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.

Neutron-scattering length density maps of [4Fe-4S] ferredoxins.

The Fo–Fc neutron-scattering length density omits maps for the hydrogen/deuterium at 2.0 σ contour levels. The pink and green cages represent the +(Fo–Fc) and –(Fo–Fc) map, respectively, and the final model of BtFd is superposed on the maps. The maps clearly indicate that the densities derived from the deuterium and hydrogen atoms are clearly visible in the neutron crystallography.

DFT calculation based on the neutron structure of [4Fe-4S] BtFd Calculated IP values of each model.

The absorption changes of various 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.

UV-Vis absorption spectroscopy of BtFd and its mutant proteins.

The spectra of reduced BtFds are indicated by the orange line and those of completely air-oxidated BtFds are indicated by the blue line. Wild-type BtFd and its mutated proteins showed very similar spectra.

Conserved amino acid residues of [4Fe-4S] ferredoxins from several species.

Cysteine residues for the ligand for the Fe-S cluster are completely conserved (orange-highlighted) and several residues around the cysteines are also highly conserved (blue-highlighted). The corresponding residues of Asp64 in BtFd are conserved in negatively charged residues (Asp or Glu: red-highlighted). This figure is prepared using ESpript ver3.0 server (https://espript.ibcp.fr/ESPript/ESPript/index.php).

Sequence alignment of [4Fe-4S], [4Fe-4S]x2 and [3Fe-4S] ferredoxins from various organisms.

Cysteine residues for the ligand for the Fe-S cluster are completely conserved (yellow-highlighted) and several residues around the cysteines are also highly conserved (blue-highlighted). The corresponding residues of Asp64 in BtFd are conserved in negatively charged residues (Asp or Glu: pink font). This figure is prepared using the Consurf server (https://consurf.tau.ac.il/index_proteins.php) and ESpript ver3.0 server (https://espript.ibcp.fr/ESPript/ESPript/index.php).

The pKa estimation of the Asp64 in BtFd

(A) A close-up view of an overlay of the of the CACO experiments (Cβ offset) of NMR. The assignments were indicated. For Asp64 signals, corresponding pH values are shown in blue as a representative of titrations. Chemical shift changes are indicated by black dotted line for each residue. Detail information were described in the supplemental method and text. (B)Titration curves obtained from the CACO experiments (Cβ offset). The points correspond to pH 7.0 and 7.5 are missing in a series of Asp64, and the point corresponds to pH 6.5 is missing in a series of Asp74. (C) The oxidation rates of BtFd wild-type and D64A under the different pH conditions.

Stability of [4Fe-4S] cluster in BtFd under the different pH conditions.

The UV-Vis absorbance ratios of 304 nm/280 nm, 388 nm/280 nm and 304 nm/388 nm are shown in the table below each spectral panel.

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.

Cartesian coordinate of CMH used for the DFT calculations

Examined charge/spin states with CMNA model for (A) oxidized and (B) reduced states. Definition of Fe1–Fe4 is illustrated using CM below. o12 and r5 (indicated by yellow highlighted) were most stable states for Ox and Red states, respectively.

Cartesian coordinate (in Å) of before and after the geometry optimization. (The Cartesian coordinate before the geometry optimization is the neutron structure)

Calculated IP values of the CMH and CM models with and without the environment effect.

The distance between the acidic residues and the [4Fe-4S] cluster.*