Characterization of the Fe site in iron-sulfur cluster-free hydrogenase (Hmd) and of a model compound via nuclear resonance vibrational spectroscopy (NRVS)

Abstract

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron site in the iron-sulfur cluster-free hydrogenase Hmd from the methanogenic archaeon Methanothermobacter marburgensis. The spectra have been interpreted by comparison with a cis-(CO)2-ligated Fe model compound, Fe(S2C2H4)(CO)2(PMe3)2, as well as by normal mode simulations of plausible active site structures. For this model complex, normal mode analyses both from an optimized Urey-Bradley force field and from complementary density functional theory (DFT) calculations produced consistent results. For Hmd, previous IR spectroscopic studies found strong CO stretching modes at 1944 and 2011 cm(-1), interpreted as evidence for cis-Fe(CO)2 ligation. The NRVS data provide further insight into the dynamics of the Fe site, revealing Fe-CO stretch and Fe-CO bend modes at 494, 562, 590, and 648 cm(-1), consistent with the proposed cis-Fe(CO)2 ligation. The NRVS also reveals a band assigned to Fe-S stretching motion at approximately 311 cm(-1) and another reproducible feature at approximately 380 cm(-1). The (57)Fe partial vibrational densities of states (PVDOS) for Hmd can be reasonably well simulated by a normal mode analysis based on a Urey-Bradley force field for a five-coordinate cis-(CO)2-ligated Fe site with additional cysteine, water, and pyridone cofactor ligands. A "truncated" model without a water ligand can also be used to match the NRVS data. A final interpretation of the Hmd NRVS data, including DFT analysis, awaits a three-dimensional structure for the active site.

Figure 1

(Left) NRVS PVDOS (—), far infrared (), and resonance Raman () for Fe(S₂C₂H₄)(CO)₂(PMe₃)₂. Right: Molecular structure of Fe(S₂C₂H₄)(CO)₂(PMe₃)₂. Thermal ellipsoids are set at the 50% probability level. Hydrogen atoms are omitted for clarity. Key distances (Å) and angles (°): Fe(1)–C(1) 1.766(3), Fe(1)–C(2) 1.773(3), Fe(1)–P(2) 2.2628(9), Fe(1)–P(1) 2.2780(9), Fe(1)–S(2) 2.3181(11), Fe(1)-S(1) 2.3355(9); C(1)–Fe(1)–C(2) 97.56(13), C(1)–Fe(1)–P(2) 91.03(9), C(2)–Fe(1)–P(2) 92.22(9), C(1)–Fe(1)–P(1) 89.37(9), C(2)–Fe(1)–P(1) 87.27(9), P(2)–Fe(1)–P(1) 179.38(3), C(1)–Fe(1)–S(2) 174.70(10), C(2)–Fe(1)–S(2) 87.68(11), P(2)–Fe(1)–S(2) 87.92(3), P(1)–Fe(1)–S(2) 91.73(3), C(1)–Fe(1)–S(1) 86.22(9), C(2)–Fe(1)–S(1) 176.18(11), P(2)–Fe(1)–S(1) 87.17(4), P(1)–Fe(1)–S(1) 93.32(3), S(2)–Fe(1)–S(1) 88.53(3).

Figure 2

Left: (top to bottom) NRVS PVDOS (), Vibratz simulation (—), and DFT simulations using LACV3P**+ basis and PWPW91 functional () or B3LYP functional (– –) for Fe(S₂C₂H₄)(CO)₂(PMe₃)₂. Right: atomic motion in normal modes at (a) 626 cm⁻¹ in Vibratz simulation, (b) 636 cm⁻¹ in DFT simulation using PWPW91 functional and LACV3P**+ basis.

Figure 3

NRVS PVDOS for (top-to-bottom): (a) Hmd in 50 mM Tricine/NaOH buffer at pH 8.0, (b) Hmd 50 mM Tricine/NaOH buffer exchanged in ¹⁸O water at pH 8.0, (c) Hmd + methenyl-H₄MPT⁺ + H₂ in 50 mM Mes-NaOH buffer at pH 6.0.

Figure 4

Left: Fe partial vibrational density of state functions for Hmd at pH 8 () in natural abundance water and simulations () for (top to bottom): ‘tetrahedral’ model, 4-coordinate ‘truncated’ model, 5-coordinate ‘trigonal bipyramidal’ model with ¹⁶O water ligand. Right: Ball and stick models of the structures employed in the calculations. Additional cysteine and pyridone atoms have been omitted for clarity.

Chart 1

Top: the reaction catalyzed by Hmd. Bottom: Hmd cofactor breakdown product formed after UV-A irradiation [9].