Redox Properties and Coordination Structure of the Heme in the CO-sensing Transcriptional Activator CooA*

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys⁷⁵ and His⁷⁷ are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro² is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876–880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were −320 mV and −260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys⁷⁵ and His⁷⁷ is thought to cause the difference between the reduction and oxidation midpoint potentials.