A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP⁺ reductase

Abstract

In order to elucidate the importance of a ferredoxin (Fd) Arg-Glu pair involved in dynamic exchange from intra-to intermolecular salt bridges upon complex formation with ferredoxin-NADP + oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair-lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe^2S] cluster. The catalytic constant (k cat ) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of k cat to a level comparable to that for FdI, demonstrating directly that the Arg-Glu pair is important for e⁄cient electron transfer between Fd and FNR.

A Structural Basis of Equisetum arvense Ferredoxin Isoform II Producing an Alternative Electron Transfer with Ferredoxin-NADP+ Reductase

“…K m values of both FdI and FdII were ϳ0.6 M, and this similarity is in contrast to our previous measurements of electron transfer in the opposite direction (from FNR to Fd) where the K m value of FdI was around 7 times greater than that of FdII (13). Consistent with our previous measurements, V max for electron transfer with FNR was greater with FdI than FdII, although it was 1.3 times greater in electron transfer from Fd to FNR, as compared with 5.5 times greater in the opposite direction (13).…”

“…Preparation of Recombinant Fds-E. arvense recombinant FdII, FdI, and the FdII mutants were expressed and accumulated as the apo-form in Escherichia coli cells and successfully converted to the holo-form by chemical reconstitution of the [2Fe-2S] cluster as described previously (13). Further purification of the Fds was carried out as described previously (15,16).…”

“…Further purification of the Fds was carried out as described previously (15,16). Site-specific and insertion mutants of FdII R22T, FdII with Tyr 32 newly inserted (FdII(Y32)) and FdII(Y32)Q38R/S28E were prepared with the Quikchange site-directed mutagenesis kit (Stratagene) (13), and these mutation sites were confirmed by DNA sequencing. The concentration of Fd was determined spectrophotometrically based on a molar extinction coefficient of 9.68 mM Ϫ1 cm Ϫ1 at 422 nm (17).…”

“…In Equisetum arvense the Arg-Glu pair is conserved in Fd isoform I (FdI) but missing from isoform II (FdII), and a comparison of electron transfer with FNR under excess NADPH shows obvious differences between the K m and k cat values of FdI and FdII. Electron transfer rates of FdII mutants, in which the Arg-Glu pair is introduced, indicate that these residues are crucial to these differences (13). These findings suggest that E. arvense FdII might vary from FdI in its electron transfer activity around photosystem I, because of its different interaction with FNR.…”

“…These findings suggest that E. arvense FdII might vary from FdI in its electron transfer activity around photosystem I, because of its different interaction with FNR. When the Arg-Glu pair of E. arvense FdI is substituted with the corresponding noncharged residues of FdII (R39Q/E28S), the [2Fe-2S] cluster became unstable (13), indicating a crucial role for these residues in stabilizing Fd in the native state. However, it should be noted that the [2Fe-2S] cluster of wild type FdII is stable even in the absence of a salt bridge between Arg 39 and Glu 28 .…”