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, 51 (15), 8617-28

EPR/ENDOR, Mössbauer, and Quantum-Chemical Investigations of Diiron Complexes Mimicking the Active Oxidized State of [FeFe]hydrogenase

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EPR/ENDOR, Mössbauer, and Quantum-Chemical Investigations of Diiron Complexes Mimicking the Active Oxidized State of [FeFe]hydrogenase

Alexey Silakov et al. Inorg Chem.

Abstract

Understanding the catalytic process of the heterolytic splitting and formation of molecular hydrogen is one of the key topics for the development of a future hydrogen economy. With an interest in elucidating the enzymatic mechanism of the [Fe(2)(S(2)C(2)H(4)NH)(CN)(2)(CO)(2)(μ-CO)] active center uniquely found in [FeFe]hydrogenases, we present a detailed spectroscopic and theoretical analysis of its inorganic model [Fe(2)(S(2)X)(CO)(3)(dppv)(PMe(3))](+) [dppv = cis-1,2-bis(diphenylphosphino)ethylene] in two forms with S(2)X = ethanedithiolate (1edt) and azadithiolate (1adt). These complexes represent models for the oxidized mixed-valent Fe(I)Fe(II) state analogous to the active oxidized "H(ox)" state of the native H-cluster. For both complexes, the (31)P hyperfine interactions were determined by pulse electron paramagnetic resonance and electron nuclear double resonance (ENDOR) methods. For 1edt, the (57)Fe parameters were measured by electron spin-echo envelope modulation and Mössbauer spectroscopy, while for 1adt, (14)N and selected (1)H couplings could be obtained by ENDOR and hyperfine sublevel correlation spectroscopy. The spin density was found to be predominantly localized on the Fe(dppv) site. This spin distribution is different from that of the H-cluster, where both the spin and charge densities are delocalized over the two Fe centers. This difference is attributed to the influence of the "native" cubane subcluster that is lacking in the inorganic models. The degree and character of the unpaired spin delocalization was found to vary from 1edt, with an abiological dithiolate, to 1adt, which features the authentic cofactor. For 1adt, we find two (14)N signals, which are indicative for two possible isomers of the azadithiolate, demonstrating its high flexibility. All interaction parameters were also evaluated through density functional theory calculations at various levels.

Figures

Figure 1
Figure 1
Schematic representation of the H-cluster in the Hox state and model compounds used in this study. An Arrow points to the vacant apical site on the distal Fe, which in the case of the enzyme, is the site for binding H2. Insert: midpoint potentials for the Hred/Hox and [Fe2(SX)2]0/+ redox transition for the H-cluster and the models 1edt and 1adt, correspondingly. For the H-cluster it is specified for pH 8 in Tris buffer with KCl electrolyte, for the model compounds the E0 values are given for [(C4H9)4N]PF6 electrolyte in CH2Cl2 and values are converted from vs Fc0/+ to vs NHE scale by using E0(Fc0/+) = +0.528 vs NHE estimation.
Figure 2
Figure 2
First derivative (pseudomodulated) Q-band two pulse echo-detected EPR spectra (blue) of frozen solutions of 1edt (A) and 1adt (B) and corresponding simulations (red) using principal g-values: A g1edt=(2.009, 2.028, 2.139), B g1adt=(2.006, 2.030, 2.124). Experimental conditions: T = 20 K, νmw = 33.8560 GHz (A), 33.918 GHz (B), τ= 340 ns. The asterisks on B indicate minor contaminations of unknown origin.
Figure 3
Figure 3
Q-band Davies ENDOR spectra (blue) of 1edt measured at T = 20K and 1197.8 mT. Simulations were performed using P1,2,3 parameters from Table 1. The 1H ENDOR region is shaded blue for clarity. Experimental conditions: A, tRF = 35 µs; νmw = 33.8708 GHz; tinv = 200ns. B, tRF = 5.5 µs; νmw = 33.8708 GHz; tinv = 80 ns; see also Figure S2 in SI for complete field dependence.
Figure 4
Figure 4
Q-band VMT (Davies) ENDOR of 1edt measured for various tmix delay times (see SI) at maximum absorption of the EPR spectrum (gy). Both low (A) and high (B) frequency ENDOR signals show a clear dependence on tmix delay times (indicated in the figure). Experimental conditions: A, T = 25 K; tRF = 20 µs; νmw = 33.8269 GHz; B0 = 1192.3 mT; tinv = 100 ns. B, T = 25 K; tRF = 7 µs; νmw = 33.8865 GHz; B0 = 1194.4 mT; tinv = 80 ns.
Figure 5
Figure 5
Mössbauer spectra of the fully 57Fe enriched 1edt measured at three magnetic field strengths at a temperature of 4.2 K (black) and respective simulations (red) accounting for two contributing 57Fe nuclei: Fe1 (blue) and Fe2 (green) with parameters presented in Tables 1 and 2.
Figure 6
Figure 6
Experimental (A–D) Q-band HYSCORE spectra of 1edt measured at the indicated field positions, and corresponding simulations (E–H) that account for one 57Fe HF coupling (A(Fe2), Table 1). Experimental conditions: T = 20 K; τ = 420 n; νmw = 33.8883 GHz; t(π/2) = 40 ns; ΔT1,2 = 52 ns.
Figure 7
Figure 7
W-band 3-pulse ESEEM spectra as function of the applied magnetic field B0 of 57Fe-enriched 1edt, measured at two different τ-values: 164 ns (A) and 288 ns (C), together with the respective simulations (B, D) accounting for two contributing 57Fe HF couplings (Table 1). Experimental conditions: T = 10 K; τ = 164 ns; νmw = 94.0928 GHz; t(π/2) = 24 ns; ΔT = 12 ns. The color coding of the signal relative intensity is represented by the color bar.
Figure 8
Figure 8
Q-band ENDOR spectra of 1adt measured at 20 K and field position 1192.8 mT (gy). Low frequency spectra (A) were recorded using the refocused Mims ENDOR sequence with τ = 108 ns and t(π/2) = 16 ns, whereas the high frequency part (B) was recorded using Davies ENDOR, tinv = 44 ns (suppressing matrix 1H signals), t(π/2) = 16 ns, τ = 400 ns. Simulations were performed using the HF coupling constants listed in Table 1. See also Figure S3 in SI for complete field dependence.
Figure 9
Figure 9
X-band HYSCORE spectra of 1adt (top) and corresponding simulations (bottom) accounting for two independent 14N signals that correspond to two isomeric forms of the amine group (see also Figure S6). Experimental parameters: B0 = 342.5 mT; T = 20 K; νmw = 9.720 GHz; t(π/2) = 8 ns; τ = 86 ns. For clarity the presented spectra are cropped to the low frequency range, neglecting the 1H signals. Asterisks denote instrumental artifacts.
Figure 10
Figure 10
Geometry optimized isomers of 1edt (A) and 1adt (B). Fe1-Fe2 distances are indicated in Å. "bas"/"ap" stands for basal or apical position of the PMe3 ligand, respectively. "flat"/"twist" stands for flat (square-pyramidal) configuration of dithiolate and dppv ligands and twisted configuration. "on"/"off" indicate the position of the amine with respect to the dppv ligand.
Figure 11
Figure 11
57Fe Aiso coupling constants of 1edtflat-bas as calculated using various basis sets (see Supporting Information for detailed description) in conjunction with the B3LYP functional and TZVPP basis set on all other atoms not taking into account second order spin orbit coupling contributions. The red dashed lines indicate the experimentally obtained values from Table 1. Brown and blue bars identify TZVPP and Wachters basis sets, respectively. The white dashed lines in the blue bars indicate spin-orbit coupling corrections to the isotropic hyperfine coupling.

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