Abstract
The ferredoxin (Fd) from Haloarcula japonica possesses a plant-type [2Fe-2S] cluster and is stable at high salt concentrations. Ha. japonica Fd (HjFd) includes an N-terminal additional domain rich in acidic amino acids, as well as a common core domain that contains the Fe-S cluster. The N-terminally HAT-tagged intact HjFd (HAT/HjFd) and spinach/Ha. japonica chimeric Fd (HAT/Sp/HjFd) were prepared and characterized. Escherichia coli-produced HAT/Sp/HjFd and Ha. japonica-produced HAT/HjFd were produced as holoproteins. On the other hand, E. coli-produced HAT/HjFd did not incorporate the Fe-S cluster. These results suggested that the N-terminal domain of HjFd contributed to the polypeptide folding and successive Fe-S cluster incorporation under high salt conditions. Both Ha. japonica-produced HAT/HjFd and E. coli-produced HAT/Sp/HjFd were stable at high salt concentrations (≥1.5 M NaCl), although a reduction in stability was observed at lower concentrations. Lack of the N-terminal domain did not affect the stability of HjFd, indicating that the core domain mainly contributed to the stability of HjFd at high salt concentrations. Solubility of E. coli-produced HAT/Sp/HjFd under high salt conditions was significantly lower than that of Ha. japonica-produced HAT/HjFd. It was revealed that substitution of the N-terminal domain of HjFd to that of spinach Fd injured the solubility of HjFd. Thus, it was concluded that the N-terminal domain of HjFd should perform the essential functions for halophilic adaptation from the folding process through the folded state.
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