Expression of Maize Ferredoxin cDNA in Escherichia coli: Comparison of Photosynthetic and Nonphotosynthetic Ferredoxin Isoproteins and their Chimeric Molecule.
Maize (Zea mays L.) has two types of ferredoxin (Fd) differentially expressed in photosynthetic and nonphotosynthetic organs. A cDNA fragment encoding the mature polypeptide of Fd III, an Fd isoprotein of the nonphotosynthetic type, was expressed in Escherichia coli, and the Fd was synthesized as a holo-form assembled with the [2Fe-2S] cluster, which was completely identical with authentic Fd III prepared from maize roots. This expression system made it possible to prepare Fd present at fairly low levels in plants in amounts sufficient for functional and structural studies. Comparison of electron transfer activity of Fd III with that of Fd I, an Fd isoprotein of the photosynthetic type, showed that Fd III was superior as an electron acceptor from NADPH, and Fd I was superior as an electron donor for NADP(+), in reactions catalyzed by Fd-NADP(+) reductase from maize leaf. The circular dichronism spectra of the two Fds also indicated a subtle difference in the geometry of their iron-sulfur clusters. These results are consistent with the view that photosynthetic and nonphotosynthetic Fds may be functionally differentiated. An artificial chimeric Fd, Fd III/Fd I, whose amino-terminal and carboxylterminal halves are derived from the corresponding regions of Fd III and Fd I, respectively, showed an activity and CD spectrum significantly similar to those of Fd I. This suggests that 18 amino acid substitutions between Fd III and Fd III/Fd I alter the properties of Fd III so that they resemble those of Fd I.
Hase
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Department or Agricultural Chemistry, School of Agriculture, Nagoya University, Nagoya 464-01, Japan.
Mizutani
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S
Mukohata
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Y