Carboxylate as the Protonation Site in (Peroxo)diiron(III) Model Complexes of Soluble Methane Monooxygenase and Related Diiron Proteins

Abstract

Addition of H⁺ to a synthetic (µ-1,2-peroxo)diiron(III) model complex results in protonation of a carboxylate rather than the peroxo ligand. This conclusion is based on spectroscopic evidence from UV-vis, ⁵⁷Fe Mössbauer, resonance Raman, infrared, and ¹H/¹⁹F NMR studies. These results suggest a similar role for protons in the dioxygen activation reactions in soluble methane monooxygenase and related carboxylate-bridged diiron enzymes.

Dioxygen activation by carboxylate-bridged diiron enzymes is involved in essential biological processes^1,2 ranging from DNA synthesis and hydrocarbon metabolism to cell proliferation.³ The carboxylate-bridged diiron superfamily of proteins includes ribonucleotide reductase (RNR),⁴ Δ⁹ desaturase,⁵ bacterial multicomponent monooxygenases (BMMs),^6,7 and most recently human deoxyhypusine hydroxylase (hDOHH).³ In all of these systems, the O₂ reduction step proceeds through a (peroxo)diiron(III) intermediate in which the resulting peroxo ligand is proposed to bridge two iron atoms in a µ-1,2 or µ-η² η² coordination mode.^8–10 Extensive studies of soluble methane monooxygenase (sMMO), a BMM family member that oxidizes methane to methanol, reveal that the generation and activation of Fe₂O₂ units requires protons.^11,12 Given the complexity of protein environments, identifying the sites involved in such proton translocation processes and their effect on O₂ activation is not a trivial undertaking.

To shed light on the possible role of protons in the dioxygen activation chemistry at carboxylate-bridged diiron enzyme active sites, we investigated the reaction of H⁺ with a well-characterized synthetic (µ-peroxo)(µ-carboxylato)diiron(III) complex, [Fe₂(µ-O₂)(N-EtHPTB)(µ-PhCO₂)]²⁺ (1a·O₂).^13,14 The dinucleating N-EtHPTB ligand provides kinetic stabilization of the Fe₂O₂ core and the benzoate group serves as a good mimic of the Asp and Glu carboxylate side chains in the protein diiron centers. By application of several spectroscopic methods, we show that the reaction of H⁺ with 1a·O₂ results in protonation at the carboxylate unit rather than the peroxo ligand (Scheme 1). This work provides experimental support for recent theoretical studies suggesting that (hydroperoxo)-diiron(III) species of non-heme diiron enzymes are too reactive to be isolable protein intermediates.¹⁵

Scheme 1.

Reaction of dioxygen with 1a and H⁺. A possible structure for [1a·O₂]H⁺ is depicted.

To aid spectral interpretation of results obtained during studies of the parent [Fe₂(N-EtHPTB)(µ-PhCO₂)]²⁺ complex (1a), two related diiron(II) precursors were synthesized (Supporting Information). One is [Fe₂(N-EtHPTB)-(Ph¹³CO₂)]²⁺ (1b), which contains a ¹³C-enriched carboxylate ligand, and the other is [Fe₂(N-EtHPTB)(C₆F₅CO₂)]²⁺ (2), in which the benzoate ring is fluorinated.

Exposure of 1a to O₂ in CH₃CN at −30 °C generates a deep blue-green solution (1a·O₂) with λ_max at 590 nm.¹³ Addition of an acetonitrile solution of [H(OEt₂)₂][3,5-(CF₃)₂C₆H₃)₄B] (H[BAr^F₄]) to 1a·O₂ red-shifts the peroxo-to-iron(III) charge transfer band to ~600 nm (Fig. 1). This absorption is assigned to the formation of a new [1a·O₂]H⁺ species that maximizes with addition of ~1.5 equiv of H[BAr^F₄]. The spectrum of 1a·O₂ is restored upon addition of 2.0 equiv of NEt₃ (Fig. 1, inset), indicating that protonation does not lead to irreversible decomposition of the 1a·O₂ unit. Reaction of 2 with O₂ affords [Fe₂(µ-O₂)(N-EtHPTB)(µ-C₆F₅CO₂)]²⁺ (2·O₂), which exhibits a broad absorption feature centered at ~600 nm. When H[BAr^F₄] was titrated into a solution of 2·O₂, a small bathochromic shift to ~610 nm occurs (Fig. S1). Unlike 1a·O₂, 2·O₂ requires ~3.0 equiv of H[BAr^F₄]. to fully generate the protonated species [2·O₂]H⁺. Given that pentafluorobenzoate, for which the acid has a pK_a of 1.2, is more electron deficient than benzoate (acid pK_a = 4.6), the greater amount of H⁺ necessary to produce [2·O₂]H⁺ from 2·O₂ compared to [1a·O₂]H⁺ from 1a·O₂ suggests either that the carboxylate ligand influences the basicity of the protonation site or that it is itself the proton acceptor.

Figure 1.

UV-vis absorption spectra of [1a·O₂](BF₄)₂ (112 µM in CH₃CN, −30°C, downward arrow) when up to 2.0 equiv of H[BAr^F₄] were added. Inset: restoration of initial spectrum (orange trace, upward arrow) upon treatment with 2.0 equiv of NEt₃.

To determine whether a (hydroperoxo)diiron(III) species may form upon addition of H⁺ to 1a·O₂ or 2·O₂, ⁵⁷Fe Mössbauer and resonance Raman (RR) spectra were recorded to examine possible changes in the Fe₂O₂ core. In the absence of H⁺, the Mössbauer spectrum of a frozen solution of 1a·O₂ in CH₃CN could be fit to a single iron site, with δ = 0.53(2) mm/s and ΔE_Q = 0.71(2) mm/s (Fig. S2A). Addition of H[BAr^F₄]. to 1a·O₂ gives [1a·O₂]H⁺ having the same isomer shift (δ = 0.53(2) mm/s) and a slightly larger quadrupole splitting parameter (ΔE_Q = 0.80(2) mm/s) (Fig. S2B). For comparison, the Mössbauer spectra of 2·O₂ and [2·O₂]H⁺ were also recorded. The (peroxo)diiron(III) complex of 2 has δ = 0.53(2) mm/s and ΔE_Q = 0.77(2) mm/s (Fig. S2C), whereas the protonated [2·O₂]H⁺ form exhibits parameters of δ = 0.54(2) mm/s and ΔE_Q = 0.84(2) mm/s (Fig. S2D). The similar isomer shifts obtained for 1a·O₂, [1a·O₂]H⁺, 2·O₂, and [2·O₂]H⁺ are indicative of iron(III) centers and the small increase in ΔE_Q values for the protonated forms implies that only minor changes occur in the coordination environment.

To investigate more directly the nature of the peroxo moiety, Fe–O and O–O vibrations were measured by RR spectroscopy for species generated with both ¹⁶O₂ and ¹⁸O₂. As previously reported,¹³ 1a·O₂ exhibits Fe–O and O–O stretching vibrations with Fermi splitting (hereafter “/”) centered at 470 and 897 cm⁻¹, respectively (Fig. S3). Also observed are weaker bands at 513/532 cm⁻¹ that downshift to 500 cm⁻¹ with ¹⁸O₂, which we therefore assign to the asymmetric Fe–O stretch of the Fe₂O₂ core. Addition of H⁺ to 1a·O₂ only marginally affects its RR spectrum, with small upshift in Fe–O and downshift in O–O vibrations (Fig. S3, Table 1). These shifts in RR frequencies upon H⁺ addition may reflect subtle changes in Fe–O–O–Fe angles,¹⁶ but are too small to support the conclusion that a (µ-1,2-peroxo)diiron(III) unit has been converted to a (hydroperoxo)diiron(III) species. The RR spectrum of 2·O₂ is practically identical to that of 1a·O₂, with symmetric and asymmetric Fe–O modes at 466/475 and 513/532 cm⁻¹, respectively, and Fermi-coupled O–O stretches centered at 897 cm⁻¹ (Fig. 2). Addition of up to 2.0 equiv of H[BAr^F₄] to generate [2·O₂]H⁺ primarily affects the symmetric Fe–O stretch, which upshifts only a few wavenumbers compared to the spectrum of 2·O₂ (Table 1). From the RR data and Mössbauer parameters for 1a·O₂, [1a·O₂]H⁺, 2·O₂, and [2·O₂]H⁺, we conclude that protonation does not lead to formation of a (hydroperoxo)diiron(III) species.

Table 1.

UV-Vis, Mössbauer, RR, and FTIR data for 1a·O₂, [1a·O₂]H⁺, 1b·O₂, [1b·O₂]H⁺, 2·O₂, and [2·O₂]H⁺.

Complex	λmax, nm (ε, M−1 cm−1)	δ, mm/s	ΔEQ, mm/s	ν (Fe–O), cm−1 (Δ18O)	ν (O–O), cm−1 (Δ18O)	νas(COO−) cm−1 (Δ13C)	νs(COO−) cm−1 (Δ13C)	Δνas-s cm−1
1a·O2	590 (3100)	0.53(2)	0.71(2)	466/474 (−18)	897 (−50)	1607/1572 (>−22)	1358 (−29)	>200
[1a·O2]H+	600 (2360)	0.53(2)	0.80(2)	467/478 (−20)	896 (−53)	1553 (−32)	-	-
2·O2	600 (3300)	0.53(2)	0.77(2)	466/475 (−18)	897 (−50)	-	-	-
[2·O2]H+	610 (2700)	0.54(2)	0.84(2)	478 (−19)	897 (−50)	-	-	-

Figure 2.

RR spectra of 2·¹⁶O₂ (black) and 2^·18O₂ (red) after addition of 0, 0.5, 1.0, 1.5, and 2.0 equiv of H[BAr^F₄]. Each spectrum was normalized based on the solvent CH₃CN bands at 392, 400, and 920 cm⁻¹.

Since the benzimidazole and propoxy groups of N-EtHPTB are kinetically inaccessible due to the multidentate nature of the ligand, we assign the carboxylate unit as the site of protonation. To test this hypothesis, we examined the carboxylate stretches of 1a and 1b, and their peroxo complexes by FTIR spectroscopy. The assignment of frequencies in terms of coordination geometry are complicated by mixing of the COO⁻ symmetric stretch with the O–C–O bend and C–C stretch.¹⁷ Nevertheless, if the asymmetric and symmetric COO⁻ stretches can be identified, the binding geometry of the carboxylate ligand can be derived from the difference in the two, Δν_as-s.¹⁸−²⁰ Specifically, Δν_as-s should be close to that of the free ionic form, 150 cm⁻¹ for PhCOO⁻, for carboxylates bridging two metal ions, larger in unidentate coordination geometry, and smaller in bidentate mononuclear complexes. As expected, 1a and 1b exhibit Δν_as-s values of 149 and 166 cm⁻¹, respectively, consistent with µ-1,3 bridging carboxylate groups (Fig. S4B). In 1a·O₂, ν_as and ν_s are at 1572/1607 and 1358 cm⁻¹, respectively, and in 1b·O₂ are at 1550 and 1329 cm⁻¹ (Fig. 3A). These frequencies give Δν_as-s > 200 cm⁻¹ and suggests a switch from bridging to unidentate coordination for the carboxylate groups in 1a·O₂ and 1b·O₂. A bridging carboxylate geometry was demonstrated by X-ray crystallography in a similar (µ-peroxo)diiron(III) species,²¹ but facile conversion in carboxylate geometry might occur in solution. Generation of [1a·O₂]H⁺ and [1b·O₂]H⁺ is associated with downshift of the ν_as modes by at least 20 cm⁻¹, whereas ν_s modes are not observed, possibly shifting below 1300 cm⁻¹ (Fig. 3B). Most importantly, the FTIR spectra show no evidence of free carboxylic acid vibrations, i.e. ν_C=O > 1700 cm⁻¹.

Figure 3.

Solution FTIR spectra of 1a·O₂ (black) and 1b·O₂ (red) before (A, top) and after (B, bottom) the addition of 1.5 equiv of H[BAr^F₄]. The spectra were acquired in CH₂Cl₂ at approx. −30 °C with a diiron concentration of ~55 mM. The intense peaks at 1356 and 1420 cm⁻¹ are due to the [BAr^F₄]⁻ anion and solvent, respectively.

A comparison of the ¹H NMR spectra of the benzoate and pentafluorobenzoate diiron complexes allows the phenyl ring protons of the former to be identified in 1a·O₂ as paramagnetically broadened peaks at 7.0, 8.7, and 11.4 ppm (Fig. S5). Upon addition of H[BAr^F₄], these resonances shift to 7.5, 8.0, and 9.8 ppm (Fig. S9), in support of the protonation of the benzoate ligand. This conclusion is confirmed by analysis of the ¹⁹F NMR spectra of 2·O₂ and [2·O₂]H⁺. The fluorine resonances of the pentafluorobenzoate ring in 2·O₂ occur at −134.4, −154.4, and −159.0 ppm (Fig. 4A) and shift to −111.3, −143.0, and −154.8 ppm upon addition of 3 equiv of H[BAr^F₄] (Fig. 4B). These results demonstrate that the C₆F₅CO₂H ligand is bound to iron, since the resonances of free pentafluorobenzoic acid appear at −139.5, −150.3, and −162.0 ppm.

Figure 4.

¹⁹F NMR spectra (470 MHz, CD₂Cl₂, −30°C) of [2·O₂](OSO₂CF₃)₂ (A); and [2·O₂](OSO₂CF₃)₂/ H[BAr^F₄] (1:3) (B).

In conclusion, the spectroscopic evidence (Table 1) clearly indicate that the carboxylate is preferred over the peroxo ligand as the site of protonation in these (peroxo)diiron(III) model complexes, a possible structure for which is depicted in Scheme 1. Our results suggest that, during the O₂ activation steps in the catalytic cycle of sMMO and related enzymes, protons might generate and/or transform the (peroxo)diiron(III) core by inducing a carboxylate shift,^22,23 possibly increasing the electrophilicity of the diiron unit and facilitating substrate access to the active site. Future work with synthetic analogs will address these important questions.