Substrate binding preferences and pka determinations of a nitrile hydratase model complex: variable solvent coordination to [(bmmp-TASN)Fe]OTf

Abstract

The five-coordinate iron-dithiolate complex (N,N'-4,7-bis-(2'-methyl-2'-mercatopropyl)-1-thia-4,7-diazacyclononane)iron(III), [LFe]+, has been isolated as the triflate salt from reaction of the previously reported LFeCl with thallium triflate. Spectroscopic characterization confirms an S = 1/2 ground state in non-coordinating solvents with room temperature microeff = 1.78 microB and electron paramagnetic resonance (EPR) derived g-values of g1 = 2.04, g2 = 2.02 and g3 = 2.01. [LFe]+ binds a variety of coordinating solvents resulting in six-coordinate complexes [LFe-solvent]+. In acetonitrile the low-spin [LFe-NCMe]+ (g1 = 2.27, g2 = 2.18, and g3 = 1.98) is in equilibrium with [LFe]+ with a binding constant of Keq = 4.7 at room temperature. Binding of H2O, DMF, methanol, DMSO, and pyridine to [LFe]+ yields high-spin six-coordinate complexes with EPR spectra that display significant strain in the rhombic zero-field splitting term E/D. Addition of 1 equiv of triflic acid to the previously reported diiron species (LFe)2O results in the formation of [(LFe)2OH]OTf, which has been characterized by X-ray crystallography. The aqueous chemistry of [LFe]+ reveals three distinct species as a function of pH: [LFe-OH2]+, [(LFe)2OH]OTf, and (LFe)2O. The pKa values for [LFe-OH2]+ and [(LFe)2OH]OTf are 5.4 +/- 0.1 and 6.52 +/- 0.05, respectively.

Figure 1

Representation of the active site of nitrile hydratase.

Figure 2

UV/visible spectrum of the synthesis of [(LFe)₂OH]OTf (3 mM) in acetonitrile.

Figure 3

ORTEP view of [(LFe)₂OH]⁺ showing 30% probability displacement ellipsoids. Calculated H atoms, solvent and triflate counter ion have been omitted.

Figure 4

EPR spectrum (77 K) of [LFe]OTf in dichloromethane with simulation. Experimental Parameters: Microwave power = 6.3 mW, Modulation Amplitude = 5.35G. Simulation parameters: g₁ = 2.06, g₂ = 2.03, g₃ = 2.02, W₁ = 21.80, W₂ = 39.87, W₃ = 21.23.

Figure 5

Variable temperature (40 °C to −43 °C) UV/visible spectra of [LFe]OTf (3 mM) in acetonitrile. Arrows denote changes in the spectra as the temperature is lowered.

Figure 6

EPR spectra of [LFe]OTf in acetonitrile (A) with simulation (B) and [LFe]OTf in benzonitrile (C) with simulaton (D). Experimental parameters: (A) microwave Power = 1.5 mW, modulation amplitude = 8.09 G; (C) microwave power = 1.99 mW, modulation amplitude = 6.00 G. Simulation parameters: (B) for [LFe]⁺ g₁ = 2.04, g₂ = 2.01, g₃ = 2.02, W₁ = 19.40, W₂ = 28.15, W₃ = 25.80 and [LFe-NCMe]⁺ g₁ = 2.27, g₂ = 2.18, g₃ = 1.98, W₁ = 17.95, W₂ = 91.21, W₃ = 33.17; (D) for [LFe]⁺: g₁ = 2.06, g₂ = 2.03, g₃ = 2.03, W₁ = 21.73, W₂ = 40.05, W₃ = 21.08 and [LFe-NCPh]⁺: g₁ = 2.28, g₂ = 2.18, g₃ = 2.00, W₁ = 64.78, W₂ = 26.50, W₃ = 46.33.

Figure 7

EPR spectra of [LFe]OTf in various solvents. All spectra recorded at 20 K except DMF (77 K).

Figure 8

Calculated High-Spin Spectra. High-spin spectra were calculated assuming S = 5/2, g_iso = 1.995, and (A) D = 0.45 cm⁻¹, E/D = 0.165, σE/D = 0.08; (B) D = 0.45 cm⁻¹, E/D = 0.165, σE/D = 0; (C) D = 0.45 cm⁻¹, E/D = 0.125, σE/D = 0; (D) D = 0.45 cm⁻¹, E/D = 0.085, σE/D = 0; (E) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0; (F) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.010; (G) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.015; (H) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.020; (I) D = 0.45 cm^{− 1}, E/D = 0.045, σE/D = 0.025; (J) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.035; (K) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.045; (L) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.100; (M) D = 0.45 cm⁻¹, E/D = 0.045, σE/D = 0.320; (N) D = 0.45 cm⁻¹, E/D = 0.130, σE/D = 0.320.

Scheme 1

Scheme 2