An emerging class of novel heme-based oxygen sensors containing a globin fold binds and senses environmental O2 via a heme iron complex. Structure-function relationships of oxygen sensors containing a heme-bound globin fold are different from those containing heme-bound PAS and GAF folds. It is thus worth reconsidering from an evolutionary perspective how heme-bound proteins with a globin fold similar to that of hemoglobin and myoglobin could act as O2 sensors. Here, we summarize the molecular mechanisms of heme-based oxygen sensors containing a globin fold in an effort to shed light on the O2-sensing properties and O2-stimulated catalytic enhancement observed for these proteins.

• MeSH
• Azotobacter vinelandii enzymologie   MeSH
• Bordetella pertussis enzymologie   MeSH
• chemotaxe MeSH
• Escherichia coli enzymologie   MeSH
• globiny chemie   MeSH
• hem chemie   MeSH
• hemoglobiny chemie   MeSH
• katalytická doména MeSH
• katalýza MeSH
• kyslík chemie   MeSH
• lyasy štěpící vazby P-O chemie   MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• myoglobin chemie   MeSH
• proteinkinasy chemie   MeSH
• proteiny z Escherichia coli chemie   MeSH
• regulace genové exprese enzymů * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

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.

• MeSH
• biokatalýza účinky léků   MeSH
• fosforylace účinky léků   MeSH
• hem chemie   metabolismus   MeSH
• histidinkinasa chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• kyslík chemie   metabolismus   MeSH
• molekulární struktura MeSH
• mutageneze cílená MeSH
• Myxococcales enzymologie   MeSH
• sulfan chemie   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH

• Publikační typ
• abstrakt z konference MeSH

Heme-based oxygen sensors allow bacteria to regulate their activity based on local oxygen levels. YddV, a globin-coupled oxygen sensor with diguanylate cyclase activity from Escherichia coli, regulates cyclic-di-GMP synthesis based on oxygen availability. Stable and active samples of the full-length YddV protein were prepared by attaching it to maltose binding protein (MBP). To better understand the full-length protein's structure, the interactions between its domains were examined by performing a kinetic analysis. The diguanylate cyclase reaction catalyzed by YddV-MBP exhibited Michaelis-Menten kinetics. Its pH optimum was 8.5-9.0, and catalysis required either Mg2+ or Mn2+; other divalent metal ions gave no activity. The most active form of YddV-MBP had a 5-coordinate Fe(III) heme complex; its kinetic parameters were KmGTP 84 ± 21 μM and kcat 1.2 min-1. YddV-MBP with heme Fe(II), heme Fe(II)-O2, and heme Fe(II)-CO complexes had kcat values of 0.3 min-1, 0.95 min-1, and 0.3 min-1, respectively, suggesting that catalysis is regulated by the heme iron's redox state and axial ligand binding. The kcat values for heme Fe(III) complexes of L65G, L65Q, and Y43A YddV-MBP mutants bearing heme distal amino acid replacements were 0.15 min-1, 0.26 min-1 and 0.54 min-1, respectively, implying that heme distal residues play key regulatory roles by mediating signal transduction between the sensing and functional domains. Ultracentrifugation and size exclusion chromatography experiments showed that YddV-MBP is primarily dimeric in solution, with a sedimentation coefficient around 8. The inactive heme-free H93A mutant is primarily octameric, suggesting that catalytically active dimer formation requires heme binding.

• MeSH
• hem chemie   MeSH
• katalytická doména MeSH
• kinetika MeSH
• ligandy MeSH
• lyasy štěpící vazby P-O chemie   genetika   metabolismus   MeSH
• oxidace-redukce MeSH
• proteiny z Escherichia coli chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• vazba proteinů MeSH
• železo chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The nonradioactive method, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of Phos-tag (Phos-tag electrophoresis), is used to evaluate a kinase autophosphorylation and/or phosphotransfer reaction from a kinase/ATP to its protein substrate. This method outperforms radioisotope methods using [32P]ATP for detecting trace amounts of phosphorylated protein in fresh protein preparations. Phos-tag electrophoresis has been used to perform detailed analyses of the kinase activity of a heme-based oxygen sensor-specifically, a globin-coupled histidine kinase from the soil bacterium Anaeromyxobacter sp. Fw109-5 (AfGcHK).

• MeSH
• adenosintrifosfát metabolismus   MeSH
• Bacteria metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• hem * metabolismus   MeSH
• kyslík metabolismus   MeSH
• ligandy MeSH
• proteiny * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The heme-based oxygen sensor protein AfGcHK is a globin-coupled histidine kinase in the soil bacterium Anaeromyxobacter sp. Fw109-5. Its C-terminal functional domain exhibits autophosphorylation activity induced by oxygen binding to the heme-Fe(II) complex located in the oxygen-sensing N-terminal globin domain. A detailed understanding of the signal transduction mechanisms in heme-containing sensor proteins remains elusive. Here, we investigated the role of the globin domain's dimerization interface in signal transduction in AfGcHK. We present a crystal structure of a monomeric imidazole-bound AfGcHK globin domain at 1.8 Å resolution, revealing that the helices of the WT globin dimer are under tension and suggesting that Tyr-15 plays a role in both this tension and the globin domain's dimerization. Biophysical experiments revealed that whereas the isolated WT globin domain is dimeric in solution, the Y15A and Y15G variants in which Tyr-15 is replaced with Ala or Gly, respectively, are monomeric. Additionally, we found that although the dimerization of the full-length protein is preserved via the kinase domain dimerization interface in all variants, full-length AfGcHK variants bearing the Y15A or Y15G substitutions lack enzymatic activity. The combined structural and biophysical results presented here indicate that Tyr-15 plays a key role in the dimerization of the globin domain of AfGcHK and that globin domain dimerization is essential for internal signal transduction and autophosphorylation in this protein. These findings provide critical insights into the signal transduction mechanism of the histidine kinase AfGcHK from Anaeromyxobacter.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• globiny chemie   metabolismus   MeSH
• histidinkinasa chemie   metabolismus   MeSH
• konformace proteinů, alfa-helix MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• Myxococcales chemie   metabolismus   MeSH
• proteinové domény MeSH
• signální transdukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• fosforylace MeSH
• hem chemie   MeSH
• histidinkinasa chemie   metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• kyslík metabolismus   MeSH
• molekulární modely MeSH
• Myxococcales metabolismus   MeSH
• oxidace-redukce MeSH
• proteinové domény MeSH
• signální transdukce MeSH
• vodík-deuteriová výměna MeSH
• železité sloučeniny chemie   MeSH
• železnaté sloučeniny chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The globin-coupled oxygen sensor, YddV, is a heme-based oxygen sensor diguanylate cyclase. Oxygen binding to the heme Fe(II) complex in the N-terminal sensor domain of this enzyme substantially enhances its diguanylate cyclase activity which is conducted in the C-terminal functional domain. Leu65 is located on the heme distal side and is important for keeping the stability of the heme Fe(II)-O2 complex by preventing the entry of the water molecule to the heme complex. In the present study, it was found that (i) Escherichia coli-overexpressed and purified L65N mutant of the isolated heme-bound domain of YddV (YddV-heme) contained the verdoheme iron complex and other modified heme complexes as determined by optical absorption spectroscopy and mass spectrometry; (ii) CO was generated in the reconstituted system composed of heme-bound L65N and NADPH:cytochrome P450 reductase as confirmed by gas chromatography; (iii) CO generation of heme-bound L65N in the reconstituted system was inhibited by superoxide dismutase and catalase. In a concordance with the result, the reactive oxygen species increased the CO generation; (iv) the E. coli cells overexpressing the L65N protein of YddV-heme also formed significant amounts of CO compared to the cells overexpressing the wild type protein; (v) generation of verdoheme and CO was also observed for other mutants at Leu65 as well, but to a lesser extent. Since Leu65 mutations are assumed to introduce the water molecule into the heme distal side of YddV-heme, it is suggested that the water molecule would significantly contribute to facilitating heme oxygenase reactions for the Leu65 mutants.

• MeSH
• hem chemie   MeSH
• hemová oxygenasa (decyklizující) metabolismus   MeSH
• kyslík metabolismus   MeSH
• leucin genetika   MeSH
• lyasy štěpící vazby P-O genetika   metabolismus   MeSH
• mutace * MeSH
• oxid uhelnatý metabolismus   MeSH
• proteiny z Escherichia coli genetika   metabolismus   MeSH
• voda chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH