Burstyn Lab
The UW-Madison Heme Team

CO-Mediated Allostery in CooA

A variety of bacteria found in oceans, soils and freshwater environments utilize carbon monoxide (CO) as a source of energy and/or carbon. These bacteria accomplish this feat through expression of carbon monoxide dehydrogenase enzymes that catalyze CO oxidation along with accessory enzymes that help to assemble the CODH catalytic metallocofactors. To ensure efficient transcriptional regulation of these complex CO oxidation systems, these organisms employ CO-sensing transcription factors such as CO oxidation activator, CooA.

CooA is a member of the well-studied CRP/FNR structural superfamily and uses the iron-containing cofactor, heme, to sense the presence of CO. Several crystallographic structures of CooA demonstrate that this protein exists as a dimer in solution with a coiled-coil dimer interface, a helix-turn-helix DNA-binding domain and a heme-binding sensory domain that binds one heme b per monomer.

Figure 1. Three functional states of CooA shown with corresponding structures of the Fe(II)-“ready-off” state in RrCooA (PDB: 1FT9) and the Fe(II)-CO “on” state in ChCooA (PDB: 2HKX). The effector-binding domain, DNA-binding domain and DNA-binding helices are shown in purple, magenta, and orange, respectively.

The most-studied homolog of CooA from the purple photosynthetic bacterium, Rhodospirillum rubrum, senses both the presence of CO and the redox potential in the cell to regulate transcription. CooA has three functionally relevant coordination states. In the ferric state, the heme iron is axially ligated by a protein-derived cysteine thiolate and the N-terminal proline. Upon reduction to the ferrous state, CooA undergoes a redox-mediated ligand switch and the thiolate ligand is replace by an adjacent histidine residue. In the ferrous state, CO can displace the axial proline ligand, allosterically activating CooA to bind its promoter sequence and initiate transcription.

Figure 2. Three functionally relevant oxidation and coordination states in RrCooA. These states map precisely onto the functional states of CooA shown in Figure 1.

Our best understanding of structure-function relationships in heme-containing gas sensors is based on studies of CooA. In our group, we are working to address the many questions about allosteric regulation in CooA and heme-dependent sensors including how the signaling molecule cooperates with protein structural elements to transmit the allosteric signal and modulate protein function.  As a group we are probing allosterically altered protein dynamics in CooA with isothermal titration calorimetry (ITC), fluorescence anisotropy, and site-directed spin-label electron paramagnetic resonance (SDSL-EPR).

Figure 3. An overview of SDSL-EPR. Cysteine residues are incorporated site-specifically into a CooA. These cysteines are labeled with nitroxides via michael addition of the cysteine thiol into a maleimide functionalized nitroxide. SDSL-EPR spectra collected on several spin-labelled Fe(III) CooA variants demonstrate that CooA exists in solution as a mixture of two dynamic states.