Characteristic features of the heterologous functional synthesis in Escherichia coli of a 2[4Fe-4S] ferredoxin

Different strategies have been used to express synthetic genes all encoding Clostridium pasteurianum 24Fe-4S] ferredoxin (Fd) in Escherichia coli. The polypeptide can be produced as the C-terminal addition to a hybrid Cro::Protein A fusion protein lacking the metallic centers. The incorporation of the 4Fe-4S] clusters into the cleaved apoFd cannot be carried out in the same conditions as those affording holoFd from purified C. pasteurianum apoFd. In contrast, fully functional Fds can be produced from non-fused synthetic genes under the dependence of strong promoters. The yields of recombinant Fd, although sufficient to purify significant quantities of protein, are limited by the very short half-life of the 24Fe-4S] Fd in E. coli, irrespective of the expression system used. These features are characteristic of 24Fe-4S] Fds when compared with the far more stable recombinant rubredoxin, and probably other small iron-sulfur proteins which have already been produced in high yields. The reasons for the high turnover of 24Fe-4S] Fds are discussed.