X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin.

In chloroplasts, ferredoxin (Fd) is reduced by Photosystem I (PSI) and oxidized by Fd-NADP(+) reductase (FNR) that is involved in NADP(+) reduction. To understand the structural basis for the dynamics and efficiency of the electron transfer reaction via Fd, we complementary used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. In the NMR analysis of the formed electron transfer complex with Fd, the paramagnetic effect of the [2Fe-2S] cluster of Fd prevented us from detecting the NMR signals around the cluster. To solve this problem, the paramagnetic iron-sulfur cluster was replaced with a diamagnetic metal cluster. We determined the crystal structure of the Ga-substituted Fd (GaFd) from Synechocystis sp. PCC6803 at 1.62 Šresolution and verified its functional complementation using affinity chromatography. NMR analysis of the interaction sites on GaFd with PSI (molecular mass of ∼1 MDa) and FNR from Thermosynechococcus elongatus was achieved with high-field NMR spectroscopy. With reference to the interaction sites with FNR of Anabaena sp. PCC 7119 from the published crystal data, the interaction sites of Fd with FNR and PSI in solution can be classified into two types: (1) the core hydrophobic residues in the proximity of the metal center and (2) the hydrophilic residues surrounding the core. The former sites are shared in the Fd:FNR and Fd:PSI complex, while the latter ones are target-specific and not conserved on the residual level.

Crystallization and preliminary X-ray studies of an electron-transfer complex of ferredoxin and ferredoxin-dependent glutamate synthase from the cyanobacterium Leptolyngbya boryana.

Ferredoxin (Fd) dependent glutamate synthase (Fd-GOGAT) is a key enzyme involved in nitrogen assimilation that catalyzes the two-electron reductive conversion of Gln and 2-oxoglutarate to two molecules of Glu. Fd serves as an electron donor for Fd-GOGAT and the two proteins form a transient electron-transfer complex. In this study, these two proteins were cocrystallized using the hanging-drop vapour-diffusion method. Diffraction data were collected and processed at 2.65 Å resolution. The crystals belonged to space group P4(3), with unit-cell parameters a = b = 84.95, c = 476.31 Å.

X-ray structure of an asymmetrical trimeric ferredoxin-photosystem I complex.

Photosystem I (PSI), a large protein complex located in the thylakoid membrane, mediates the final step in light-driven electron transfer to the stromal electron carrier protein ferredoxin (Fd). Here, we report the first structural description of the PSI-Fd complex from Thermosynechococcus elongatus. The trimeric PSI complex binds three Fds in a non-equivalent manner. While each is recognized by a PSI protomer in a similar orientation, the distances between Fds and the PSI redox centres differ. Fd binding thus entails loss of the exact three-fold symmetry of the PSI's soluble subunits, inducing structural perturbations which are transferred to the lumen through PsaF. Affinity chromatography and nuclear magnetic resonance analyses of PSI-Fd complexes support the existence of two different Fd-binding states, with one Fd being more tightly bound than the others. We propose a dynamic structural basis for productive complex formation, which supports fast electron transfer between PSI and Fd.