The study on the structure and function of the transcriptionalregulator containing a heme as a signal sensor

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CooA is a heme-containing and CO-sensing transcriptional activator whose activity is regulated by CO. A protoheme acts as a CO sensor in CooA. Mutagenesis and spectroscopic studies revealed the unique coordination structure of the heme in CooA, the Pro^2/Cys^<75>, Pro^2/His^<77>, and CO/His^<77> coordinations for the ferric, ferrous, and CO-bound hemes, respectively. The ligand exchange between Cys^<75> and His^<77> took place during the change in the oxidation state of the heme. The ligand-switching process induced by the reduction of the heme was investigated by the technique of pulse radiolysis. Hydrated electron reduced the heme iron in ferric CooA within 1 μs to form the first intermediate with the Soret peak at 440 nm, suggesting that a six-coordinated ferrous heme with a thiolate axial ligand was formed initially.The first intermediate was converted into the second intermediate with the time constantjof 40 μs (k = 2.5 x 10^4 s^<-1>). In the second intermediate, the thiolate from Cys^<75> was thought to be protonated and/or the Fe-S bond was thought to be elongated. The second intermediate was converted into the final reduced form with the time constant of 2.9 ms (k = 3.5 x 102 s^<-1>) for wild-type CooA. The ligand exchange between Cys^<75> and His^<77> took place during the conversion of the second intermediate into the final reduced form. We constructed a truncated mutant, CooAΔN5, in which the first five residues in the N-terminal were deleted, to examine the functional role of Pro^2 coordination. CooAΔN5 showed a similar spectroscopic and functional properties to those of wild type, indicating the flexibility of the polypeptide chain of CooA in its N-terminal region.