Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The k(cat) values of the heme Fe(III)-OH-, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O-2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their K-m(ATP) values were 18.9-35.4 mu M. However, the active form bearing a CO-bound Fe(II) heme had a k(cat) of 1.0 min(-1) but a very high K-m(ATP) value of 357 mu M, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had k(cat) and K-m(ATP) values of 0.4 min(-1) and 78 mu M, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with k(cat) and K-m(ATP) values of 0.3 min(-1) and 33.6 mu M, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its k(cat)/K-m(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.