Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

Abstract

Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O2, and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O2 binding site at the heme distal side. The O2 dissociation rate constant, 0.10 s−1, was substantially increased up to 8.0 s−1 upon Y45L mutation. The resonance Raman frequencies representing νFe-O2 (559 cm−1) and νO-O (1149 cm−1) of the Fe(II)-O2 complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (νFe-O2, 557 cm−1; νO-O, 1141 cm−1), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.

Heme-based Globin-coupled Oxygen Sensors: Linking Oxygen Binding to Functional Regulation of Diguanylate Cyclase, Histidine Kinase, and Methyl-accepting Chemotaxis

“…Contrary to expectations, heme-free forms and heme-binding domain-truncated forms of YddV, AfGcHK, and EcDOS have sufficient catalytic activity (32,44,72,94). Instead of stimulating catalysis, the heme iron complex on the heme-based oxygen sensor serves to suppress catalysis, and oxygen association/dissociation releases this catalytic suppression.…”

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

“…Af GcHK is also distinctive in that its catalytic activity is stimulated by the binding of O 2 to its heme Fe(II) complex, whereas the catalytic activity of FixL and DosS/DosT is stimulated by the dissociation of O 2 from the heme Fe(II) complex. 22 Af GcHK has already been partially characterized, 14 but many questions regarding its function and mechanism of action remain unresolved. In particular, its mechanism of action is poorly understood because it is not clear how signals are transduced between the sensor and functional domains.…”

“…29 Cloning, site-directed mutagenesis, overexpression in E. coli, and purification of GST-tagged RR were performed in a Biochemistry Article DOI: 10.1021/acs.biochem.5b00517 Biochemistry 2015, 54, 5017−5029 manner slightly different from that of the experiments previously described. 14 The full-length wild-type and Asp52/ 169Ala mutant RR were prepared as described below. Briefly, E. coli BL21(DE3) cells (Novagen) were transformed with the corresponding pGEX-6P-2 expression vector, plated on LB agar containing 100 μg mL −1 ampicillin, and incubated at 37°C overnight.…”

A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans

“…The absorption properties clearly differ from the 6c-LS heme found in the direct oxygen sensor EcDos from E. coli (53,54), recombinant barley hemoglobin (55), CO sensor CooA from R. rubrum (6,56), and globin-coupled histidine kinase AfGcHK from Anaeromyxobacter sp. Fw109-5 (20) and from the five-coordinate high spin heme found in YddV (57) and BjFixL from Bradyrhizobium japonicum (58).…”

“…FixL, DevS, and DosT are sensor histidine kinases of two-component systems (8,19). Recently, a globin-coupled histidine kinase also was identified (20). The only known heme-based sensor from Archaea is the aerotaxis transducer Hs-HemAT from Halobacterium salinarum (2).…”