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 Phe132in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) “
Heme is an essential and functionally versatile cofactor. Our understanding of how the environment of a heme in a protein tunes its function has benefited from spectroscopic and functional investigations of heme proteins and their variants with altered heme environments. Two properties of current interest are the conformation of the heme and hydrogen bonding to heme propionates. By combining nuclear magnetic resonance experiments and density functional theory calculations, both of these characteristics have been shown to influence the distribution of the singly occupied molecular orbital on the heme of ferricytochrome
- NSF-PAR ID:
- 10238995
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Israel Journal of Chemistry
- Volume:
- 56
- Issue:
- 9-10
- ISSN:
- 0021-2148
- Page Range / eLocation ID:
- p. 693-704
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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