Sulfur K-edge XAS and DFT calculations on nitrile hydratase: geometric and electronic structure of the non-heme iron active site

Abstract

The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS(-))-, sulfenate (RSO(-))-, and sulfinate (RSO(2)(-))-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO(-) species changes upon protonation as the S-O bond is elongated (by approximately 0.1 A). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe(III) in the active site of NHase as CysS(-), CysSOH, and CysSO(2)(-) both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z(eff) of the Fe and reveals that the Fe in [FeNO](6) NHase species has a Z(eff) very similar to that of its photolyzed Fe(III) counterpart. DFT calculations reveal that this results from the strong pi back-bonding into the pi antibonding orbital of NO, which shifts significant charge from the formally t(2)(6) low-spin metal to the coordinated NO.

Figure 1

Proposed active-site structure of NHase from the 1.7-Å resolution crystal structure.

Figure 2

Schematic structures of the model complexes of NHase studied.

Figure 3

S K-edge XAS of free cysteine at pH 7 (black), OHCH₂SO₂⁻ (gray) and OHCH₂SO₂H (dashed gray), and model complexes 1 (blue), 2 (dashed blue), 3 (red), and 4 (green).

Figure 4

DFT-calculated ground-state S_3p mixing in 2 (dashed blue), 3 (red), CH₃SO₂⁻ (gray), CH₃SO₂H (dashed gray), CH₃SO⁻ (green), and CH₃-SOH (dashed green). Zero defined at 2390 eV.

Figure 5

S K-edge XAS of NHase-NO (black), NHase-photoactivated (dashed black), simulation with 1 only (blue), and simulation with 3 (red). Inset: Expanded pre-edge region of NHase-NO and NHase-photoactivated.

Figure 6

Second derivative of XAS data of 2 (dashed blue), 3 (red), 4 (green), NHase-NO (black), and NHase-photoactivated (dashed black).

Figure 7

(A) 1.6-Å resolution crystal structure of the NHase-NO complex active site. (B) Optimized geometry, gas phase. (C) Optimized geometry, PCM, ε = 4.0.

Figure 8

DFT-calculated energy level diagram (β MO’s) of the NHase-NO active site (left) and NHase-photoactivated (right) (occupied orbitals are filled diamonds and unoccupied orbitals are open diamonds). The MO’s having S_3p mixing will have pre-edge transition intensity.

Figure 9

Active-site model of NHase NO-bound form developed from S K-edge XAS and DFT calculations.

Figure 10

(Left) H₂O binding at a hypothetical NHase site where cysteines are not oxidized; Fe–O = 3.4 Å. (Right) H₂O binding at the actual enzymatic site (Fe–O = 2.1 Å).