The structural identity of the product-inhibited complexTo visualize the atomic identity of the product-inhibited complex and the corresponding active site protonation states, a cryocooled neutron structure of perdeuterated Trp161Phe MnSOD soaked with deuterium peroxide (D2O2) was solved at 2.30 Å resolution. We pursued the neutron structure to visualize Mn-ligand interactions without radiation-induced effects29. The crystallization at pH 7.8 allowed the identification of all the proton positions in the active site at the physiological pH of mitochondria. Enzymes that use proton transfers to facilitate catalysis, like MnSOD, are expected to undergo changes in protonation states depending on the presence of substrate or product56,57,58. We chose the Trp161Phe variant to study product inhibition for its propensity to accumulate and retain the product-inhibited complex26,37,50,51,59. First, we solved the all-atom structure of the entire enzyme excluding the active site. Then, with these phases, we carefully interrogated the active site nuclear density maps for visually distinct peroxide-derived species and protons on O or N atoms.The first |Fo | - |Fc| nuclear scattering length density we examined immediately showed that the two subunits of the crystallographic asymmetric unit contained different species bound to the manganese ion, opposite His26 (Fig. 2a). Differences between the two subunits are often observed for the MnSOD P6122 crystal form because their solvent accessibility is not the same27. The density of chain A has the characteristic oval shape previously seen in reduced wildtype MnSOD corresponding to the metal-bound water molecule WAT127. For chain B, the density is more elongated and in an orientation nearly parallel with the Mn-Asp159 bond. We interpreted this density as a dioxygen species that has displaced WAT1 to bind the Mn ion (denoted as LIG for ligand, Fig. 2a). LIG refined well with full occupancy. The corresponding X-ray structures (Supplementary Fig. 1) and a previous peroxide-soaked Escherichia coli wildtype MnSOD structure support this interpretation of the nuclear density59,60. The dioxygen molecules in these X-ray structures are different in orientation and partially occupied due to the effects of X-ray irradiation. They demonstrate the benefit of using neutrons for radiation-free structural analysis. At physiological temperatures, thermochromism spectra also suggest a displacement of WAT1 for binding of a dioxygen species in agreement with our cryocooled crystallographic data61. From the nuclear density, we conclude that a dioxygen species displaced the metal-bound solvent molecule to form a five-coordinate complex.Fig. 2: Neutron structures and protonation states at the active site of D2O2-soaked, reduced, and oxidized Trp161Phe MnSOD.a D2O2-soaked Trp161Phe MnSOD for chain B with a singly-protonated dioxygen ligand (denoted LIG). Chain A is displayed as an inset to highlight differences in the difference density shapes that led to the identification of LIG in chain B. b D2O2-soaked Trp161Phe MnSOD for chain B. c Trp161Phe Mn2+SOD for chain A. d Active site overlay of wildtype Mn2+SOD and Trp161Phe Mn2+SOD demonstrating differences in hydrogen bond strength of the Gln143 amide anion and movement of WAT1. Inset highlights the position of the residue 161 mutation. e Trp161Phe Mn3+SOD for chain A. f Active site overlay of wildtype Mn3+SOD and Trp161Phe Mn3+SOD. Inset highlights the position of the residue 161 mutation. Green, orange, and magenta omit |Fo | - |Fc| difference neutron scattering length density of protons displayed at 2.5σ, 3.0σ, and 3.5σ, respectively. Light blue 2|Fo | - |Fc| density is displayed at 1.0σ. Distances are in Å. Dashed lines indicate typical hydrogen bonds and hashed lines indicate SSHBs that are hydrogen bonds <1.8 Å. All neutron structures were solved to 2.3 Å resolution, including those of wildtype MnSOD from our previous work27. For the resting state structures, only one active site is shown due to high structural similarities; see Supplementary Fig. 2 for the other active site. All neutron structures were solved to 2.3 Å resolution.Next, the protonation states near the Mn ion and the dioxygen ligand that correspond to product inhibition were examined. The dioxygen species has a single proton (labeled as D2, Fig. 2b) on O2 and points toward the Nε1 of Trp123, but not at a hydrogen bonding distance. Given that the crystal was soaked with D2O2 before data collection, it can be presumed that the molecule was deprotonated first and then displaced WAT1 to bind the Mn ion. For Gln143, rather than being deprotonated to an amide anion that as seen in wildtype Mn2+SOD27 and Trp161Phe Mn2+SOD (Fig. 2c, d), a neutral amide is present. Dε22(Gln143) and O1(LIG) form a 1.7 Å SSHB. This SSHB may be a possible conduit for proton transfer to occur, especially since a similar proton transfer was directly observed in wildtype MnSOD (Fig. 1b, c). The product-inhibited complex consists of a singly-protonated dioxygen ligand forming a SSHB with neutral Gln143.The coordination number of the Mn ion for product inhibition has long been debated35,37,52,62,63,64. One proposed mechanism from QM calculations was that a dioxygen species binds to Mn3+ at the sixth ligand position opposite Asp15961,62,63. An elegant study from Sheng and colleagues on Saccharomyces cerevisiae MnSOD observed the formation of a six-coordinate Mn3+ ion complex from electron paramagnetic resonance (EPR) and supported the possibility that Mn3+ may become six-coordinated during inhibition47. A second proposal was that a dioxygen species replaces the position of the solvent ligand as seen in optical absorption spectra and in an H2O2-soaked wildtype E. coli MnSOD X-ray crystal structure60,61. However, such a five-coordinate inhibited complex in the E. coli structure has been interpreted to be less likely as the dioxygen species was partially occupied and introduced steric crowding that led to higher computational energies37,62,63. X-ray structures are further complicated by radiation effects that lead to bond distances that are not representative of catalytic conditions29. Our radiation-free neutron diffraction suggests that product inhibition in human MnSOD is a five-coordinate complex with a singly-protonated dioxygen ligand displacing the metal-bound solvent molecule. Although sterically crowded, this complex prohibits catalysis during its lifetime by blocking the fast back-and-forth proton transfer between WAT1 and Gln143 during PCET redox cycling (Fig. 1b, c)27. We explore the valency of the Mn ion and the identity of the dioxygen species in later sections.The Gln143-WAT1 SSHB of Mn2+SOD limits product inhibitionNext, we look for clues as to what structural features lead to product inhibition, why the Trp161Phe variant is highly product-inhibited, and why Trp161 is needed for catalysis. To begin to understand the effects of the point mutation, we obtained a cryocooled 2.30 Å resolution neutron structure of Trp161Phe Mn2+SOD where the metal ion was chemically reduced with dithionite (Fig. 2c). Dithionite reduces the Mn ion without entering the active site27,65. The Mn bond distances of five-coordinate Trp161Phe Mn2+SOD are similar to wildtype Mn2+SOD (Supplementary Table 1). Moreover, the protonation states resemble that of wildtype Mn2+SOD, the bound solvent molecule is D2O (not -OD) and Gln143 is deprotonated to an amide anion (Figs. 1c, 2d). Deprotonated amino acids are confirmed when attempts to model and refine a proton result in negative |Fo | - |Fc| difference neutron scattering length density and all the other protons of the amino acid can be placed. The Trp161Phe variant and wildtype contrast at the Gln143-WAT1 interaction. In wildtype, the Gln143 amide anion forms a 1.6 Å SSHB with WAT1 (Fig. 2d)27. For the variant, there is a weaker 2.2 Å hydrogen bond between Gln143 and WAT1. These observations suggest that for the reduced enzyme, the role of Trp161 is to hold Gln143 and WAT1 in a tight hydrogen bonding interaction.We next measured a room temperature Trp161Phe Mn3+SOD neutron structure at 2.30 Å resolution (Fig. 2e). Cryocooling and chemical treatment were not pursued due to a limited supply of perdeuterated crystals during beamtime. However, purified human MnSOD is ~90% oxidized66 and the resultant Trp161Phe Mn-ligand bond distances strongly resemble that of the chemically oxidized wildtype Mn3+ counterpart (Supplementary Table 2). Furthermore, the hydrogen bonding and protonation states between variant and wildtype are identical, where WAT1 is a -OD molecule and Gln143 is of the neutral amide form (Fig. 2f). Due to the high similarity in structure between Trp161Phe and wildtype in the oxidized state, we conclude that the catalytic consequence of the variant for the Mn3+ to Mn2+ redox transition is not related to the interactions between the molecules of WAT1, Gln143, and Trp123. Instead, catalytic differences may be related to subtle electronic differences or alterations elsewhere in the active site (vide infra).The Trp161Phe variant has historically been puzzling as it profoundly affects PCET catalysis without directly removing a hydrogen bond donor or acceptor26,48,51. The fast redox transition of Mn2+ to Mn3+ is nearly ablated (k2, Table 1) while the formation of the inhibited complex is enriched (k2 « k3, Table 1) with a slow disassociation (k4, Table 1)26,37,48. From the cryocooled neutron structure of Trp161Phe Mn2+SOD (Fig. 2c), it is apparent that the Gln143-WAT1 hydrogen bonding interaction is perturbed compared to wildtype Mn2+SOD (Fig. 2d) and may be the primary cause of the observed kinetic effects. The bulky Trp161 residue likely constrains Gln143 and WAT1 to form a SSHB for proton transfers. We previously established that a strong O(WAT1)-D2(WAT1)-Nε2(Gln143) interaction is needed during the Mn2+SOD oxidation state for a proton transfer to occur during the Mn2+ to Mn3+ PCET27. Mutation of glutamine 143 to asparagine similarly ablates catalysis for the Mn2+ to Mn3+ half-reaction, and a longer WAT1-Gln interaction distance among isoforms of MnSODs correlate with decreased catalytic rates35,67. In summary, the structural features that lead to product inhibition include (1) the displacement of WAT1 by a dioxygen species (Figs. 2a), (2) when WAT1 is easier to displace product inhibition is increased (Fig. 2d, Table 1), and (3) a major role for Trp161 is to promote a strong Gln143-WAT1 interaction for proton transfer.The electronic identity of the product-inhibited complexThe coordination of dioxygen species and the oxidation state of the Mn ion for product inhibition has long been under debate35,37,52,62,63,64. Therefore, we pursued Mn K-edge XAS of MnSOD to cross-validate our crystallographic data and inform our refined structure. K-edge XAS can be divided into two regions: (1) the extended X-ray absorption fine structure (EXAFS) region at higher energies that contains structural information and (2) the X-ray absorption near edge structure (XANES) region that is reflective of the metal oxidation and geometric state. EXAFS of the peroxide-soaked Trp161Phe MnSOD complex was sought to obtain Mn covalent bond distance information for comparison to the neutron structure and QM density functional theory (DFT) calculations (Table 2). XANES was similarly pursued to identify the oxidation state and cross-validate the Mn ion’s geometry corresponding to the inhibited complex. X-ray irradiation was carefully monitored so that two subsequent scans of the same spot did not have photoreduction differences, and different positions on the sample were used for each scan.Table 2 Comparison of peroxide-soaked Trp161Phe MnSOD bond lengths from EXAFS fits, neutron structures, and DFT calculationsaWe first address the EXAFS structure of peroxide-soaked Trp161Phe MnSOD. The Fourier transform of the raw EXAFS spectra yields the atomic radial distribution around the absorbing Mn ion and is referred to as R space (Fig. 3a). The EXAFS spectrum exhibits a peak centered at ~2.1 Å that is best fit by two sets of scatterers, three nitrogen atoms at 2.20 Å and two oxygen atoms at 2.04 Å (Supplementary Table 2). These distances agree with those of Mn ion bond lengths seen in the neutron structure and DFT geometry optimizations using a divalent Mn2+ ion (Table 2)27. The largest contributor to the second peak observed at ~2.9 Å are seven carbon atoms that correspond to the Cδ2 and Cε1 of the three Mn-bound histidines and the Cγ of the Mn-bound aspartate (Supplementary Table 2). For the second oxygen of the dioxygen ligand (O2, Table 2), the best distance fit for its scattering is at 2.52 Å, which is in slight contrast to the neutron structure and DFT distances of 2.62 Å and 2.70 Å, respectively. This difference may be due to the atom having more degrees of freedom than those directly bound to the Mn ion. Overall, the structural parameters of the Mn-bound atoms derived from the EXAFS spectrum agree with the crystallographic and QM counterparts.Fig. 3: X-ray Absorption Spectroscopy of Trp161Phe MnSOD.a Fourier transform of Mn K-edge EXAFS data [k2χ(k)] from hydrogen peroxide-soaked Trp161Phe MnSOD with the raw EXAFS spectrum seen in the inset. Due to the scattering phase shift, the distance found by the Fourier Transformation (R) is ~0.5 Å shorter than the actual distance, and a 0.5 Å correction (α) was implemented. The black line represents the experimental data, while the red line is simulated EXAFS spectra from the neutron structure fit to the experimental data. b Kα HERFD-XANES of Trp161Phe MnSOD treated with hydrogen peroxide, potassium dichromate, and sodium dithionite to isolate the product-inhibited, Mn3+SOD resting, and Mn2+SOD resting states, respectively. The inset corresponds to a zoom-in of the pre-edge. c Fit of HERFD-XANES spectra of Trp161Phe MnSOD treated with hydrogen peroxide. The black line represents the experimental data, while the red line is simulated XANES spectra from the neutron structure fit to the experimental data. d Overlay of the neutron and XANES fit structures that are colored magenta and cyan, respectively. Data from this figure are provided within the Source Data file.We used Kα high-energy resolution fluorescence detected absorption (HERFD) to measure the XANES region. In K-edge XANES, the oxidation state and coordination geometry can be inferred from the spectra’s rising edge and pre-edge regions (Fig. 3b). The energy of the rising edge increases as the oxidation state of the metal increases while staying in the same spin state. When comparing the rising edges in the XANES region between oxidized and reduced Trp161Phe MnSOD, a shift to lower energy is observed that indicates a one-electron difference of oxidation state and coincides with Mn3+SOD and Mn2+SOD resting forms, respectively (Fig. 3b)68. With hydrogen peroxide, the rising edge is most like the reduced counterpart, indicative of a Mn2+ ion. The feature around 6554 eV of peroxide-soaked Trp161Phe MnSOD is of lower intensity than the reduced form while a higher intensity is observed for peroxide-soaked Trp161Phe MnSOD at ~6570 eV. Additionally, a noticeable variation in shape resonance (6540–6590 eV) is seen between the two Mn2+ complexes. These differences are caused by the replacement of the solvent molecule in reduced Trp161Phe MnSOD with a dioxygen species in peroxide-soaked Trp161Phe MnSOD. The HERFD-XANES spectra for wildtype MnSOD demonstrate the same trends; however, the dioxygen-bound species from peroxide soaking is not as well isolated (Supplementary Fig. 3). This is because wildtype has less product inhibition than Trp161Phe (Table 1). For the pre-edge region, the intensity (i.e., area of the pre-edge feature) corresponds to metal 1s to 3d transitions. These transitions gain intensity through 4p character mixing into the 3d orbitals from symmetry distortions and a loss of inversion symmetry69,70,71. A six-coordinate Mn complex is expected to have a different pre-edge intensity compared to a five-coordinate complex. For Trp161Phe with peroxide treatment, the pre-edge intensity is comparable to both the oxidized and reduced and indicates the coordination number is the same between the three chemical species (Fig. 3b). Like the rising edge, the pre-edge also shifts in energy depending on the oxidation state of the metal. A higher energy pre-edge for oxidized Trp161Phe MnSOD coincides with a Mn3+ ion, while a lower energy pre-edge for reduced and peroxide-soaked Trp161Phe MnSOD represents a Mn2+ ion. From the HERFD-XANES data, we can conclude that the introduction of hydrogen peroxide leads to a Mn2+ ion that is five-coordinate, though it differs in structure compared to the reduced resting state.Next, we performed structural refinement using the shape resonance of the HERFD-XANES spectra between 6545 and 6590 eV using combined QM chemistry simulations and machine learning72,73. With this method, systematic deformations of an input neutron structure were applied, and the corresponding XANES spectra of each deformation were simulated. The simulated spectra of the deformed structures then served as a training set for assigning a structure-to-spectra relationship for structural refinement. For the peroxide-soaked Trp161Phe complex, the simulated spectra of the best-fit structure capture nearly all features and relative intensities of the experimental spectra, with the exception of the 6550 eV feature that is found by the fit but underestimated (Fig. 3c, d). These trends are also the case for oxidized and reduced counterparts for the variant and wildtype (Supplementary Fig. 4). The Mn bond distances of the XANES structural refinement are in agreement with the structures from EXAFS, neutron diffraction, and DFT (Table 2 and Supplementary Table 3). Overall, the structural information of the inhibited complex extracted from the shape resonance of the HERFD-XANES spectra confirms the structure derived by the other methods.Clear evidence for the oxidation state of the Mn during product inhibition has previously been challenging to obtain due to ambiguities in optical spectra, large zero-field splitting values for the Mn using conventional EPR, and efforts to capture the inhibited complex with the wildtype enzyme leading to a mixture of species35,37,50,51,52,67. Historically, the product-inhibited oxidation state has generally been accepted to be Mn3+ as the oxidative addition of dioxygen species to low-valent metal ions has been described26,35,37,50,51,52. However, the HERFD data resemble a divalent Mn ion in both the rising edge and pre-edge regions with the introduction of hydrogen peroxide to the Trp161Phe variant (Fig. 3b). Furthermore, the metal bond distances observed by neutron diffraction and EXAFs support a divalent Mn ion (Table 2). Previous wildtype and Trp161Phe studies that coupled kinetics and visible spectra demonstrate that the inhibited complex lacks the characteristic 480 nm absorbance of the five-coordinate trivalent Mn oxidation state, although they were unable to rule out a six-coordinate Mn3+ complex26,50,51. These past observations can be reconciled with our data that indicate a five-coordinate Mn2+ complex.It was surprising that H2O2 reduces the Mn ion. The reduction potential of wildtype Mn3+SOD to Mn2+SOD is 0.4 V, and the oxidation potential of H2O2 to O2●− is −0.85 V66. These values indicate that the reduction of Mn3+SOD by H2O2 is not thermodynamically favorable. However, previous reports have noted that mixing Mn3+SOD and stoichiometric excesses of H2O2 leads to a slow reaction yielding Mn2+SOD26,50,51,52. The process was best fit by second-order kinetics, where reduction of Mn3+SOD and H2O2 results in Mn2+SOD and O2•− (a backward reaction), and then a subsequent reaction of O2•− with a second Mn3+SOD to yield another Mn2+SOD site (a forward reaction)52. While H2O2 reducing the Mn ion is thermodynamically unfavorable, past investigations have observed that excesses of H2O2 reduce the Mn ion.Several studies have further observed that introducing high concentrations of H2O2 to Mn3+SOD leads to the formation of the inhibited complex that decays to Mn2+SOD26,50,51,52. For our neutron diffraction and XAS experiments, 0.28 M D2O2/H2O2 was used to ensure full occupancy and unambiguously isolate the product-inhibited Trp161Phe MnSOD complex in the crystal or in the 3 mM protein solutions used in XAS. Scanning stopped-flow spectrophotometry studies by Hearn et al. noted that mixing H2O2 and Mn3+SOD at a 10:1 ratio led to rapid formation of the inhibited complex, though observation of a O2●− intermediate is lost during the dead time of the instrument (1.4 ms)50. Interestingly, these studies correlate product inhibition achieved through mixing H2O2 to the measured kinetics in the forward direction (i.e., reactions with O2●−, Table 1). For example, variants with k2 « k3 (Table 1), like Trp161Phe, accumulate the inhibited complex with excessive molar ratios of H2O2. Indeed, mixing Mn3+SOD with either O2●− or excessive concentrations of H2O2 leads to the same characteristic UV spectra of product inhibition50. These past observations suggest that the product-inhibited achieved through mixing H2O2 with Mn3+SOD may be a combination of reverse and forward reactions.Thus far, we have not addressed the electronic identity of the singly-protonated dioxygen ligand. Is it a protonated superoxide (HO2•) or hydroperoxyl anion (HO2−) bound to Mn2+ ion? At this time, we cannot experimentally differentiate between HO2• and HO2−. Given that H2O2 soaking leads to the reduction of the Mn ion26,51,52, a [Mn2+-•OOH] complex could form. Our DFT calculations indicate that a high spin [Mn2+-•OOH] complex has a Mn-O1(LIG) distance that is 0.32 Å or longer compared to distances found from our experimental methods (Table 2). Broken-symmetry DFT calculations of a low-spin [Mn2+-•OOH] complex collapses to a [Mn3+–OOH] complex, which differs from our XAS data indicating a Mn2+-containing complex (Fig. 3b). Another possibility is that a H2O2 molecule is deprotonated and binds the Mn2+ ion. Our DFT calculations most closely resemble our experimental data when the identity of the dioxygen species is HO2− (Table 2). HO2− may be more chemically likely to form a stable product-inhibited complex due to its ionic rather than radical character. While we cannot definitively ascribe an electronic identity to the dioxygen species, our quantum chemistry calculations (Table 2) support the interpretation of an HO2− molecule.Determination of the molecular orbitals that define redox reactivityTo investigate the molecular orbitals that determine the enzyme’s activity, ground-state and excited-state DFT calculations were performed along with analysis of the HERFD-XANES pre-edge (Fig. 4). Three complexes were studied as they reflect the most physiologically relevant complexes that had experimental data: (1) wildtype Mn3+SOD, (2) wildtype Mn2+SOD, and (3) Trp161Phe Mn2+SOD bound to HO2−. Unambiguous structural data of wildtype MnSOD bound to HO2− is unavailable, so the Trp161Phe variant was used instead. For our calculations, we used high spin S = 2 and S = 5/2 for the Mn3+ and Mn2+ states, respectively, as indicated by experimental data74.Fig. 4: The molecules orbitals involved in enzyme activity.a Energy diagram of the unoccupied valence orbitals of Mn3+SOD. The energy axis is shifted to set the z2 α orbital at zero. The isosurface plots of the unoccupied orbitals are contoured at 0.05 au. Percentages of orbital character are derived from Löwdin population analysis where the C3v symmetry-related xz and yz orbitals were averaged for the eπ values, and the xy and x2-y2 orbitals were averaged for the eσ values. b The HOMO of Mn2+SOD. c The HOMO of Trp161Phe Mn2+SOD bound to HO2−. d–f TDDFT simulated spectra where the simulated spectrum is colored black, and the corresponding experimental HERFD-XANES spectrum is colored gray. The green, red, and blue vertical lines correspond to eπ, eσ, and z2 transitions, respectively. Simulated intensities were uniformly scaled to experimental intensities, and the energy axis was shifted. Due to a misestimation of the Mn3+SOD pre-edge spectra from the TDDFT simulation (see text), a pseudo-Voight peak fit was performed with identical color-coding of the transitions and is included as an inset for (d). Unclear peak identities are colored purple.For the ground-state DFT calculations on Mn3+SOD, the unoccupied metal 3d orbitals largely determine the redox reactivity of the enzyme. The MnSOD active site has a distorted local C3v symmetry where the z-axis is along the Mn-O bond of the Mn-WAT1 interaction (Fig. 4a). High spin (S = 2) Mn3+ has one unoccupied α-spin orbital and five unoccupied β-spin orbitals. The lowest unoccupied molecular orbital (LUMO) is composed predominately of the 3dz2 α orbital due to strong spin polarization from the partially occupied α-manifold and interacts strongly with −OH. The LUMO is composed of 57% d character while the −OH contribution to the orbital is 17%, indicating that the -OH molecule is a contributor towards the reactivity of the orbital. The eπ (xz/yz) orbitals of the β-manifold have strong d character of 81%, though are tilted along the axis suggesting mixing with other d orbitals. The similarly tilted eσ (xy/x2-y2) β orbitals have 74% d character and bond strongly with the amino acid ligands leading to 1.5% mixing of the Mn 4p orbitals with the eσ orbitals. Due to the 4p mixing, the eσ orbitals are expected to lead to significant electric dipole character even with these small percentages of mixing69,70,75. Lastly, the 3dz2 β orbital is strongly σ-interacting with ligands along the z-axis. The 2.1 eV splitting between the z2 α and β orbitals is explained by spin polarization differences between the α- and β-manifolds reflected by a partially occupied α-manifold and an entirely unoccupied β-manifold. From the ground-state DFT calculations of Mn3+SOD, the 3dz2 α orbital is expected to be the orbital participating in enzymatic redox reactions, and the −OH molecule contributes towards reactivity.Ground-state DFT calculations were performed to analyze the highest occupied molecular orbitals (HOMOs) of Mn2+SOD and Trp161Phe Mn2+SOD bound to HO2−. The HOMOs are the orbitals participating in catalytic activity for Mn2+-containing complexes. In both complexes, the 3dz2 α orbital is the HOMO and interacts with the H2O or HO2− ligand along the z-axis (Fig. 4b, c). For wildtype Mn2+SOD, the d character of 58% strongly resembles that of the unoccupied 3dz2 α orbital of Mn3+SOD with 57% d character (Fig. 4a). Given the protonation of the solvent ligand in Mn2+SOD (Fig. 4b), the 2p character from the solvent contributes less density to the 3dz2 α orbital compared to that of Mn3+SOD. In the case of Trp161Phe Mn2+SOD bound by HO2−, the HO2− contributes a large portion of density to the orbital, with Mn 3d and HO2− contributions of 43% and 38%, respectively (Fig. 4c). The density localized on the HO2− is reflective of the 2p π-antibonding orbital. Since the z2 α orbital performs redox reactions, oxo or dioxo species involved in catalysis would bind in an orientation favoring interaction along the z-axis as it leads to the greatest orbital overlap. Analysis of the HOMOs potentially explains why HO2− prefers to displace the metal-bound solvent rather than binding opposite the aspartate residue.Next, we calculated the Mn K-pre-edge XAS for the three complexes using time-dependent density functional theory (TDDFT), an excited-state simulation, to assign the metal 3d orbital contributions to the experimental HERFD-XANES spectra of the pre-edge peak. The simulated spectra and corresponding experimental spectra are plotted in Fig. 4d–f. The vertical stick heights correspond to the intensity of the 1s to 3d transitions with the sum of quadrupole and dipole contributions. Overall, the spectral shapes of the divalent complexes (Fig. 4e, f) are well reproduced by the simulation. For the trivalent complex, the interpretation of the TDDFT simulation is trickier because the splitting of the orbital transitions is underestimated, while the intensity is overestimated (Fig. 4d).The TDDFT simulation of wildtype Mn3+SOD (Fig. 4d) identifies the eσ transitions as the primary contributors of intensity, though it has a misestimation of the overall spectral intensity and shape. Significant dipole intensity is found in the eσ and z2 transitions. The eσ orbitals bear the most dipole character which is reflective of the higher 4p mixing seen in the ground-state DFT calculations (Fig. 4a). The valence 3d orbital splitting of the simulation is underestimated and the contribution of the transitions to the spectra is better represented by pseudo-Voigt peak fitting of the experimental data that supports an increased splitting energy (inset, Fig. 4d). Four peaks were required to fit the spectrum and attempts to fit five or more peaks did not lead to a better solution. The two larger peaks found in the fit potentially stem from the splitting of the eσ orbitals (xy/x2-y2) due to the distorted C3v symmetry where one of the orbitals may have more overlap with those of ligands. The higher energy peak found in the fit at ~6542.5 eV is best identified as the z2 β transition supported by both ground and excited-state DFT. A fourth peak is capable of being fit as a small shoulder at ~6539 eV, though the orbital contribution to the peak is unclear as it could be of eπ or z2 α character (or both). The eπ or z2 α orbitals are probably underneath the stronger eσ orbitals. For the TDDFT simulation, the overestimation of the transition intensities and underestimation of the valence 3d orbital splitting has been previously noted for other high spin S = 2 d4 complexes with similar symmetry75,76. In brief, the Mn 1s core hole exchange interaction with the valence 3d orbitals is overestimated by DFT, and its effects in the spectra are most prominent when there are both α and β transitions. This overestimation explains why the misestimations are less pronounced in the divalent S = 5/2 d5 complexes (Fig. 4e, f) as they contain only β transitions. Nevertheless, the TDDFT simulation suggests that the eσ transitions are the predominant contributors to pre-edge spectral intensity for wildtype Mn3+SOD.For Mn2+SOD, the assignment of the metal 3d orbital contributions to the experimental spectra is more straightforward. The TDDFT simulation of wildtype Mn2+SOD (Fig. 4e) suggests the eσ orbitals have the most dipole character from 4p mixing and thus predominantly contribute to the intensity. The eπ orbital transitions are mostly of quadrupole character, leading to a weak low-energy tail. Note that the eπ orbitals are allowed to mix with the 4p orbitals, though have small effects due to π-bonding71. The z2 transition consists of low dipole intensity leading to a high energy tail. The trend of transition energy (eπ, eσ, z2) mirrors that of Mn3+SOD (Fig. 4d) and suggests the order of the metal 3d orbital energies is maintained between the two resting redox states. Overall, the TDDFT simulation of wildtype Mn2+SOD matches the experimental data.For the TDDFT simulation of Trp161Phe Mn2+SOD bound to HO2− (Fig. 4f), the overall spectra shape agrees well with that of the experimental data. Here, all transitions bear significant dipole intensity, the eπ and eσ transitions overlap in energy, and the z2 transition contributes a tail to the spectra. The observations coincide with further distortion of C3v symmetry introduced from the binding of the HO2− molecule, causing the metal 3d orbitals to mix further. This mixing distributes the dipole character over the five unoccupied β orbitals, leading to an overlap of the eπ and eσ sets of orbitals. Importantly, the mixing permits the distinction of a dioxygen species bound complex over that of the solvent molecule-bound complexes seen in Mn3+ and Mn2+ resting states (Fig. 4d, e). Overall, the TDDFT simulation of Trp161Phe Mn2+SOD bound to HO2− agrees with the experimental data.The ground-state and excited-state DFT simulations help explain the catalytic mechanism of MnSOD. The S = 2 d4 configuration of Mn3+SOD and the distorted trigonal ligand field leads to spin polarization of the 3dz2 α orbital and its large splitting with the 3dz2 β orbital (Fig. 4a). This splitting is reflected in the experimental pre-edge spectra though is underestimated in the TDDFT simulations and is documented by studies of similar complexes (Fig. 4d)75,76. Importantly, the splitting leads to the 3dz2 α becoming the LUMO that is the putative electron acceptor orbital for reaction with O2●−. The 3dz2 α orbital mixes strongly with antibonding molecular orbitals of the metal-bound ligands. In particular, the −OH molecule bound along the z-axis contributes a relatively large portion of density towards the LUMO, suggesting that it is a determinant in redox activity. For Mn2+SOD that has an S = 5/2 d5 configuration, ground-state DFT calculations (Fig. 4b) indicate that the 3dz2 α orbital is the HOMO, the putative electron donor orbital for reaction with O2●−. The TDDFT simulation (Fig. 4e) matches the HERFD-XANES spectra to suggest that our calculations are recapturing the experimental data for the resting Mn2+ complex. For the Trp161Phe Mn2+SOD complex bound to HO2−, the HOMO is again the 3dz2 α orbital though the 2p π-antibonding character of HO2− contributes a large portion of density to the orbital (Fig. 4c). Jackson and colleagues have previously hypothesized that dioxygen species may preferentially displace a metal-bound solvent for Mn ion binding since the dioxygen π-antibonding orbitals would overlap with the Mn 3dz2 α orbital77. Additionally, the experimental and TDDFT simulations of the pre-edge (Fig. 4f) permit spectral discrimination of a dioxygen-bound Mn2+SOD complex against a resting state Mn2+SOD complex. Altogether, the DFT simulations indicate that (1) the z2 α orbital is the orbital performing redox reactions, (2) reactivity is influenced by the species bound along the z-axis of the Mn, and (3) displacement of the solvent molecule for dioxygen species binding is chemically preferred due to orbital overlaps.Unexpected second-sphere hydrogen peroxide binding siteWe previously demonstrated that residues Gln143, Tyr166, His30, and Tyr34 have unusual pKas and undergo changes in protonation states during the MnSOD PCET cycle (Fig. 1)27. The active site gains two protons during the Mn3+ → Mn2+ redox transition where one proton is acquired by Tyr34 and the other by His30. These protons are lost during the Mn2+ → Mn3+ transition, which led us, at the time, to hypothesize that Tyr34 and His30 bind and protonate O22− to H2O2. This was supported by the fact that the gateway between these two residues is the only solvent-accessible entrance/exit of the active site. When investigating the active site of the D2O2-soaked structure at chain B, a strong omit |Fo | - |Fc| difference density peak is seen between His30 and Tyr34 with an oblong shape that is longer than a water molecule (orange density, Fig. 5a). This is the same active site where the dioxygen ligand is bound to the Mn2+ ion (Fig. 2a). We interpret the omit nuclear density as D2O2 (denoted as PEO, Fig. 5a) given (1) the crystal was soaked with D2O2 before data collection, (2) modeling and refining a D2O (water) molecule leads to unoccupied residual density (Supplementary Fig. 5), and (3) the density has two protruding features indicating two protons. The locations of the D2O2 protons are further verified by their nuclear density peaks (green density of PEO, Fig. 5a), with the D1(PEO) proton forming a 1.4 Å SSHB with a deprotonated Tyr34. The D2(PEO) forms a longer 2.2 Å hydrogen bond with anionic Tyr34 and may explain the unusual 17° dihedral angle of PEO. Furthermore, the D2O2 molecule forms a second 1.4 Å SSHB between atoms Dδ1(His30) and O1(PEO). The proton between Oη(Tyr166) and Nε2(His30) that we previously observed participating in a LBHB for the resting Mn2+SOD structure (Fig. 1g) is instead fully localized onto Oη(Tyr166) and participates in a 1.6 Å SSHB with Nε2(His30). The experimental data suggest a strong network of hydrogen bonds is present at physiological pH involving Tyr34-PEO-His30-Tyr166 and support our hypothesis that Tyr34 and His30 bind and protonate O22− to H2O2.Fig. 5: Neutron structures and protonation states of second sphere active site residues in D2O2-soaked, reduced, and oxidized Trp161Phe MnSOD.a D2O2-soaked Trp161Phe MnSOD at the active site of chain B. b D2O2-soaked Trp161Phe MnSOD at the active site of chain A. Inset highlights the structure near the divalent Mn ion. Chain B is more accessible to solvent than chain A and helps explain differences in ligand binding. c Divalent resting state of Trp161Phe MnSOD at the active site of chain A. d Trivalent resting state of Trp161Phe MnSOD at the active site of chain A. Blue, green, orange, and magenta omit |Fo | - |Fc| difference neutron scattering length density of protons are displayed at 2.0 σ, 2.5σ, 3.0σ, and 3.5σ, respectively. Light blue 2|Fo | - |Fc| density is displayed at 1.0σ. Distances are in Å. Dashed lines indicate typical hydrogen bonds and hashed lines indicate SSHBs, hydrogen bonds <1.8 Å. For the resting state structures, only one active site is shown due to high structural similarities, see Supplementary Fig. 2 for the other active site. All neutron structures were solved to 2.3 Å resolution.The active site of chain A for the D2O2-soaked Trp161Phe MnSOD, which does not have binding of a dioxygen species in either the first or second spheres, was next investigated (Fig. 5b). The active site has a divalent Mn2+ ion with Mn bond distances and protonation states reflecting that of Trp161Phe and wildtype Mn2+SOD (Supplementary Table 1). Without treatment of redox reagents or D2O2/H2O2, human MnSOD is ~90% trivalent66 (Fig. 2e and Supplementary Fig. 3) and suggests peroxide reduced the metal ion, and the resulting species vacated the active site before flash-freezing. Here, it is also evident that the variant destabilized the Gln143-WAT1 interaction as they are not in a hydrogen bond (Fig. 5b, inset). The omit |Fo | - |Fc| difference density shows that Tyr34 is protonated (Fig. 5b) and contrasts with the deprotonated Tyr34 of chain B, supporting the idea that Tyr34 may gain a proton from D2O2. For His30 of chain A, omit |Fo | - |Fc| difference density is observed for protons on both nitrogen atoms at contours of 2.5σ or higher, indicating His30 is positively charged and Tyr166 is negatively charged. The Dε2(His30) proton forms a 1.4 Å SSHB with Oη(Tyr166) and also differs with chain B, where the proton is localized on His30 rather than Tyr166. Given that the proton between Nε2(His30) and Oη(Tyr34) is seen altering its position, a back-and-forth proton transfer may occur between the heteroatoms depending on the ligand environment, such as the binding of a dioxygen species. The observation also reinforces the model originated from the room temperature wildtype neutron structures where a proton is either transiently shared between His30 and Tyr166 or a back-and-forth proton shuffle occurs during the catalytic cycle27. Overall, the neutron structure of D2O2-soaked Trp161Phe MnSOD at the active site of chain A, in conjunction with the active site of chain B, provides insight into the role of Tyr34, His30, and Tyr166 during product binding.We next sought mechanistic clues from the neutron structures of resting state Trp161Phe Mn2+SOD and Trp161Phe Mn3+SOD. Both chains of each structure have high structural similarity (Supplementary Fig. 2). For Trp161Phe Mn2+SOD, the protonation states of Tyr34, His30, and Tyr166 resemble those of the wildtype Mn2+SOD though there are notable structural features. Tyr34 engages in a 1.6 Å SSHB with WAT2 where Oη(Tyr34) acts as a hydrogen bond acceptor (Fig. 5c). It is apparent that Tyr34 forms SSHBs with water molecules regardless of its protonation state and Mn oxidation state, supporting the notion that Tyr34 is central to proton turnover during catalysis27. Indeed, its mutation predominately affects the Mn2+ to Mn3+ redox transition of the enzyme35,37,48. Besides Tyr34, the other notable structural feature is the hydroxyl proton of Tyr166 that points away from His30 (Fig. 5c). It is unclear whether the orientation of the proton has catalytic significance, is somehow related to the Trp161Phe variant, or is a consequence of cryocooling. Regardless, the Trp161Phe Mn2+SOD and the D2O2-bound neutron structures (Fig. 5a) provide evidence that Tyr34 is involved in SSHBs with both D2O2 and water molecules and supports the interpretation that Tyr34 is a central player in proton transfer.Surprisingly, Tyr34 and His30 are both protonated in the neutron structure of Trp161Phe Mn3+SOD, which is the opposite of wildtype Mn3+SOD protonation states (Fig. 1d, f). Strong omit |Fo | - |Fc| difference density is observed for Dη(Tyr34), Dδ1(His30), and Dη(Tyr166) (Fig. 5d). A potential explanation for the different protonation states are alterations of residue pKas due to small changes in the active site. Past studies across different MnSOD isoforms have established that subtle changes in hydrogen bonding and residue orientation lead to significant changes in catalysis, mainly due to shifts of the net electrostatic vectors35,37,64,78,79. Other changes in the Trp161Phe variant include the previously discussed Gln143 and WAT1 molecules (Fig. 2f) and a slight 1.7 Å movement of Tyr34 toward His30 (Supplementary Fig. 6). Our neutron diffraction data of Trp161Phe Mn3+SOD suggest that subtle movement of active site residues affect the pKa of residues involved in proton transfer.Our Trp161Phe MnSOD neutron structures indicate that D2O2 forms strong hydrogen bonds with His30 and Tyr34 and that residues Tyr34, His30, and Tyr166 undergo protonation changes. While the D2O2 binding site between His30 and Tyr34 was predicted, the neutron structure is the first experimental evidence for this binding27,34,35,37,64,80. The binding site has been thought to be where an O22− or HO2− species gain protons during the Mn2+ to Mn3+ redox transition (k2). However, our neutron structure of D2O2-soaked Trp161Phe MnSOD brings the possibility that it may also play a role during product inhibition, with three possible interpretations of the data. First, D2O2 binding occludes the only entrance to the active site and may potentially block substrate from entering since the Tyr166-His30-PEO-Tyr34 network is composed of several SSHBs (Fig. 5a). Second, the life of the network is short-lived, and a PEO → Tyr34 proton transfer would create a HO2− with high electrostatic affinity towards the Mn ion. Third, a HO2− molecule already bound to Mn2+ may stabilize H2O2 species binding between His30 and Tyr34 and block the product from exiting. Altogether, the neutron structures provide considerable insight into MnSOD catalysis by revealing both binding orientations of dioxygen species and protonation states.Summary of active site configurations solvedThe neutron structures, XAS data, and simulations of D2O2-soaked reduced, and oxidized Trp161Phe MnSOD present four primary configurations of active sites. For chain B of the D2O2-soaked neutron structure, two dioxygen species are found within the active site (Fig. 6a). One of the dioxygen species is singly-protonated and bound directly to the metal ion, replacing the position of WAT1, and forming a SSHB with Gln143 that is in the neutral form (Fig. 2a). Our XAS measurements and DFT simulations support the interpretation that the Mn ion is divalent and the singly-protonated dioxygen species is HO2− (Figs. 3 and 4, Table 2). In the same active site, D2O2 is observed binding tightly between an ionic Tyr34 and neutral His30 (Fig. 5a). For chain A of the D2O2-soaked neutron structure, the active site is reduced, but a dioxygen ligand is not present, and Gln143 is deprotonated (Fig. 6b). Since high peroxide concentrations reduce the Mn ion (Fig. 3b)26,50,51, we presume that the active site metal ion underwent reduction and resulting dioxygen species left before cryocooling (Fig. 5b). For the Trp161Phe reduced resting state, both chains have the same structure, and WAT1 is forming a hydrogen bond with the amide anion form of Gln143 (Fig. 6c). For the Trp161Phe oxidized resting state, the structure is the also same between chains where WAT1 is −OH and Gln143 is in the canonical amide form (Fig. 6d). Overall, our data indicate that a H2O2 molecule undergoes deprotonation prior to binding Mn2+, and His30, Tyr166, Gln143, and Tyr34 are capable of changing protonation states.Fig. 6: Summary of active site configurations observed for D2O2-soaked, reduced, and oxidized Trp161Phe MnSOD.a Dioxygen-bound Trp161Phe MnSOD is seen in chain B. The singly-protonated dioxygen species bound to the metal ion (Fig. 2b) is probably hydroperoxyl anion as supported by our XAS measurements (Fig. 3) and DFT calculations (Fig. 4, Table 2). A D2O2 molecule is also coordinated between His30 and Tyr34 with SSHBs (Fig. 5a). b Active site of Trp161Phe MnSOD chain A was treated with D2O2, though cryocooling did not capture any dioxygen species (Fig. 5b). c Reduced resting state of Trp161Phe MnSOD (Figs. 2c, 5c). d Oxidized resting state of Trp161Phe MnSOD (Figs. 2e, 5d). All neutron structure active sites are shown in Supplementary Fig. 2. Dashed lines represent normal hydrogen bonds, and wide dashed lines are SSHBs. The portrayal of structures and bond lengths in 2D are not representative of those seen experimentally in 3D.Altogether, the present work reveals key features of product inhibition and the related active site proton environment. Through combined neutron diffraction, XAS, and computational chemistry calculations, we find that (1) the inhibited complex is a five-coordinate Mn2+ complex where a HO2− replaces WAT1 found in the resting states; (2) how easily WAT1 may be displaced correlates with the extent of product inhibition; (3) preference for dioxygen species binding in place of the WAT1 position is a result of the overlap with the Mn 3dz2 orbital; (4) H2O2 strongly binds to an anionic Tyr34 and His30; (5) an anionic Tyr166 and cationic His30 forms at least transiently with H2O2 treatment, and (6) slightly different orientations of active site residues can alter their pKas due to the strong electrostatic vectors provided by the Mn ion. With this knowledge of product binding, we propose a mechanism for how product inhibition and relief may occur.Mechanism for MnSOD product inhibition and reliefFrom our use of high H2O2/D2O2 concentrations, we postulate that the inhibited complex we observe is from first a backward reaction of H2O2 with Mn3+ to form a O2●− species and Mn2+, and then a forward reaction of O2●− with another Mn3+ site in the presence of H2O2 to form the inhibited complex. Here, H2O2 is seen as a means to generate O2●− and study the forward reactions. We have framed our proposed mechanism only in the forward direction to better represent what would occur in physiological conditions.We propose a mechanism of product inhibition dependent on a molecule of O2●− arriving and reducing Mn3+ before the departure of a recently formed H2O2. O2●− reduces Mn3+ to Mn2+ in conjunction with anionic Tyr34 accepting a proton from H2O2 to form neutral Tyr34 and HO2− (Fig. 7a). Note that reaction of Mn3+ with O2●− to form Mn2+ and O2 is represented by a gain of an electron due to the lack of experimental evidence for whether O2●− requires coordination to the Mn ion for electron transfer. After the PCET, HO2− immediately displaces WAT1 to bind the Mn2+ ion and forms a SSHB with Gln143 to make the inhibited complex (Fig. 7b). Decay of the inhibited complex involves a Gln143 to HO2− proton transfer that yields a Gln143 in the amide anion form, and the H2O2 product is subsequently replaced by a water molecule (Fig. 7c, d).Fig. 7: A suggested mechanism for MnSOD product inhibition and relief.a Product inhibition is dependent on the presence of H2O2 (denoted as PEO) coordinated between His30 and Tyr34 during the Mn3+ → Mn2+ redox transition. Due to the lack of experimental evidence for O2●− binding and uncertainty in whether it requires coordination with the Mn ion for redox catalysis, the redox reaction is instead represented by a gain of an electron. For the formation of the inhibited complex to proceed, the gain of an electron by Mn3+ coincides with the deprotonation of H2O2 by Tyr34. Note that His30 has been shown to change protonation states on both of its nitrogen atoms and could potentially extract a proton from H2O2 instead of Tyr34. b After the PCET, HO2− replaces the WAT1 solvent molecule to form the inhibited complex characterized by the elimination of a Gln143-WAT1 interaction while the Mn ion is in the divalent redox state. c The relief of the inhibited complex involves protonation of HO2− by Gln143 to form H2O2 and an ionized Gln143 and subsequent replacement of the original WAT1 position by a water molecule. d After H2O2 leaves the active site, the Mn2+SOD is formed that is characterized by an ionized Gln143 forming a SSHB with WAT1, and Tyr34, His30, and Tyr166 in the neutral states. Dashed lines represent normal hydrogen bonds, and wide dashed lines are SSHBs. The portrayal of the displayed structures and bond lengths in 2D are not representative of those seen experimentally in 3D.Altogether, the proposed mechanism of product inhibition is dependent on the presence of H2O2 within the active site during the Mn3+ to Mn2+ redox transition, deprotonation of H2O2 by Tyr34, and subsequent displacement of WAT1 by HO2−. Inhibition is characterized by the elimination of the back-and-forth WAT1 and Gln143 proton transfer that is central for PCET catalysis27,67, where dioxygen species replacing the WAT1 position is a result of favored molecular orbital overlap with the Mn 3dz2 α orbital. Relief of the complex involves the protonation of HO2− to form H2O2, which is replaced by a water molecule. The combination of neutron diffraction and XAS has revealed, to our knowledge, the first direct experimental evidence for the identity of the inhibited complex and how it is formed and relieved. Most published mechanistic models of MnSOD product inhibition have presumed that inhibition proceeds through a Mn2+SOD and O2●− reaction (k3, Table 1)18,35,37, without the involvement of the Gln143 proton transfer, and independent of the Mn3+ → Mn2+ half-reaction, though our data are best described by the contrary. We also show with cryo-neutron crystallography that Tyr166 and His30 are unambiguously capable of being ionized (Fig. 5b), which presents further evidence that the active site metal ion environment leads to unusual pKas and biochemical states. In total, we conclude that human MnSOD achieves product inhibition through an unusual PCET mechanism. High product inhibition is achieved from the precise arrangement of active site residues and likely arose evolutionarily to regulate the many roles H2O2 plays in human cell physiology35,37,47. Abnormalities in cellular H2O2 concentrations are hallmarks of disease43,44, and MnSOD product inhibition helps prevent cellular dysfunction.
