Metalloprotein enabled redox signal transduction in microbes

Abstract

Microbes utilize numerous metal cofactor-containing proteins to recognize and respond to constantly fluctuating redox stresses in their environment. Gaining an understanding of how these metalloproteins sense redox events, and how they communicate such information downstream to DNA to modulate microbial metabolism, is a topic of great interest to both chemists and biologists. In this article, we review recently characterized examples of metalloprotein sensors, focusing on the coordination and oxidation state of the metals involved, how these metals are able to recognize redox stimuli, and how the signal is transmitted beyond the metal center. We discuss specific examples of iron, nickel, and manganese-based microbial sensors, and identify gaps in knowledge in the field of metalloprotein-based signal transduction pathways.

Fig. 1:. Metalloproteins in signal transduction pathways can be represented as switches or logic gates reacting to redox events at their input nodes.

Heme iron in DosS/DosT enzymes acts as an OR operator and senses the presence of NO, CO, or hypoxia to activate the dosR regulon. Iron-sulfur cluster in Rhizobium acts as a AND operator to signal the presence of low Fe and high O₂, and activates the IRO box in response. Finally, the Ni-Fe cluster in proteobacterium R. eutrophus switches on hox genes in response to the presence of H₂ in the environment.

Fig. 2.. Heme iron dependent signal transduction pathways.

A. Heme sensors sense redox stimuli through various mechanisms including (i) stimuli binds directly to ferrous heme as in Mtb DosS/DosT (ii) stoichiometric NO causes displacement of His proximal ligand and excess NO results in minor species with NO on proximal side, as in H-NOX, (iii) displacement of Met distal ligand to enable O₂ binding as in Ec DosP, or (iv) three-phase binding where the proximal ligand switches from Cys to His with heme reduction followed by distal Pro dissociation allowing CO to bind as in CooA. In these figures, black outline on heme parallelogram represents an inactive protein sensor, while green outline represents a protein sensor that is active and signaling. B. From sensing at the heme center, the signal is communicated through various mechanisms such as (i) a hydrogen-bonding network in Mtb DosS (PDB 2W3G) [adapted from Cho et al.] (ii) The signal is further propagated through a phosphorylation pathway in which DosS/T is autophosphorylated, the phosphoryl group is transferred to DosR, and DosR binds to DNA to facilitate transcription of dormancy genes. (iii) In H-NOX, the signal is communicated through a series of conformational changes such as the heme flattening, dissociation of His and Pro residues, and movement of the distal domain [adapted from Hespen et al.]. (iv) CooA binds directly to the DNA and (v) Ec DosP hydrolyzes the secondary messenger c-di-GMP to pGpG.

Fig. 3.

Chemical structures of non-heme iron-based cofactors involved in signal transduction, which include (i) [4Fe-4S] clusters, (ii) [2Fe-2S] clusters, or (iii) di-nuclear iron motifs, among others. These motifs are useful for sensing dissolved gasses, such as NO or O₂, in the bacterial environment. B (i) WhiB1 sensing mechanism uses an [4Fe-4S] cluster that gets disintegrated upon nitrosylation. (ii) The degradation of WhiB1’s [4Fe-4S] cluster allows the protein to bind to DNA and regulate the expression of Mtb virulence factors. C (i) ODP contains a di-nuclear iron motif that binds to diatomic oxygen. (ii) ODP sensing mechanism involves two states of the protein. When Td is oxygen deplete, both irons are ferrous and ODP stimulates CheA auto-phosphorylation. When ODP is O₂-bound, both irons become ferric, and facilitate de-phosphorylation of CheA.

Figure 4:. Signal transduction pathways involving Ni/Fe and Mn cofactors.

(A) Crystal structures of [NiFe] active site in the hydrogenase of Ralstonia eutropha (PDB: 3RGW) and Mn center in the peroxide sensor PerR of Bacillus subtilis (PDB: 3F8N). (B) Postulated mechanism of H₂ sensing in the regulatory hydrogenase [NiFe] center (adapted from Kaur-Ghumaan and Stein). H₂ attaches to the Ni center and subsequent heterolytic cleavage of the gaseous ligand leads to the bridging hydride between the two metal centers. Next, Ni(III) Fe(II) state is formed by electron transfer and concomitant loss of the generated proton. In the final step, a second round of electron and proton loss occurs to recover the initial state of Ni(II) Fe(II) with the open bridge. (C) Biomolecular pathway of the H₂-sensor HoxB/C in the regulatory [NiFe] hydrogenase. (i) In the absence of H₂, lack of protein-protein interaction between the dimeric heterodimer HoxB/C and the histidine kinase HoxJ enables HoxJ to undergo autophosphorylation. HoxJ, in turn, transfers the phosphoryl group to the response regulator HoxA, which then binds to the DNA and inactivates hydrogenase gene transcription. (ii) In contrast, with H₂ present, HoxB/C directly binds to HoxJ and inhibits HoxJ kinase activity. This results in non-phosphorylated form of HoxA, which releases the DNA and initiates the transcription. (D) Biomolecular pathway of the H₂O₂-sensor PerR. (i) H₂O₂ leads to the oxidation of coordinating histidines in metalated PerR, which releases the metal and induces a conformational change in PerR such that it is unable to associate with DNA. This DNA release allows RNA polymerase to bind, instead and upregulates the expression of an array of oxidative stress resistance genes. (ii) Without H₂O₂, PerR binds directly to DNA and acts as a transcription repressor.