Abstract
The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site-directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs-ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild-type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe132 in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) "locked-off" CooA variants labeled with either MTSL or MAL-6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs-ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) "locked-off" CooA. This observation suggests that, in direct contrast to effector-free CRP, Fe(III) "locked-off" CooA undergoes conformational exchange on the μs-ms timescale. Importantly, we establish MAL-6 as a spin label with a redox-stable linkage that may be utilized to compare conformational dynamics between functional states of CooA.