Heme environmental structure of CooA is modulated by the target DNA binding. Evidence from resonance Raman spectroscopy and CO rebinding kinetics.
In order to investigate the gene activation mechanism triggered by the CO binding to CooA, a heme-containing transcriptional activator, the heme environmental structure and the dynamics of the CO rebinding and dissociation have been examined in the absence and presence of its target DNA. In the absence of DNA, the Fe-CO and C=O stretching Raman lines of the CO-bound CooA were observed at 487 and 1969 cm-1, respectively, suggesting that a neutral histidine is an axial ligand trans to CO. The frequency of nu(Fe-CO) implies an open conformation of the distal heme pocket, indicating that the ligand replaced by CO is located away from the bound CO. When the target DNA was added to CO-bound CooA, an appearance of a new nu(Fe-CO) line at 519 cm-1 and narrowing of the main line at 486 cm-1 were observed. Although the rate of the CO dissociation was insensitive to the additions of DNA, the CO rebinding was decelerated in the presence of the target DNA, but not in the presence of nonsense DNA. These observations demonstrate the structural alterations in the heme distal site in response to binding of the target DNA and support the activation mechanism proposed for CooA, which is triggered by the movement of the heme distal ligand to modify the conformation of the DNA binding domain.
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
T
T
Uchida
H
H
Ishikawa
S
S
Takahashi
K
K
Ishimori
I
I
Morishima
K
K
Ohkubo
H
H
Nakajima
S
S
Aono
D001426:Bacterial Proteins / Q000737:chemistry; D002248:Carbon Monoxide / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry; D005786:Gene Expression Regulation / Q000502:physiology*; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D012247:Rhodospirillum rubrum / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D015534:Trans-Activators / Q000737:chemistry*; D015533:Transcriptional Activation / Q000235:genetics*
D001426:Bacterial Proteins; C108776:CooA protein, Rhodospirillum rubrum; D004268:DNA-Binding Proteins; D006420:Hemeproteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D015534:Trans-Activators; D006418:Heme; D002248:Carbon Monoxide; D004247:DNA