 14 ± 0.3 μm, as well as the Hill coefficient of 0.56 ± 0.05 (<1). The first binding guarantees the high specificity of the inducer by the high affinity, although the second binding turns on the gene expression at a 200-fold higher concentration, a more suitable switching point for the catabolism of d-camphor.
© 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.