Generation of a μ-1,2-hydroperoxo FeIIIFeIII and a μ-1,2-peroxo FeIVFeIII Complex

μ-1,2-Peroxo-diferric intermediates (P) of non-heme diiron enzymes are proposed to convert upon protonation either to high-valent active species or to activated P′ intermediates via hydroperoxo-diferric intermediates. Protonation of synthetic μ-1,2-peroxo model complexes occurred at the μ-oxo and not at the μ-1,2-peroxo bridge. Here we report a stable μ-1,2-peroxo complex {FeIII(μ-O)(μ-1,2-O2)FeIII} using a dinucleating ligand and study its reactivity. The reversible oxidation and protonation of the μ-1,2-peroxo-diferric complex provide μ-1,2-peroxo FeIVFeIII and μ-1,2-hydroperoxo-diferric species, respectively. Neither the oxidation nor the protonation induces a strong electrophilic reactivity. Hence, the observed intramolecular C-H hydroxylation of preorganized methyl groups of the parent μ-1,2-peroxo-diferric complex should occur via conversion to a more electrophilic high-valent species. The thorough characterization of these species provides structure-spectroscopy correlations allowing insights into the formation and reactivities of hydroperoxo intermediates in diiron enzymes and their conversion to activated P′ or high-valent intermediates.


Protonation of [(susan 6-Me ){Fe III (-O)(-1,2-O2)Fe III }] 2+
In a UV-Vis cuvette under N2 protecting atmosphere, 1 °C for 5 days resulting in brownish suspension, which was diluted with the fourfold volume of CH2Cl2. This suspension was filtered over a short silica column with CH2Cl2 as eluent.
Volatiles were removed at reduced pressure resulting in a colorless liquid (18 mg). 1 H NMR spectroscopy revealed acetaldehyde and DL-2-phenylpropionaldehyde as the major ingredients with a ratio of 1 : 8±2.

Electrophilic Reactivity of [(susan 6-Me ){Fe IV (-O)(-1,2-O2)Fe III }] 3+
A solution of thianthrenium perchlorate (1.0 eq.) in CH3CN/CH2Cl2 (1:1) was added to a solution of [(susan 6  structures were solved and refined with the programs SHELXT/L 10-12 using OLEX2. 13 Crystal data and details concerning data collections and structure refinements are given in where J is the exchange coupling constant and g is the average electronic g value. Magnetic moments were obtained from numerically generated derivatives of the eigenvalues of eq. 1, and summed up over 16 field orientations along a 16-point Lebedev grid to account for the powder distribution of the sample. 57 Fe Mössbauer spectra were recorded on an alternating constant-acceleration spectrometer. The minimal line-width was 0.24 mm s -1 full-width at half-height. The sample temperature was maintained constant in a bath cryostat (Wissel MBBC-HE0106). 57 Co/Rh was used as the radiation source. Isomer shifts were determined relative to iron at room temperature.
Resonance Raman spectroscopy was performed with a confocal Raman microscope The fiber transfers the signals into a spectrometer (Princeton Instruments, Acton 2300i) acting also as a pinhole to gain better focal sectioning in the sample. The wavelengths separation is performed in the spectrometer by selecting a 600 groves /mm reflecting grating (blaze wavelength 500 nm). A spectroscopy CCD-camera (Andor, DU401-BR-DD) is detecting the final spectrum. The initial calibration was acquired using a toluene sample at room temperature by selecting four prominent Raman peaks in the range of 520 -1003 cm -1 . Samples were prepared with a typical concentration of 20mM in MeCN and directly frozen onto an aluminum cold finger in thermal contact to liquid nitrogen reservoir. In order to avoid ice accretion dry nitrogen was flowed over the sample. All spectra were acquired over 10 seconds and 10 single spectra are averaged for noise reduction using the cosmic ray removal function of the camera acquisition software.
Further measurements were performed to obtain resonance Raman spectra of the 3+ and the protonated complex S15 [(susan 6-Me ){Fe III (-O)(-1,2-OOH)Fe III }] 3+ using a different setup. For these experiments 3-5 l of a precooled sample (-60 °C, ~20 mM) were pipetted onto a quartz plate cooled with liquid N2 under a dry N2 atmosphere and were inserted into a pre-cooled THMS600 Linkam cryostat (80K). Measurements were conducted using a LabRam HR-800 (Jobin Yvon) confocal Raman spectrometer equipped with a Symphony II CCD camera (Horiba) cooled with liquid N2. Kr laser (tuned to either 568 nm or 647 nm) or Ar laser (tuned to 514 nm) were used as excitation sources. The Raman shift was subsequently calibrated using an external standard (toluene) recorded before and after the measurement of the samples.

Computational Details
All calculations were performed using ORCA 4.2 [14][15][16] with the ZORA scalar relativistic method. 17 Relativistically recontracted versions of the Karlsruhe def2-TZVP basis sets for iron and the coordinating atoms were used together with the auxiliary basis set def2/J. 18 For the C and H atoms the according SVP basis set 19,20 together with the auxiliary basis set def2/J was used. 18 The solvation model CPCM with a dielectric constant  = 36.6 (CH3CN) 21 along with D3BJ for dispersions correction were used. 22,23 The geometry optimizations were performed using the RIJCOSX approximation (RI approximation for non-hybrid functionals). 24 Broken-symmetry solutions of the diiron complexes were found S16 by first converging to hypothetical ferromagnetically coupled high-spin solutions and then converging to a broken symmetry state using the ORCA command "brokensym".

Evaluation of functionals in geometry optimizations for [(susan 6-
Geometry optimizations were then conducted on the energetically lower solutions. In all cases, the broken symmetry solutions were energetically favored and persisted throughout the geometry optimization processes. The results of these calculations are summarized in Figure S2 and Table S2 including the functionals employed.  In order to evaluate the site of protonation, the molecular structure, and the properties of the protonated complex, a proton was added to the experimental molecular structure of 1) at the peroxo oxygen atom O1 leading to the hypothetical 2) at the peroxo oxygen atom O2 leading to the hypothetical

S22
The Mulliken spin populations (  In order to get more insight into these effects, we calculated  and EQ for all other diiron complexes of the ligand susan 6-Me yet obtained (two diferrous, one diferric, and one mixed-valence Fe II Fe III complex) based on their crystallographically determined molecular structures. All calculated isomer shifts underestimate the experimental values roughly by 0.1 mm s -1 , while EQ were calculated within the known limited accuracy. For prediction of EQ we tested in some cases a different protocol 32 but observed only small effects. Therefore, EQ was calculated at the B3LYP/CP(PPP) level of theory. 18 However, to gain more accuracy in the computation of  we decided to use a linear dependence between the calculated total electron densities at the iron nuclei  and the experimental isomer shifts, which is adapted to the diiron complexes of the ligand susan 6-Me . In order to complement the linear dependence to higher valent cases, we incorporated six additional mononuclear iron complexes (Table S4).   Table 1 of the main manuscript.

Supplementary
As the stated above, the Mulliken spin populations (Table S3)  However, these Mössbauer parameters were calculated using a well-established protocol based the B3LYP CP(PPP)/def2-TZVP level of theory. 29,30 Interestingly, these calculations provided Mulliken spin populations (Table S5) that change only minorly to those of the TPSSh/def2-TZVP calculations thus also indicating a localized mixed valence class II Fe IV Fe III description. This at first glance conflicting results -localized from Mulliken spin populations vs a description from isomer shifts in which both iron atoms are effected by oxidation -reflects the difference between spin densities and total electron densities at the nuclei. Moreover, these results indicate a relaxation of the electron density of the whole molecule to compensate for the creation of one electron hole.  Only at base temperature (1.7 K) the data could be reasonably well analyzed by using the usual spin-Hamiltonian formalism for the cluster spin ground state St = 1/2, since even at 5 K the spectra showed significant broadening due to the onset of intermediate paramagnetic relaxation (Fig. S18). The Zeeman g values g = (2.02, 2.14, 2.27) were taken from EPR, and two subspectra of equal intensities were adopted with either the nested model (1) of isomer shifts and quadrupole splitting (left panel), or the stacked model (2) (right panel). Isomer shifts and quadrupole splittings were taken from the zerofield spectra shown in Figure S14 and slightly adapted for the lower temperatures. The signs of ΔE Q and the asymmetry parameters η were optimized, as well as the hyperfine coupling tensors for both iron sites, A/gNβN, given in Tesla. For the sake of less ambiguity all tensor axes have been taken to coincide. The parameters used are provided in Supplementary Table 6.  In the slow relaxation limit at low temperature, each site of each configuration provides its own Mössbauer doublet. Due to symmetry, the spectra of the reduced site on A and on B are the same. Thus, the overall spectrum is the sum of two localized doublets.

Supplementary
In the fast relaxation limit at high temperature, the electron hopping is faster than the Mössbauer time scale of 10 -7 s, so that for each site an averaged Mössbauer spectrum is observed. Due to symmetry, the two sites A and B are identical so that the overall spectrum is one doublet with averaged Mössbauer parameters.
Such a temperature-dependent electron hopping was observed in a symmetrical class II system of Fe II low spin and Fe III low spin with two localized doublets at 5 K and one averaged doublet at 353 K. 38 In a class II In the slow relaxation limit at low temperature, each site of each configuration provides its own Mössbauer doublet. Due to asymmetry, all four spectra are different. Due to the low temperature, the configuration with the lower energy is more populated (here ).
Thus, the overall spectrum is the sum of four localized doublets with the two doublets of  with the higher ratio a and the doublets of the energetically higher  with the smaller ratio b.
In the fast relaxation limit at high temperature, the electron hopping is faster than the At 180 K, the system is in the fast hopping limit and two different averaged Mössbauer doublets are observed. At lower temperatures, the Mössbauer spectra broaden ( Figure   S10) indicating at least one temperature-dependent process that is fast at 180 K relative to the Mössbauer timescale of 10 -7 s. This can be ascribed to a slower electron hopping or to a combination with slower electronic relaxation.