title: Structural and functional characterization of the ga-substituted ferredoxin from Synechocystis sp. PCC6803, a mimic of the native protein.
abstract: In photosynthetic organisms, ferredoxin (Fd) interacts with many proteins, acting as a shuttle for electrons from Photosystem I to a group of enzymes involved in NADP(+) reduction, sulfur and nitrogen assimilation, and the regulation of carbon assimilation. The study of the dynamic interactions between ferredoxin and these enzymes by nuclear magnetic resonance is severely hindered by the paramagnetic [2Fe-2S] cluster of a ferredoxin. To establish whether ferredoxin in which the cluster has been replaced by Ga is a suitable diamagnetic mimic, the solution structure of Synechocystis Ga-substituted ferredoxin has been determined and compared with the structure of the native protein. The ensemble of 10 structures with the lowest energies has an average root-mean-square deviation of 0.30 +/- 0.05 A for backbone atoms and 0.65 +/- 0.04 A for all heavy atoms. Comparison of the NMR structure of GaFd with the crystal structure of the native Fd indicates that the general structural fold found for the native, iron-containing ferredoxin is conserved in GaFd. The ferredoxin contains a single gallium and no inorganic sulfide. The distortion of the metal binding loop caused by the single gallium substitution is small. The binding site on Fd for binding ferredoxin:NADP(+) reductase in solution, determined using GaFd, includes the metal binding loop and its surroundings, consistent with the crystal structures of related complexes. The results provide a structural justification for the use of the gallium-substituted analogue in interaction studies.
authors.affiliation: Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
authors.forename: Xingfu
authors.initials: X
authors.lastname: Xu
authors.forename: Sandra
authors.initials: S
authors.lastname: Scanu
authors.forename: Jung-Sung
authors.initials: JS
authors.lastname: Chung
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hirasawa
authors.forename: David B
authors.initials: DB
authors.lastname: Knaff
authors.forename: Marcellus
authors.initials: M
authors.lastname: Ubbink
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D005708:Gallium / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D013329:Structure-Activity Relationship; D046939:Synechocystis / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D005708:Gallium