Hydroxo-bridged active site of a flavodiiron NO reductase revealed by spectroscopy and computations
NO and O2 are detoxified in many organisms using flavodiiron proteins (FDPs). The exact coordination of the iron centre in the active site of these enzymes remains unclear in spite of numerous structural studies. Here, we used 57 Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in Escherichia coli FDP. This data combined with density functional theory (DFT) and 57 Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)-(µOH -)2-Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)-(µOH -)-Fe(II) site upon elimination of one bridging OHligand, thereby providing an open coordination site for NO binding. Prolonged NRVS data collection of the oxidised FDP sample resulted in photoreduction and formation of a partially reduced diiron centre with two bridging hydroxo ligands. These results have crucial implications for studying and understanding the mechanism of FDP as well as other non-haem diiron enzymes.
Introduction<!>Conclusions<!>Conflicts of interest<!>Sample Preparation<!>Mössbauer Spectroscopy<!>Nuclear Resonance Vibrational Spectroscopy<!>Resonance Raman Spectroscopy<!>Density Functional Theory Calculations<!>Supplementary Results and Discussion<!>Mössbauer Spectroscopy of a Photoreduced State<!>Fig. S3
<p>Nitric oxide (NO) inhibits multiple cellular processes including aerobic respiration and energy metabolism. 1 Therefore, NO is produced in response to pathogens by the human innate immune system. In order to survive this threat, microbes utilise several enzymes to detoxify NO. Among these are the flavodiiron proteins (FDPs), which reduce it to the innocuous N2O. 2,3 FDPs have a minimal core constituted by a metallo-β-lactamase-like domain, containing the catalytic diiron centre, and a flavodoxin-like domain with a flavin mononucleotide (FMN). The FDP from Escherichia coli (EcFDP) has an extra rubredoxin-like domain at its C-terminus, which is important for electron transfer as the electron entry point. 3,4 Although some FDPs are able to reduce O2 to H2O or NO to N2O, most of them cannot accomplish these reactions at the same rate, showing preference to one of the substrates. 5 The EcFDP is the only known FDP with a clear preference for NO over O2, exhibiting a rate of ca. 10 times higher for the NO reduction. Electrons for this reaction are transferred from NADH via an NADH:flavorubredoxin oxidoreductase. 5,7 Several three-dimensional structures of FDPs have been determined by X-ray crystallography, which disclosed that the diiron active site is coordinated by four histidines, two aspartates, one glutamate and one µ-hydroxo bridge (Figs. 1 and S8). 6,8,9 With two histidines at each Fe and one bridging bidentate carboxylate of Asp166, the FDP metal-ligand core bears an approximate reflection symmetry. Similar non-haem diiron centres are found in a variety of other proteins, such as soluble methane monooxygenase, Δ9-desaturase, ribonucleotide reductase, (bacterio)ferritin, toluene monooxygenase, haemerythrin, and rubrerythrin. 10 Fig. 1 Representation of the active site of the E. coli flavodiiron protein obtained by X-ray crystallography. A: structure in the as-isolated (oxidised) form (PDB 4D02). B: structure of the dithionite-reduced crystal (PDB 5LLD). Orange spheres represent iron. The structure represented in A shows a µ-hydroxo (µOH -) ligand bridging the two iron ions, while the structure represented in B shows a dioxygen molecule in the vicinity of the diiron centre. 6 The phosphate ion present in the as-isolated protein structure was omitted for clarity. Fig. 2 Zero-field Mössbauer spectra (dots) and fittings (lines) of as-isolated FDP-D OX (red) and reduced FDP-DRED (blue) samples, recorded at 13 K. Mössbauer parameters are presented in Table 1.</p><p>The Mössbauer parameters were also compared to other non-haem diiron enzymes (Table S1). The most similar to FDP-DOX were those of soluble methane monooxygenase (sMMO), which contains two hexacoordinated iron ions connected by two bridging hydroxo ligands in its oxidised state. 17,12 Reduction with dithionite leads to a significant change in the spectrum (Figs. 2 and S3).</p><p>The zero-field Mössbauer spectrum of FDP-DRED showed quadrupole doublets with parameters indicative of high-spin (S = 2) Fe 2+ (Table 1) as observed for reduced TmFDP. 15 Besides the contribution of the two main species consistent with pentacoordinated iron centres, the spectrum also presents a third minor feature (13 %) that shows a higher EQ value, possibly due to a hexacoordinated centre indicating a modified amino acid coordination or free iron in solution. 18 The spectra at 120 K and at 13 K in the presence of an external magnetic field of 37 mT, applied parallel to the γ-ray direction, were nearly identical to the zero-field Mössbauer spectrum of FDP-DRED at 13 K (Fig. S2). A comparison of the Mössbauer parameters of iron species #1 and #2 with other non-haem diiron enzymes (Table S1) suggests a pentacoordinated structure with one µ-hydroxo ligand such as in reduced ribonucleotide reductase or sMMO. 12,13,17,19 Table 1:  In order to unequivocally determine the structure of the FDP active site, vibrational spectroscopy was employed. Initial resonance Raman (RR) spectroscopic experiments on FDP-D lacked resonance enhanced metal ligand vibrations 20 and gave only signals arising from intrinsic FMN (Fig. S6). In contrast to the traditional infrared (IR) and RR techniques prone to their selection rules, a distinct advantage of NRVS is that it detects all vibrational modes with 57 Fe nuclei motion, thus having high specificity and sensitivity to the iron centres. 21,22 Our first NRVS data of FDP-DOX showed changes to the spectra during the 20 h of data collection (Fig. S7). Mössbauer spectra recorded after the 20 h NRVS measurements (Fig. S5) revealed two new additional iron species with a total of 12% contribution (Table S2). The fitted isomer shifts (δ = 1.28 and 1.24 mm/s) and quadrupole splittings (ΔEQ = 2.03 and 2.70 mm/s) correspond to high-spin Fe 2+ , which indicates partial photoreduction of the active site by the 14.4 keV X-rays. Here, the two Fe 2+ Mössbauer species are tentatively associated with either iron site photoreduced.</p><p>Three other oxidised FDP-D samples from the same preparation were investigated by NRVS for 4, 6, and 10 h data collection times, respectively, and their Mössbauer spectra were subsequently recorded (Fig. S4). No changes were identified after 4 h, while after 6 h the Mössbauer spectrum changed slightly and after 10 h a total intensity of approximately 8% of two high-spin Fe 2+ species was detected (Table S2). Thus, only the first NVRS spectra measured up to 4 h were considered for calculating the 57 Fe partial vibrational density of states (PVDOS) of FDP-DOX (Fig. 3).</p><p>The NRVS of FDP-DOX displays three major bands in the 200-300 cm -1 vibrational energy region, broader features in the 300-400 cm -1 region, and smaller intensity peaks at 471 cm -1 and 527 cm  to the bands between 200 and 300 cm -1 . 23 The corresponding Fe-O stretches of mono-oxo, peroxo and di-oxo/hydroxo bridging ligands were identified between 350 and 600 cm -1 .</p><p>Remarkably, our NRVS data of FDP-DOX correspond better with the di-oxo/hydroxo than with the mono-oxo compounds. 23 The reduction of FDP-D resulted in a red shift of the bands below 350 cm -1 (Fig. 3). The Mössbauer spectra for the pre-NRVS and post-NRVS FDP-DRED samples were nearly identical (not shown), which indicates that no radiation damage occurred. The red shift is consistent with the Fe(III)Fe(III) to Fe(II)Fe(II) reduction, resulting in longer metal-ligand bond lengths. 21 The reduced FDP-D shows more intense features mostly in the lower-energy region at 163 cm -1 and between 185 cm -1 and 275 cm -1 . At higher energies, a feature with two shoulders appears at 420 cm -1 and a distinct peak at 579 cm -1 . The two high-energy features are analogous to Fe(III)-μO-Fe(III) compounds showing symmetric Fe-O stretches in the ~530-550 cm -1 region, and anti-symmetric Fe-O stretches in the ~710-750 cm -1 region. [23][24][25][26] A recent NRVS study of a series of trigonal bipyramidal Fe(III)-hydroxo and -oxo complexes with varying H-bonding networks showed that even subtle changes in the H-network resulted in significant shifts of the Fe-O(H) vibrational frequencies. 27 Protonation of an oxo ligand caused a large ~180 cm -1 red shift in the energy of the stretching Fe-O band to ~480 cm -1 and elongation of the Fe-O(H) bond. The lower energy bands of FDP-DRED can therefore be explained by the reduction of the diiron centre resulting in longer bonds, a bridging hydroxo instead of an oxo species, or a change in the hydrogen bond network.</p><p>A broad set of in total 28 alternative DFT models of the FDP active site has been computationally explored, invariantly containing seven side chains binding the two Fe ions as described in the ESI methods section, targeting the reproduction of the NRVS-observed FDP-DOX/RED spectra. The structural alternatives involved modifications of the bridging oxygenous ligands, their number, and protonation level. These included primarily hydroxo OHspecies as well as water H2O, peroxo O2 2-, superoxo O2 •-, and oxo O 2-Fe ligands, or a vacant bridging site.</p><p>Following X-ray structural data (Fig. S8), 6,8 O2 and H2O molecules weakly bound in the FDP active site pocket have been further considered, as well as a bidentate phosphate anion PO4 3-coordination observed in the crystal structure of the oxidised state of EcFDP (Fig. S8). The calculations generally relied on broken-symmetry 28 (BS) solutions implying antiferromagnetic coupling between the two high-spin homovalent iron sites and approximating the diamagnetic (total S = 0) state. Furthermore, alternative electronic states as well as mixed-valence (total S are coplanar with the reduced Fe(II)-(µOH -)-Fe(II) core (Fig. 4). While the FDP-DOX state shows two hexacoordinate Fe(III) sites, reductive elimination of one µOHligand produces the FDP-DRED state with two pentacoordinate Fe(II) sites and the remaining µOHposition in qualitative agreement with the X-ray structural reference (Fig. S8). 6,8 This redox-dependent active site transformation largely follows a recent rationalisation by Weitz et al. for an O2-selective FDP, 15 and agrees with our Mössbauer results outlined above. An important redox-invariant determinant, presently deduced for both the FDP-DRED/OX structures, is the hydrogen bonding between the bridging hydroxo ligand and the Asp83 carboxylate oxygen (Figs. 4 and 5). The characteristic µO(H) region, all contributing to the 57 Fe-PVDOS. Another difference here is the prediction of mixed Fe-µO-H bending modes in the ~600-800 cm -1 region exclusively in FDP-DOX (vs. >800 cm -1 in FDP-DRED) with their small 57 Fe-PVDOS intensities at the level of the experimental error. The effects listed above can be traced in the 57 Fe-µOH -/O(Asp,Glu)/N(His) KED profiles shown in Fig. S19 as well as in the DFT-based normal mode animations provided as part of the ESI.</p><p>In the high-intensity region below 300 cm -1 , two prominent and matching bands in the FDP-DRED NRVS/DFT spectra are correspondingly at 245/220 (global maximum) and 164/161 cm -1 (Fig. S18a). In contrast, NRVS/DFT 57 Fe-PVDOS of FDP-DOX shows a blue-shifted and diminished-intensity global maximum at correspondingly 271/268 cm -1 , with a 2 nd -highest intensity band at 218/237 cm -1 (Fig. S18b). Regardless of the oxidation states, the aforementioned bands represent Fe-His/Asp/Glu stretches and bends, where the entire imidazole and carboxylate groups participate in vibration.</p><p>In the region above 300 cm -1 , the matching NRVS/DFT 57 Fe-PVDOS features of FDP-DRED are correspondingly at (i) ~400-440/420-450 cm -1 (with maxima at 419/434 cm -1 ) and (ii) at 581/574 cm -1 . Employing the FDP active site approximately mirror plane symmetry, DFT rationalises these bands as correspondingly (i) in-plane and (ii) out-of-plane iron-hydroxo Fe-µOHvibrations (Fig. 4, left). The two 57 Fe-PVDOS features are produced by several normal modes each, due to the vibrational coupling between µOHand carboxylates from the terminal Asp83 and bridging Asp166 Fe ligands. These couplings have correspondingly (i) Fe-O(Asp)</p><p>stretching and (ii) Fe-O-C(Asp) bending character. In comparison, FDP-DOX shows a tentatively matching NRVS/DFT band at 471/453 cm -1 , with the underlying DFT normal mode of mostly in- plane character (i above), where the two µOHligands displace in-phase (Fig. 4, right panel).</p><p>From the DFT calculations, the out-of-plane (µOH -)2 modes (ii above) of FDP-DOX appear at lower energies, 421 cm -1 (symmetric) and 354 cm -1 (asymmetric), where they become mixed with the Fe-O(Asp,Glu)/N(His) vibrations having higher 57 Fe-PVDOS intensities compared to the isolated Fe-µOHmotions. The vibrational energy order of modes type (i) and (ii) is therefore opposite in FDP-DOX and FDP-DRED, but in both states the type (i) in-plane µOHnormal modes produce higher individual 57 Fe-PVDOS intensities (Fig. S18).</p><p>For the photoreduced species upon prolonged NRVS data collection (see above), alternative DFT models of the FDP active site were examined. Indeed, the presence of a 24% contribution of a mixed-valence Fe(III)-(µOH)2-Fe(II) state (FDP-DMV) is in a good agreement with the changes of the experimental NRVS data based on the 17-20 h collection interval (Figs.</p><p>S7 and S20b), as well as the post-NRVS Mössbauer data (Fig. S5). Although red-shifted global spectral maxima at vibrational energies below 220 cm -1 might be explained by a contribution of a diferrous Fe(II)-(µOH -) 2 -Fe(II) species (Fig. S20a), the better performance of the (partially) reduced models with two rather than one bridging hydroxo ligands (Fig. S20) indicate an active site structure similar to that of the oxidised state (Fig. S11 bottom). This is logical, considering how unlikely it is for ligand dissociation to occur under cryogenic conditions. A mixed-valence species, that has also been observed by EPR spectroscopy upon reduction with 1 equivalent of menadiol, 7 might represent an intermediate during catalysis (Fig. 5).</p><!><p>In conclusion, the first combined NRVS, DFT and Mössbauer characterisation of a nonhaem diiron enzyme has addressed the active site composition and redox-dependent changes in the NO-converting EcFDP. We demonstrated the presence of two bridging hydroxo ligands in the oxidised state, and one bridging hydroxo ligand in the reduced state (Fig. 5). This finding unravels a discrepancy between the Fe-ligand configurations found in the crystal structures of EcFDP as compared with the now determined solution structures of this enzyme. We identified photoreduction of the as-isolated (oxidised) sample upon prolonged NRVS data collection. Our data suggest the formation of a partially reduced species (the mixed-valence state) with two bridging hydroxo ligands (Fig. 5). Future studies in terms of NRVS-monitored X-ray photoreduction may provide additional spectroscopic insights on non-haem diiron metalloenzymes. Furthermore, our approach provided access to the redox-dependent vibrational signature of the FDP active site. Vibrational bands arising from the µOHmotions, predominantly either parallel or perpendicular to the Fe•••Fe vector, were identified.</p><p>Alternative diiron cores with ligands either different to hydroxo, or with the vacant bridging site, are clearly disfavoured as significant components in our EcFDP-D samples. 57 Fe-NRVS spectroscopy, applied here in conjunction with DFT modelling, proves itself as a useful method for addressing metalloenzyme´s iron coordination in its fine detail and demonstrated the pioneering resolution of protein Fe-OH vibrational bands. We conclude that a di-μ-hydroxo diferric species (in the as-isolated enzyme) is reduced to a mono-μ-hydroxo diferrous state, the functional form ready to react with the NO substrate. An extension of this approach to catalytic intermediates in flavodiiron enzymes will provide valuable information about their structure, thereby opening novel perspectives in gas-converting chemistry.</p><!><p>There are no conflicts to declare.</p><!><p>The enzyme construct, FDP-D (residues 1 to 400 of the norV gene product, lacking the rubredoxin domain), was expressed as previously described. 1 To obtain 57 Fe enriched samples, the growth media was supplemented with 0.1 mM 57 FeCl both at the beginning of the growth and at the time of induction. The cells were disrupted by 3 cycles in a French press at 16000 psi (Thermo) in the presence of DNAse (Applichem) and the crude extract was clarified by low-speed centrifugation at 25,000 g for 25 min and at 138,000 g for 1 h and 30 min and at 4 o C to remove cell debris and the membrane fraction, respectively. The supernatant was dialysed overnight at 4 o C against 20 mM Tris-HCl, pH 7.5 containing 18% glycerol (buffer A). The soluble extract was then loaded onto a Q-Sepharose Fast-Flow column (50 mL, GE Healthcare) previously equilibrated with buffer A. FDP-D was eluted with a linear gradient from buffer A to 20 mM Tris-HCl, pH 7.5 containing 18% glycerol and 500 mM NaCl. The eluted fractions were analysed by 15% SDS-PAGE and UV-visible spectroscopy. Fractions containing pure protein were pooled and concentrated.</p><p>FDP-D sample was quantified and the iron and flavin content was evaluated as previously described. 2 The determined Fe/flavin/protein ratio was 2 (0.2)/0.8 (0.2)/1.</p><p>Reduced FDP-D samples used for NRVS, resonance Raman, and Mössbauer spectroscopy measurements were obtained anaerobically, inside a glovebox, by addition of a buffered sodium dithionite solution. Both as prepared and reduced samples were 2 mM in protein concentration.</p><!><p>Zero-field Mössbauer spectra were recorded on a SEECO MS6 spectrometer that comprises the following instruments: a JANIS CCS-850 cryostat, including a CTI-CRYOGENICS closed cycle 10 K refrigerator, and a CTI-CRYOGENICS 8200 helium compressor. The cold head and sample mount are equipped with calibrated DT-670-Cu-1.4L silicon diode temperature probes and heaters. The temperature was controlled by a LAKESHORE 335 temperature controller. Spectra were recorded using a LND-45431 Kr gas proportional counter with beryllium window connected to the SEECO W204 γ-ray spectrometer that includes a high voltage supply, a 10 bit and 5 μs ADC and two single channel analysers. Motor control and recording of spectra was taken care of by the W304 resonant γ-ray spectrometer. For the reported spectra a RIVERTEC MCO7.114 source ( 57 Co in Rh matrix) with an activity of about 1 GBq was used. All spectra were recorded as frozen solutions at 13 K and data were accumulated for about 24 hours. Mössbauer data were processed and fitted using the WMOSS4 program (www.wmoss.org). Isomeric shifts are referenced to α-iron at room temperature.</p><!><p>Nuclear resonance vibrational spectroscopy (NRVS) measurements were performed at SPring-8 BL19LXU with a 0.8 meV energy resolution at 14.4125 keV as described previously. 3 A 2×2 elements avalanche photodiode detector array was used to detect delayed nuclear fluorescence and K fluorescence by 57 Fe atoms. All measurements were performed at a 10 K reading of the cryostat sensor. The real sample temperature, as obtained from the spectral analysis, was 30-80 K. To enhance the S/N ratio in the Fe-(OH)x-Fe range of the spectra, sectional measurements of these regions were performed. While for the usual scans the time for every point was 5 s, in the case of sectional scans the acquisition time for every data point in the region of interest, i.e.</p><p>from 300 to 400 cm -1 was 8 s, from 600 to 800 cm -1 was 20 s, and for the remaining regions was 2 s. The raw data were analysed with the PHOENIX software to obtain the iron partial vibrational density of states ( 57 Fe-PVDOS). 4, 5</p><!><p>A LabRam HR-800 Jobin Yvon confocal Raman spectrometer coupled to a liquid-N2-cooled charge-coupled device (CCD) was applied to accumulate resonance Raman spectra of pure FMN (2 mM and 50 mM potassium iodide at pH 8 in Tris-HCl buffer) and FDP samples (as described in the sample preparation section). The 458 nm or 514 nm emission lines of an Ar + ion laser beam at a power of 1-2 mW was focussed on a 2-4 µm spot on the surface of the sample drop to induce Raman scattering. The temperature was set to 80 K using a liquid-N2-cooled cryostage (Linkam Scientific instruments). Toluene was used as an external standard for frequency calibration of each spectrum. Experimental data were analysed and processed using the Bruker OPUS software version 6.5 or higher.</p><!><p>E. coli FDP oxidised active site topology employed in the present density functional theory (DFT) modelling was initially based on the crystal structure of Moorella (M.) thermoacetica FDP in its reduced and subsequently NOreacted state, which corresponds to an oxidised sate (PDB 1YCH) 6 . This structure shows a bridging mono-oxo coordination in the active site. This selection of the X-ray reference based on a protein different from E. coli FDP is justified by (i) the absence of a co-crystallised species resolved in the active site of M. thermoacetica FDP, and and Discussion, subsection (viii).</p><p>The DFT calculations were done mostly using GAUSSIAN 09 Revision D.01, 8 based on a high-quality initial guess from single point calculations using JAGUAR 9.4. 9 The qualified initial guess approach was specifically employed to ensure broken-symmetry (BS) 10</p><!><p>Alternative DFT Models While no major transformations were produced upon the reduction level shifts in both structures (Fig. S11), the predicted 57 Fe-PVDOS spectra (Fig. S20) generally display notable variations as described below.</p><p>Our primary cross-check addressed the two structures with their oxidation levels interchanged. At the Fe(II)Fe(II) level, a dihydroxo differous system Fe(II)-(µOH -)2-Fe(II) produced strong conflicts to the observed FDP-DRED spectrum (Fig. S20a) throughout the entire 0-600 cm -1 range, e.g. an absence of the ~580 cm -1 feature (attributed to the Fe(II)-µOHstretch in the monohydroxo system). At the Fe(III)Fe(III) level, a monohydroxo diferric system Fe(III)-(µOH -)-Fe(III) is in lesser conflict with the FDP-DOX NRVS data (Fig. S20c). With its (i) too sharp bands in the ~300-600 cm -1 and (ii) lack of intensity in the ~200-230 cm -1 regions, Fe The 57 Fe-PVDOS signature of the Fe(II)-( )-Fe(II)(O2)[S=1] model (Fig. S23) is apparently similar to that of the Fe(II)-( )-Fe(II) model (Fig. S22a). In summary, while the above-considered dioxygen species spectra are not reminiscent to any of the presently collected FDP-DRED/OX NRVS data, these results are relevant to the chemistry by FDPs, which often display both O2 and NO reductase activities.</p><p>(vi) Phosphate anion coordination. Potential occupancy of the FDP active site by a phosphate anion, resolved in the as-isolated (oxidised) E. coli FDP (PDB 4D02) 7 structure additionally to the mono hydroxo bridge (Fig. S8), was investigated in alternative DFT models shown in Fig. S15 with their computed 57 Fe-PVDOS spectra in (vii) Bridging hydroxyl interaction with a solvent water molecule. Following an earlier iron-oxido/hydroxido 57 Fe-NRVS and DFT study, 17 an importance of hydrogen bonding interactions to the iron-bound hydroxo ligand has been presently examined by inclusion of a solvent water molecule as found in the FDP active site pocket of, e.g., the reduced state of M. thermoacetica FDP (Fig. S8). The corresponding DFT models Fe(II)-(µOH -)- Fe(III)(H2O) models. The implicit solvent SCRF / IEF-PCP scheme otherwise employed here, in contrast to the explicit H2O inclusion, is therefore empirically concluded to serve as an optimal approach.</p><p>(viii) Rigid protein backbone scheme. In view of notable displacements of the Fe-coordinating residues obtained for some of the models described above, a rigid ("[R]") protein backbone framework scheme has been alternatively explored in models Fe(II)-(µOH -)-Fe(II)[R] and Fe(III)-(µOH -)2-Fe(III)[R] shown in Fig. S17, with their predicted 57 Fe-PVDOS spectra in Fig. S26. In contrast to the Cα-only fixation scheme permitting rotations of the side chains, such rotations are severely restricted when the entire -CαH3 methyl terminal fixations are imposed.</p><p>Alternative side chain to backbone fixation schemes have been previously examined in DFT modeling of 57 Fe-NRVS spectra of e.g. the methane monooxygenase binuclear iron site 18 and protein [4Fe-4S] clusters. 15 Interestingly, the rigid backbone scheme results in an over-extended (Asp166)O•••Fe = 2.8 Å distance in the reduced state structure, implying the bidentate Asp166 coordination lost to one of the Fe sites (Fig. S17). In contrast, all three X-ray structures collected in Fig. S8 and the reduced/oxidised representative models from the Cα-only fixation scheme show the (Asp166)O-Fe = 2.1-2.2 Å distance within its bonding range. The (Asp166)O-Fe coordination loss in the Fe(II)-(µOH -)-Fe(II)[R] model can be rationalised by positioning of the His148 residue in the M. thermoacetica FDP PDB 1YCH 6 X-ray reference, which coordinates to the same Fe site; in the rigid backbone DFT model, the His148 imidazole displacement is restricted and the favourable Fe coordination to both the His148 imidazole and Asp166 carboxylate cannot be achieved. The His148 position notably different to that of PDB 1YCH 6 is seen in both the E. coli FDP structures (Figs. S8) and the representative DFT models (Fig. S9).</p><p>While the reduced state Fe(II)-(µOH -)-Fe(II)[R] model produces its characteristic high-frequency band ~50 cm -1 blue-shifted (Fig. S26a) as compared to that from the representative model and the FDP-DRED NRVS data (570-580 cm -1 ), the oxidised state rigid backbone model Fe(III)-(µOH -)2-Fe(III)[R] does not lead to the (Asp166)O-Fe coordination loss and performs on 57 Fe-PVDOS well in line with the experimental results (Fig. S26b); above 300 cm -1 , the performance of Fe(III)-(µOH -)2-Fe(III)[R] is somewhat superior to the otherwise representative Fe(III)-(µOH -)2-Fe(III) model. In summary, the common 'soft backbone' Cα-only fixation scheme was found optimal for a multi-state modeling of the FDP active site.</p><!><p>Mössbauer spectra recorded after the NRVS measurements (Fig. S5) revealed two new additional iron species with a total of 12% contribution (Table S2) with parameters corresponding to high-spin Fe 2+ , which indicates partial photoreduction by the 14.4 keV X-rays. Assuming reduction of only one out of two Fe centres per active site, 12% Fe 2+ couples to 12% Fe 3+ in a mixed-valence state. This provides 24% contribution of the signal assigned to the photoreduced state. Following analysis in subsection (i) above and in the main text, photoreduction of the FDP-DOX sample to a fully reduced diferrous species is a less likely alternative.   1 and S2). Fig. S2 Mössbauer spectra of the FDP-DRED sample at 13 K (black circles), at 120 K (red circles), both in the absence of an applied magnetic field, and at 13 K with a magnetic field of 37 mT applied parallel to the γ-ray direction (green circles).</p><!><p>Mössbauer subspectra of FDP-DRED (experimental: empty circles, global fit: solid red line). Solid black lines on top of experimental spectra represent the component subspectra (Tables 1 and S2).   S2.</p>