Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from Paraburkholderia xenovorans is a dimer, wherein the heme-binding domain mediates dimerization. Using bioinformatics, we show that RcoM is found in three distinct genomic contexts, in accordance with the previous literature. We propose a refined consensus DNA-binding sequence for RcoM based on sequence alignments of coxM-associated promoters. The RcoM promoter consensus sequence bears two well-conserved direct repeats, consistent with other LytTR domain-containing transcription factors. In addition, there is a third, moderately conserved direct repeat site. Surprisingly, PxRcoM-1 requires all three repeat sites to cooperatively bind DNA with a [P]1/2 of 250 ± 10 nM and an average Hill coefficient, n, of 1.7 ± 0.1. The paralog PxRcoM-2 binds to the same triplet motif with comparable affinity and cooperativity. Considering this unusual DNA binding stoichiometry, that is, a dimeric protein with a triplet DNA repeat-binding site, we hypothesize that RcoM interacts with DNA in a manner distinct from other LytTR domain-containing transcription factors.