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 Å resolution 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.
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) , Saitama 332-0012, Japan.
Risa
R
Mutoh
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Norifumi
N
Muraki
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Kanako
K
Shinmura
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) , Saitama 332-0012, Japan.
Hisako
H
Kubota-Kawai
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Young-Ho
YH
Lee
Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr University Bochum , 44780 Bochum, Germany.
Marc M
MM
Nowaczyk
Plant Biochemistry, Faculty of Biology and Biotechnology, Ruhr University Bochum , 44780 Bochum, Germany.
Matthias
M
Rögner
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Toshiharu
T
Hase
Department of Medical Life Science, Yokohama City University , 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) , Saitama 332-0012, Japan.
Takahisa
T
Ikegami
Institute for Protein Research, Osaka University , 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.|Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST) , Saitama 332-0012, Japan.
Genji
G
Kurisu
D017033:Anabaena / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005288:Ferredoxins / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D046939:Synechocystis / Q000737:chemistry*
D005288:Ferredoxins