Ternary and primary structures of low-potential [4Fe-4S] BtFd.

(A) The 3D structure of BtFd (PDB id: 1IQZ). The [4Fe-4S] cluster and aspartate residues are shown as a space-filling model, and the ligand cysteines for the Fe-S cluster are shown as yellow sticks. The surface drawing is superposed on the cartoon model. (B) Whole amino acid sequence of BtFd. The ligating manner of the [4Fe-4S] cluster is illustrated, and the ligand cysteine and aspartate residues are highlighted.

Neutron structure of low-potential [4Fe-4S] BtFd

(A) Overall neutron structure of BtFd. BtFd polypeptides are colored in rainbow colors from blue (N-terminus) to red (C-terminus). The [4Fe-4S] cluster and water molecules are shown as a space-filling model. White and cyan balls indicate hydrogen (deuterium) and oxygen atoms, respectively. The figure on the right is the left one rotated approximately 90°. (B) The 2Fo–Fc (1.0 sigma: blue cage) and Fo–Fc (3.0 sigma: green cage) neutron-scattering length density map around the [4Fe-4S] cluster and the structural model. The 2Fo–Fc map is shown within 1 Å around the [4Fe-4S] cluster for clarity. (C) X-ray electron density map of the corresponding region of (B). 2Fo–Fc (5.0 sigma) and Fo–Fc (3.0 sigma) maps are shown in blue and green, respectively. (D) Hydrogen bond with the [4Fe-4S] cluster and its ligand Sγ atoms of cysteines. The hydrogen (deuterium), oxygen, carbon, and nitrogen atoms are shown in white, red, cyan, and blue, respectively. The hydrogen bonds indicate pink broken lines and the distance between the amide hydrogen and sulfur atoms is shown in angstroms. (E) The distances and angles of the hydrogen bonds. The distance/angle between the hydrogen and the sulfur are shown on the left and the center columns, and the distance between nitrogen/oxygen and sulfur are shown on the right column. (F) Hydrogen bond between Thr63-OH and the main chain –CO of Thr10.

Statistics for Neutron and X-ray Diffraction Data

DFT calculation based on the neutron structure of [4Fe-4S] BtFd

(A) Illustration of model structures (CMN, CM, and CMH) and their calculated IP values. (B) Assumed charge/spin states of the [4Fe-4S] cluster for Ox and Red states. Fe1 is indicated by the orange underscore and dominantly changes its charge state. (C) Distribution of LUMOs of CM and CMH. The red and green colors indicate positive and negative phases, respectively.

Spectral changes upon [4Fe-4S] cluster oxidation and typical voltammogram of BtFd and its mutant proteins

(A) Time-dependent changing of UV-vis absorption spectrum of wild-type BtFd by air oxidation. The spectra are colored in rainbow colors from red (reduced BtFd) to purple (oxidated BtFd). The arrows indicate the absorbance increase at 340 nm, 420 nm, and indicate the absorbance decrease at 260 nm. (B) The absorption changes of the Asp64 mutated BtFds at 420 nm recorded every second. The standard deviations calculated from at least 3 times the measurements indicated as the line width with the light color. The T1/2 value (sec) is indicated in the inset. (C) The cyclic voltammograms obtained at the disposable screen-printed carbon electrode from a solution of as isolated 100 μM BtFd or its mutated proteins in 50 mM Tris-HCl (pH 7.0), 150 mM NaCl and 2.5 mM neomycin, at sweep rate of 50 mV/s. The CV measurement performed in the anerobic chamber (O2 < 5 ppm ).

The effect of aspartate/glutamate residue in various ferredoxins harboring the low-potential [4Fe-4S] cluster

(A) Pyrogenetic tree of ferredoxin harboring the [4Fe-4S], [4Fe-4S] x 2 and [3Fe-4S] cluster from various organism. (B) The air-oxidation absorption changes at 420 nm of various ferredoxin and its Asp/Glu mutated ferredoxin are also indicated.

Proposed putative mechanism of the low-potential ferredoxin

(i) The deprotonation state of the aspartate residue (Asp-COO-) neighboring the oxidated [4Fe-4S]/[3Fe-4S] cluster is the receivable state of an electron. (ii) When the electron-donor protein forms a complex with ferredoxin, the electrons move to the [4Fe-4S]/[3Fe-4S] cluster instantaneously. (iii) Next, the aspartate residue accepts the proton for neutralization (Asp-COOH), and the reduced state of [4Fe-4S]/[3Fe-4S] clusters is stabilized.