Table 1.
Data collection and crystallographic statistics.
Table 2.
Refinement statistics.
Fig 1.
Redox changes of HiPIP in the solution.
(A) UV-visible spectra for the oxidized and reduced states are indicated as orange and green lines, respectively. In addition, the spectrum for the non-treated sample is indicated as a gray line, while it is overlapped with the green line. The spectra were scaled by fitting to the absorbance at 280 nm. (B) Changes in the ratio A280/A380 as a function of incubation time. Green circles: reduced HiPIP in 100 mM sodium citrate (pH 4.5); orange filled circles: oxidized HiPIP in 100 mM sodium citrate (pH 4.5); orange circles: oxidized HiPIP in 1.0 M ammonium sulfate, 100 mM sodium citrate (pH 4.5) and 2 mM K3[Fe(CN)6].
Fig 2.
(A) Photograph of crystals for oxidized HiPIP. The scale bars indicate 0.1 mm. (B) Small and thin crystals of oxidized HiPIP are shown in order to distinguish crystal colors of oxidized HiPIP from those of reduced HiPIP in panel c. (C) Photograph of small and thin crystals of reduced HiPIP.
Fig 3.
Electron density maps of HiPIP in the oxidized state at 0.8 Å resolution.
(A) The electron density map around the iron-sulfur cluster of the oxidized HiPIP at 0.8 Å resolution. The hydrogen omit Fobs−Fcalc map is shown in cyan surface at a contour level of 3σ. The 2Fobs−Fcalc map is shown at contour levels of 5σ (gray mesh), 25σ (orange mesh) and 45σ (magenta surface). (B) The electron density map around Trp74. (C) The electron density map around Arg28.
Table 3.
Bond length for the Fe4S4(Cys-Sγ)4 cluster.
Table 4.
Distance between S atoms of the cluster and the peptide portion.
Fig 4.
Crystal structures of HiPIP at 0.8 Å.
(A) Differences between the reduced and oxidized forms. The structures in the oxidized and reduced states are superimposed and represented in orange and green, respectively. Hydrogen atoms are omitted from the figure for clarity. (B) Multi-conformational residues in the oxidized and reduced states are represented as orange and green sticks, respectively. Depth of colors reflects the occupancy of each conformation at the residue. Single-conformational residues and the iron-sulfur cluster are represented as gray tubes and sticks, respectively.
Fig 5.
Differences on the respective bonds of the iron sulfur cluster.
Bond sticks are colored in red (positive) or blue (negative) according to the difference values in the bond distance, (dOx—dRed)/dRed. (A) Values are from X-ray analyses of HiPIP from T. tepidum (this work). (B) X-ray analyses of HiPIP from A. vinosum [12]. Fe-(Cys-Sγ) distances are the averaged value of four bonds. (C) Theoretical calculations for [Fe4S4(SCH3)4] [33].
Fig 6.
Positional changes of the amide-hydrogen atom of Cys75 on the redox change.
(A) The hydrogen omit Fobs−Fcalc map for the oxidized state is shown as an orange mesh at a contour level of 4σ. The value without parentheses is the bond length between amide-H of Cys75 and S3, while that with parentheses is the bond length for the C-N bond. (B) The hydrogen omit Fobs-Fcalc map for the reduced state is shown as a green mesh at a contour level of 4σ.
Fig 7.
Water molecules on the surface of HiPIP.
(A) Oxygen atoms of waters for the oxidized and reduced HiPIP are colored in orange and green, respectively. HiPIP is represented as a transparent surface model. The side chain of Phe48 on the putative electron-transfer pathway [36] is colored in blue. (B) The backside surface. (C) A water cluster on the surface of HiPIP in the oxidized state. The hydrogen omit Fobs-Fcalc map is shown in orange surface at a contour level of 3σ. The 2Fobs-Fcalc map is shown in gray surface at contour levels of 2σ. (D) The same water cluster in the reduced state. The hydrogen omit Fobs-Fcalc map is shown in green surface at a contour level of 3σ.