Bacteria catalyze the oxidation of carbon monoxide (CO) to carbon dioxide to generate energy for vital life processes. While both aerobic and anaerobic organisms perform CO oxidation chemistry, the carbon monoxide dehydrogenase (CODH) enzymes differ drastically between these two classes of organisms. Anaerobic CO oxidizing organisms contain a gene cluster known as the coo operon, which encodes a Ni-dependent, oxygen-sensitive CODH. Aerobic CO oxidizing organisms contain a gene cluster known as the cox operon, which encodes a Cu-, Mo- and flavin-dependent, oxygen-tolerant CODH. The transcriptional regulation of these essential operons is critical to these organisms due to the significant energy cost in producing these CODH enzymes. Due to different transcriptional needs of aerobic and anaerobic CO oxidizing bacteria, it is no surprise that the regulatory proteins associated with the coo and cox operons differ drastically. While cooA is exclusively found as a regulator of coo operons in nature, cox operons are typically associated with another transcription factor known as regulator of COmetabolism, or rcoM.
RcoM, is a heme-containing transcription factor most commonly associated with the cox operon in aerobic CO-oxidizing bacteria. However, rcoM is also found within the context of coo operons as well as cowN, a gene associated with mitigating CO toxicity in nitrogen fixing bacteria demonstrating that this protein is used to regulate a variety of genes in a CO-dependent manner. While CooA represents the paradigm of CO-mediated transcriptional regulation, RcoM shows no structural homology to CooA. Based on sequence homology, RcoM is predicted to be a unique single component transcription factor consisting of an N-terminal, heme-binding PAS domain and a C-terminal, DNA-binding LytTR domain. While many examples of PAS and LytTR domain-containing proteins are involved in transcriptional regulation, RcoM is the only known transcription factor that combines these two domains as a single-component regulatory system.
Previous work from the Burstyn Lab studying the RcoM-1 homolog from the polychlorinated biphenyl-degrading organism Paraburkholderia xenovorans have shown that the protein exists in three functionally relevant coordination states. In the ferric state, the heme iron in RcoM is axially ligated by protein-derived histidine and cysteine thiolate ligands. Upon reduction to the ferrous state, RcoM undergoes a redox-mediated ligand switch and the thiolate ligand is replaced by methionine. In the final step of RcoM activation, Met-104 is replaced by CO. RcoM possesses an extremely high affinity for CO (g), demonstrating Kd values of approximately 100 pM (approximately six orders of magnitude greater that the CO affinity of CooA).
The Burstyn Lab is interested in understanding RcoM structure and function at an atomic level. We are currently using biophysical methods such as small angle x-ray scattering and analytical ultracentrifugation to provide insight into the structure of this protein. Additionally, bioinformatic tools have provided valuable insight into RcoM function and genetic context in nature.