Coordination and redox state-dependent structural changes of the heme-based oxygen sensor AfGcHK associated with intraprotein signal transduction.

The heme-based oxygen sensor histidine kinase AfGcHK is part of a two-component signal transduction system in bacteria. O2 binding to the Fe(II) heme complex of its N-terminal globin domain strongly stimulates autophosphorylation at His183 in its C-terminal kinase domain. The 6-coordinate heme Fe(III)-OH- and -CN- complexes of AfGcHK are also active, but the 5-coordinate heme Fe(II) complex and the heme-free apo-form are inactive. Here, we determined the crystal structures of the isolated dimeric globin domains of the active Fe(III)-CN- and inactive 5-coordinate Fe(II) forms, revealing striking structural differences on the heme-proximal side of the globin domain. Using hydrogen/deuterium exchange coupled with mass spectrometry to characterize the conformations of the active and inactive forms of full-length AfGcHK in solution, we investigated the intramolecular signal transduction mechanisms. Major differences between the active and inactive forms were observed on the heme-proximal side (helix H5), at the dimerization interface (helices H6 and H7 and loop L7) of the globin domain and in the ATP-binding site (helices H9 and H11) of the kinase domain. Moreover, separation of the sensor and kinase domains, which deactivates catalysis, increased the solvent exposure of the globin domain-dimerization interface (helix H6) as well as the flexibility and solvent exposure of helix H11. Together, these results suggest that structural changes at the heme-proximal side, the globin domain-dimerization interface, and the ATP-binding site are important in the signal transduction mechanism of AfGcHK. We conclude that AfGcHK functions as an ensemble of molecules sampling at least two conformational states.

The capacity and effectiveness of diosmectite and charcoal in trapping the compounds causing the most frequent intoxications in acute medicine: A comparative study.

The aim of the study was to compare the adsorption ability of two adsorbent materials, namely diosmectite and activated charcoal towards selected model compounds that are most commonly involved in acute intoxication. Eleven model compounds were selected: acetylsalicylic acid, α-amanitin, amlodipine, digoxin, phenobarbital, ibuprofen, imipramine, carbamazepine, oxazepam, promethazine, and theophylline. Of the tested compounds, promethazine and imipramine were the most effectively adsorbed to diosmectite. Their adsorption to diosmectite (0.356±0.029mg promethazine/mg diosmectite and 0.354±0.019mg imipramine/mg diosmectite, respectively) was significantly higher than their adsorption to activated charcoal. The effect of temperature and pH on the adsorption efficiencies was also evaluated. In the case of experiments with mixture of both adsorbents, they mostly behaved in a solution independently or in a slightly antagonistic way. Using various methods such as N2 adsorption and thermogravimetric analysis, the structure and texture of diosmectite and activated charcoal were attained.

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor AfGcHK.

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O2 or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H2S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O2, CO, and NO. To address this knowledge gap, we investigated the effects of H2S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O2-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na2S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na2S, the initially formed Fe(III)-SH heme complex was quickly converted into Fe(II) and Fe(II)-O2 complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)-SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)-OH complex), whereas in the case of EcDOS the changes in coordination caused by Na2S treatment led to remarkable increases in catalytic activity.

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.

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109-5 forms a two-component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N-terminal sensor domain causes the C-terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX-MS studies on the AfGcHK:RR complex also showed that the N-side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the ß-strand B2 area of the RR protein's Rec1 domain, and that the C-side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and ß-strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375-1389. © 2016 Wiley Periodicals, Inc.

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 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.

Catalytic enhancement of the heme-based oxygen-sensing phosphodiesterase EcDOS by hydrogen sulfide is caused by changes in heme coordination structure.

EcDOS is a heme-based O2-sensing phosphodiesterase in which O2 binding to the heme iron complex in the N-terminal domain substantially enhances catalysis toward cyclic-di-GMP, which occurs in the C-terminal domain. Here, we found that hydrogen sulfide enhances the catalytic activity of full-length EcDOS, possibly owing to the admixture of 6-coordinated heme Fe(III)-SH(-) and Fe(II)-O2 complexes generated during the reaction. Alanine substitution at Met95, the axial ligand for the heme Fe(II) complex, converted the heme Fe(III) complex into the heme Fe(III)-SH(-) complex, but the addition of Na2S did not further reduce it to the heme Fe(II) complex of the Met95Ala mutant, and no subsequent formation of the heme Fe(II)-O2 complex was observed. In contrast, a Met95His mutant formed a stable heme Fe(II)-O2 complex in response to the same treatment. An Arg97Glu mutant, containing a glutamate substitution at the amino acid that interacts with O2 in the heme Fe(II)-O2 complex, formed a stable heme Fe(II) complex in response to Na2S, but this complex failed to bind O2. Interestingly, the addition of Na2S promoted formation of verdoheme (oxygen-incorporated, modified protoporphyrin IX) in an Arg97Ile mutant. Catalytic enhancement by Na2S was similar for Met95 mutants and the wild type, but significantly lower for the Arg97 mutants. Thus, this study shows the first isolation of spectrometrically separated, stable heme Fe(III)-SH(-), heme Fe(II) and heme Fe(II)-O2 complexes of full-length EcDOS with Na2S, and confirms that external-ligand-bound, 6-coordinated heme Fe(III)-SH(-) or heme Fe(II)-O2 complexes critically contribute to the Na2S-induced catalytic enhancement of EcDOS.