Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin.

• Keywords
• globin-coupled sensors, heme, heme-based sensor proteins, hemoproteins, oxygen sensors, signal transduction,
• MeSH
• Heme * chemistry   MeSH
• Hemoglobins MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Oxygen metabolism   MeSH
• Myoglobin * metabolism   MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• diguanylate cyclase MeSH Browser
• Heme * MeSH
• Hemoglobins MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Myoglobin * MeSH

Hydrogen/deuterium exchange (HDX) is a well-established analytical technique that enables monitoring of protein dynamics and interactions by probing the isotope exchange of backbone amides. It has virtually no limitations in terms of protein size, flexibility, or reaction conditions and can thus be performed in solution at different pH values and temperatures under controlled redox conditions. Thanks to its coupling with mass spectrometry (MS), it is also straightforward to perform and has relatively high throughput, making it an excellent complement to the high-resolution methods of structural biology. Given the recent expansion of artificial intelligence-aided protein structure modeling, there is considerable demand for techniques allowing fast and unambiguous validation of in silico predictions; HDX-MS is well-placed to meet this demand. Here we present a protocol for HDX-MS and illustrate its use in characterizing the dynamics and structural changes of a dimeric heme-containing oxygen sensor protein as it responds to changes in its coordination and redox state. This allowed us to propose a mechanism by which the signal (oxygen binding to the heme iron in the sensing domain) is transduced to the protein's functional domain.

• Keywords
• Globin-coupled histidine kinase, Heme-containing oxygen sensors, Hydrogen/deuterium exchange, Ligand binding, Mass spectrometry, Protein conformational dynamics, Signal transduction,
• MeSH
• Deuterium MeSH
• Heme chemistry   MeSH
• Hemeproteins * MeSH
• Mass Spectrometry methods   MeSH
• Oxygen metabolism   MeSH
• Artificial Intelligence MeSH
• Deuterium Exchange Measurement methods   MeSH
• Hydrogen chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Deuterium MeSH
• Heme MeSH
• Hemeproteins * MeSH
• Oxygen MeSH
• Hydrogen MeSH

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.

• Keywords
• Chemotaxis, Cyclic GMP (cGMP), Heme, Hemoglobin, Histidine Kinases, Myoglobin, Oxygen Binding,
• MeSH
• Azotobacter vinelandii enzymology   MeSH
• Bordetella pertussis enzymology   MeSH
• Chemotaxis MeSH
• Escherichia coli enzymology   MeSH
• Globins chemistry   MeSH
• Heme chemistry   MeSH
• Hemoglobins chemistry   MeSH
• Histidine Kinase MeSH
• Catalytic Domain MeSH
• Catalysis MeSH
• Oxygen chemistry   MeSH
• Phosphorus-Oxygen Lyases chemistry   MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Myoglobin chemistry   MeSH
• Protein Kinases chemistry   MeSH
• Escherichia coli Proteins chemistry   MeSH
• Gene Expression Regulation, Enzymologic * MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Protein Binding MeSH
• Binding Sites MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• diguanylate cyclase MeSH Browser
• Globins MeSH
• Heme MeSH
• Hemoglobins MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Phosphorus-Oxygen Lyases MeSH
• Myoglobin MeSH
• Protein Kinases MeSH
• Escherichia coli Proteins 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.

• Keywords
• Autophosphorylation, Heme-based oxygen sensor, Histidine kinase, Hydrogen sulfide, Intramolecular catalytic regulation, Two-component signal transduction,
• MeSH
• Biocatalysis drug effects   MeSH
• Phosphorylation drug effects   MeSH
• Heme chemistry   metabolism   MeSH
• Histidine Kinase chemistry   genetics   metabolism   MeSH
• Kinetics MeSH
• Oxygen chemistry   metabolism   MeSH
• Molecular Structure MeSH
• Mutagenesis, Site-Directed MeSH
• Myxococcales enzymology   MeSH
• Hydrogen Sulfide chemistry   pharmacology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heme MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Hydrogen Sulfide 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.

• Keywords
• bacterial protein kinase, crystal structure, globin, heme-containing oxygen sensor, histidine kinase, hydrogen-deuterium exchange, signal transduction, two component signal transduction system,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Heme chemistry   MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Mass Spectrometry MeSH
• Crystallography, X-Ray MeSH
• Protein Structure, Quaternary MeSH
• Oxygen metabolism   MeSH
• Models, Molecular MeSH
• Myxococcales metabolism   MeSH
• Oxidation-Reduction MeSH
• Protein Domains MeSH
• Signal Transduction MeSH
• Deuterium Exchange Measurement MeSH
• Ferric Compounds chemistry   MeSH
• Ferrous Compounds chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Heme MeSH
• Histidine Kinase MeSH
• Oxygen MeSH
• Ferric Compounds MeSH
• Ferrous Compounds 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.

• Keywords
• Diguanylate cyclase, Globin-coupled oxygen sensor, Heme oxygenase, Heme-based oxygen sensor, NADPH:cytochrome P450 reductase, Verdoheme,
• MeSH
• Heme chemistry   MeSH
• Heme Oxygenase (Decyclizing) metabolism   MeSH
• Oxygen metabolism   MeSH
• Leucine genetics   MeSH
• Phosphorus-Oxygen Lyases genetics   metabolism   MeSH
• Mutation * MeSH
• Carbon Monoxide metabolism   MeSH
• Escherichia coli Proteins genetics   metabolism   MeSH
• Water chemistry   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Heme MeSH
• Heme Oxygenase (Decyclizing) MeSH
• Oxygen MeSH
• Leucine MeSH
• Phosphorus-Oxygen Lyases MeSH
• Carbon Monoxide MeSH
• Escherichia coli Proteins MeSH
• Water MeSH
• yddV protein, E coli MeSH Browser

The catalytic activity of a heme-based oxygen sensor phosphodiesterase from Escherichia coli (EcDOS) towards cyclic diGMP is regulated by the redox state of the heme iron complex in the enzyme's sensing domain and the association of external ligands with the iron center. Specifically, the Fe(II) complex is more active towards cyclic diGMP than the Fe(III) complex, and its activity is further enhanced by O2 or CO binding. In order to determine how the redox state and coordination of the heme iron atom regulate the catalytic activity of EcDOS, we investigated the flexibility of its isolated N-terminal heme-binding domain (EcDOS-heme) by monitoring its spectral properties at various hydrostatic pressures. The most active form of the heme-containing domain, i.e. the Fe(II)-CO complex, was found to be the least flexible. Conversely, the oxidized Fe(III) forms of EcDOS-heme and its mutants had relatively high flexibilities, which appeared to be linked to the low catalytic activity of the corresponding intact enzymes. These findings corroborate the suggestion, made on the basis of crystallographic data, that there is an inverse relationship between the flexibility of the heme-containing domain of EcDOS and its catalytic activity. The Fe(II)-CO form of the heme domain of a second heme-based oxygen sensor, diguanylate cyclase (YddV), was also found to be quite rigid. Interestingly, the incorporation of a water molecule into the heme complex of YddV caused by mutation of the Leu65 residue reduced the flexibility of this heme domain. Conversely, mutation of the Tyr43 residue increased its flexibility.

• Keywords
• heme-based oxygen sensor, intramolecular catalytic regulation, pressure effects, protein compressibility, protein flexibility,
• MeSH
• Escherichia coli chemistry   MeSH
• Phosphoric Diester Hydrolases chemistry   MeSH
• Hydrostatic Pressure MeSH
• Catalysis MeSH
• Phosphorus-Oxygen Lyases chemistry   MeSH
• Oxidation-Reduction MeSH
• Escherichia coli Proteins chemistry   MeSH
• Spectrophotometry, Ultraviolet MeSH
• Protein Structure, Tertiary MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• dosP protein, E coli MeSH Browser
• Phosphoric Diester Hydrolases MeSH
• Phosphorus-Oxygen Lyases MeSH
• Escherichia coli Proteins MeSH
• yddV protein, E coli MeSH Browser

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 β-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 β-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.

• Keywords
• analytical ultracentrifugation, heme-based oxygen sensor, histidine kinase, homology modeling, hydrogen-deuterium exchange, protein-protein docking, protein-protein interaction, two-component signal transduction system,
• MeSH
• Aeromonas salmonicida genetics   metabolism   MeSH
• Bacterial Proteins chemistry   genetics   metabolism   MeSH
• Escherichia coli genetics   metabolism   MeSH
• Phosphorylation MeSH
• Heme chemistry   metabolism   MeSH
• Histidine chemistry   metabolism   MeSH
• Histidine Kinase chemistry   genetics   metabolism   MeSH
• Cloning, Molecular MeSH
• Aspartic Acid chemistry   metabolism   MeSH
• Oxygen chemistry   metabolism   MeSH
• Myxococcales chemistry   enzymology   MeSH
• Protein Domains MeSH
• Recombinant Proteins chemistry   genetics   metabolism   MeSH
• Protein Structure, Secondary MeSH
• Signal Transduction * MeSH
• Structural Homology, Protein MeSH
• Deuterium Exchange Measurement MeSH
• Iron chemistry   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Heme MeSH
• Histidine MeSH
• Histidine Kinase MeSH
• Aspartic Acid MeSH
• Oxygen MeSH
• Recombinant Proteins MeSH
• Iron 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.

• Keywords
• bacterial protein kinase, cell signaling, crystal structure, dimerization interface, globin, heme, heme-based oxygen sensor, histidine kinase, signal transduction, two component system,
• MeSH
• Bacterial Proteins chemistry   metabolism   MeSH
• Phosphorylation MeSH
• Globins chemistry   metabolism   MeSH
• Histidine Kinase chemistry   metabolism   MeSH
• Protein Conformation, alpha-Helical MeSH
• Protein Conformation MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Protein Multimerization MeSH
• Myxococcales chemistry   metabolism   MeSH
• Protein Domains MeSH
• Signal Transduction MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• Globins MeSH
• Histidine Kinase MeSH