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A carbon monoxide dependent transcriptional regulator which contains B-type heme as a carbon monoxide sensor
362
N23
Journal of Inorganic Biochemistry
Abstracts
A CARBON MONOXIDE DEPENDENT TRANSCRIPTIONAL REGUI.ATOR WHICH CONTAINS B-TYPE HEME AS A CARBON MONOXIDE SENSOR
S. Aono, K. Okubo, T. Matsuo, M. Okada, and H. Nakajima School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, TatsunokuchL lshikawa 923-12, Japan The purple, nonsulfur, phototrophic bacterium Rhodospirillum rubrum synthesizes an enzyme sy stem for carbon monoxide (CO) oxidation which is encoded in coo regulon. Carbon monoxide induces the expression of this regulon under anaerobic conditions. CooA is responsible for the COdependent expression of coo regulon and acts as a CO-dependent transcriptional activator. In this work, we constructed the expression sy stem of CooA in E. coli and studied about biochemical and biophysical properties of the recombinant CooA. The recombinant CooA was expressed in the soluble fraction ofE. coli JM 109. The purified CooA shows a typical electronic absorption spectra of heme proteins.[1] The pyridine ferrohemochrome derived from CooA showed an a-peak at 556 nm showing that CooA contained protoheme (b-type heme). The oxidized CooA has the peak of the Soret band at 423.5 nm, and peaks at 539.5 and 570 nm in Q-band region, which is typical for low spin ferric-heme proteins. EPR spectrum of the oxidized CooA at 77K also revealed the typical signal due to low spin ferric-heme proteins. The reduced CooA has the peak of Soret band at 424.5 nm, and at 528.5 and 558.5 nm in Qband region, which is typical for six-coordinated low spin heme proteins. CO-bound CooA was formed by the introduction of CO into the reduced CooA, but not in the case of the oxidized CooA. The formation of CO-bound CooA shows that one of the axial ligands in the reduced CooA will be replaced by CO. Only CO-bound CooA will be active as a transcriptional activator in vivo because the expression of coo regulon takes place in R. rubrum only with carbon monoxide under anaerobic condition. We propose that the replacement of the axial ligand by CO is a trigger of the activation of CooA as the transcriptional activator. When the reduced CooA was reacted with oxygen, the reduced form was converted to the oxidized form without the formation of a stable oxygen-bound (oxo-) form of CooA. The mutant CooA proteins in which histidine at position 28, 77, 133, 146 or 200 is replaced by alanine are preparing to determine the axial li~_nd of CooA. The mutation at position 77 affected the electronic absorption spectrum of CooA suggesting that this histidine was one of the axial ligands. The mutants where methionine is replaced by leucine are also preparing, and their properties will be discussed. The activity of the wild type and these mutant CooA was measured by the reporter gene system in which the operon fusion of the promoter region regulated by CooA and lacZ was constructed in the chromosome in E. coli. 1) S. Aono et al., Biochim. Biophys. Res. Commun., 228, 752 (1996)
N23
Journal of Inorganic Biochemistry
Abstracts
A CARBON MONOXIDE DEPENDENT TRANSCRIPTIONAL REGUI.ATOR WHICH CONTAINS B-TYPE HEME AS A CARBON MONOXIDE SENSOR
S. Aono, K. Okubo, T. Matsuo, M. Okada, and H. Nakajima School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, TatsunokuchL lshikawa 923-12, Japan The purple, nonsulfur, phototrophic bacterium Rhodospirillum rubrum synthesizes an enzyme sy stem for carbon monoxide (CO) oxidation which is encoded in coo regulon. Carbon monoxide induces the expression of this regulon under anaerobic conditions. CooA is responsible for the COdependent expression of coo regulon and acts as a CO-dependent transcriptional activator. In this work, we constructed the expression sy stem of CooA in E. coli and studied about biochemical and biophysical properties of the recombinant CooA. The recombinant CooA was expressed in the soluble fraction ofE. coli JM 109. The purified CooA shows a typical electronic absorption spectra of heme proteins.[1] The pyridine ferrohemochrome derived from CooA showed an a-peak at 556 nm showing that CooA contained protoheme (b-type heme). The oxidized CooA has the peak of the Soret band at 423.5 nm, and peaks at 539.5 and 570 nm in Q-band region, which is typical for low spin ferric-heme proteins. EPR spectrum of the oxidized CooA at 77K also revealed the typical signal due to low spin ferric-heme proteins. The reduced CooA has the peak of Soret band at 424.5 nm, and at 528.5 and 558.5 nm in Qband region, which is typical for six-coordinated low spin heme proteins. CO-bound CooA was formed by the introduction of CO into the reduced CooA, but not in the case of the oxidized CooA. The formation of CO-bound CooA shows that one of the axial ligands in the reduced CooA will be replaced by CO. Only CO-bound CooA will be active as a transcriptional activator in vivo because the expression of coo regulon takes place in R. rubrum only with carbon monoxide under anaerobic condition. We propose that the replacement of the axial ligand by CO is a trigger of the activation of CooA as the transcriptional activator. When the reduced CooA was reacted with oxygen, the reduced form was converted to the oxidized form without the formation of a stable oxygen-bound (oxo-) form of CooA. The mutant CooA proteins in which histidine at position 28, 77, 133, 146 or 200 is replaced by alanine are preparing to determine the axial li~_nd of CooA. The mutation at position 77 affected the electronic absorption spectrum of CooA suggesting that this histidine was one of the axial ligands. The mutants where methionine is replaced by leucine are also preparing, and their properties will be discussed. The activity of the wild type and these mutant CooA was measured by the reporter gene system in which the operon fusion of the promoter region regulated by CooA and lacZ was constructed in the chromosome in E. coli. 1) S. Aono et al., Biochim. Biophys. Res. Commun., 228, 752 (1996)