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

Abstract

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 kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 μM, 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 kcat and Km(ATP) values of 0.3 min(-1) and 33.6 μM, 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 kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.

Structural characterization of the heme‐based oxygen sensor, AfGcHK, its interactions with the cognate response regulator, and their combined mechanism of action in a bacterial two‐component signaling system

“…Moreover, we can assume that the binding of the RR protein induces conformational changes in the ATP binding region (helix H11) of Af GcHK. Such induced conformational changes could increase the enzyme's P value, in keeping with recent findings . Moreover, the deuteration profiles of the peptides derived from the aforementioned areas [Fig.…”

“…HDX‐MS experiments were performed with the full‐length Af GcHK protein having an Fe(III) ion bound to its heme moiety. This form has previously been shown to be the most enzymatically active in the way of autophosphorylation of its kinase domain . The results obtained are summarized in the time‐resolved protection plot shown in Figure .…”

“…Cloning, overexpression in E. coli and purification of Af GcHK was performed as previously described . Briefly, His‐tagged Af GcHK was expressed in BL21(DE3) (Novagen, Madison, WI) harboring the pET 21c (+) plasmid.…”

“…This form has previously been shown to be the most enzymatically active in the way of autophosphorylation of its kinase domain. 32 The results obtained are summarized in the time-resolved protection plot shown in Figure 2. In addition, the deuteration levels of the globin and kinase domains at the initial time point (0.5 min) are visualized on the corresponding homology models in Figure 3, and the complete kinetic profile for the protein's deuteration is presented in Supporting Information Movie S1.…”

Mechanism and Role of Globin-Coupled Sensor Signalling

“…Fw109-5, termed Af GcHk ( A naeromyxobacter sp. F W109–5 G lobin c oupled H istidine k inase), has provided insight into signal transmission between globin and kinase domains in GCS proteins (Fojtikova et al, 2016; Fojtikova et al, 2015; Kitanishi et al, 2011; Stranava et al, 2016). Within the heme pocket, a distal tyrosine (Y45) is required for stable O 2 binding, forming a direct hydrogen bond, and H99 serves as the proximal ligand (Figure 2).…”