The role of Tyr34 in proton-coupled electron transfer of human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O2●−) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pKa due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.

De ning how PCET reactions occur in oxidoreductases bene ts the understanding of redox-mediated disease states and furthers therapeutic interventions [14][15][16][17][18] .Some oxidoreductases use their PCETs to regulate the concentrations of reactive oxygen species (ROS) in cells.These PCETs are crucial as uctuations of ROS levels stimulate mitophagy and programmed cell death, and excessive levels lead to the damage of DNA, proteins, and lipids 19 .Impairment of oxidoreductase function promotes several diseases, including cardiovascular and neurological conditions and cancer progression 20,21 .In particular, the oxidoreductase human manganese superoxide dismutase (MnSOD) is responsible for eliminating O 2 •− in the mitochondrial matrix, and dysfunction of its activity contributes to a broad range of diseases 22 .
Within the mitochondrial matrix, MnSOD uses PCETs to decrease O 2 •− concentrations rapidly and e ciently (k cat /K m > ~ 10 9 M − 1 s − 1 ).In the rst half-reaction, O 2 •− is oxidized to molecular oxygen (O 2 ) with a Mn 3+ ion (k 1 ), and in the second half-reaction, O 2 •− is reduced to hydrogen peroxide (H 2 O 2 ) with a Mn 2+ ion (k 2 ).The two half-reactions regenerate the Mn 3+ ion 23,24 .O 2 •− is endogenously produced as a byproduct of the mitochondrial electron transport chain, and MnSOD is the only means to reduce O 2 •− in the mitochondrial matrix 25,26 .Aberrations of MnSOD function play roles in psoriasis, in ammatory bowel disease, multiple sclerosis, cardiovascular disease, and breast and prostate cancer [27][28][29][30][31] .Overall, the PCETs of MnSOD contribute to health and longevity 24,32 .
For PCET catalysis, MnSOD uses a hydrogen bond network (dashed blues lines, Fig. 1a) to couple proton transfers with changes in Mn oxidation state [32][33][34] .The Mn ion is covalently bound to His26, His74, His163, Asp159, and a solvent molecule called WAT1.From WAT1, the hydrogen bond network extends to Asp159, Gln143, Tyr34, another solvent molecule denoted as WAT2, His30, and Tyr166 from the adjacent subunit.To enter the active site, the substrate and solvent pass through a gateway formed by Tyr34 and His30.Hydrophobic residues Trp123, Trp161, and Phe66 pack the hydrogen bonding atoms of Asp159, WAT1, Gln143, and Tyr34 close together 35 .In our previous work, we investigated the protonation states of the hydrogen bond network in relation to the oxidation state of the Mn ion 13 .
Using neutron crystallography, we previously observed how changing the Mn oxidation state shifts the pK a of active site amino acids and leads to several unusual protonation states 13 .Neutron diffraction is advantageous for studying PCET mechanisms because the scattering of deuterium is on par with carbon, nitrogen, and oxygen, and neutrons do not introduce photoreduction of metal ions, unlike Xrays 36,37 .The neutron structures of Mn 3+ SOD and Mn 2+ SOD revealed three important pieces of data on how PCET catalysis is facilitated.First, during the Mn 3+ to Mn 2+ redox transition, an unconventional proton transfer occurs between Gln143 and metal-bound − OH(WAT1) leading to an unusual Gln143 amide anion and H 2 O (Fig. 1b).The amide anion is stabilized by two short-strong hydrogen bonds (SSHBs) with WAT1 and Trp123.SSHBs stabilize catalytic steps and enhance kinetic rates (hashed lines, Fig. 1b) [38][39][40] .
Second, a shared proton between Tyr166 and N ε2 (His30) modulates the protonation of N δ1 (His30), with N δ1 (His30) only being protonated in the Mn 2+ oxidation state (Fig. 1c).A low-barrier hydrogen bond (LBHB) is formed between Tyr166 and His30 in the Mn 2+ state.A LBHB is a type of SSHB where the heteroatoms transiently share a proton, and the hydrogen bond distances between the donor D/H atom and the heteroatoms are nearly equivalent (1.2-1.3Å) 41 .Third, Tyr34 is deprotonated in the Mn 3+ oxidation state and becomes protonated in the Mn 2+ state.Tyr34 and N δ1 (His30) are probably the two proton donors needed during the Mn 2+ to Mn 3+ redox transition where H 2 O 2 forms from the protonation of the substrate (k 2 ) 32,34,42,43 .Overall, the neutron structures indicate that multiple proton transfers are coupled to electron transfer events and that the active site metal leads to residues with unconventional pK a s that are essential for PCET catalysis.
How O 2 •− interacts with the active site for catalysis is unclear [32][33][34] .O 2 •− may bind the Mn ion directly for an "inner-sphere" electron transfer or between His30 and Tyr34 for a long-range "outer-sphere" electron transfer with the Mn ion [32][33][34]43 . Quatum chemistry computational studies have postulated that the rst half-reaction (k 1 , Mn 3+ → Mn 2+ ) proceeds through an inner-sphere electron transfer while the second half-reaction (k 2 , Mn 2+ → Mn 3+ ) proceeds through an outer-sphere electron transfer 43 .The outersphere mechanism for the second half-reaction is a promising hypothesis because His30 and Tyr34 have been shown to lose protons during the Mn 2+ → Mn 3+ redox transition that converts O 2 •− to H 2 O 2 (Fig. 1c, d) 13 .His30 and Tyr34 could protonate O 2 •− in concert with long-range electron transfer to form the H 2 O 2 product.While there is no experimental evidence for how O 2 •− interacts with the active site for electron transfer, modeling and quantum chemical calculations support the second half-reaction proceeding through an outer-sphere mechanism 43 .
Human MnSOD is inhibited by its product, H 2 O 2 , to regulate the output of mitochondrial H 2 O 2 .
Physiologically, MnSOD product inhibition is thought to be related to H 2 O 2 acting as a secondary messenger 33,34,44 .Mitochondrially-derived H 2 O 2 plays a role in apoptosis 45,46 , mitochondrial biogenesis 44 , and protein localization and activity 47 .Furthermore, mitochondrial H 2 O 2 has been shown to modulate the activity of phosphatase and tension homolog (PTEN), a protein tyrosine phosphatase that contributes to cancer and neurological disease 48,49 .While mitochondrial H 2 O 2 may be scavenged by peroxiredoxins (PRDXs) and glutathione peroxidases (GPXs), these enzymes are dependent on nicotinamide adenine dinucleotide phosphate (NADPH) for function [50][51][52] .MnSOD product inhibition regulates mitochondrial H 2 O 2 levels independent of metabolic status.However, little is known about how MnSOD mechanistically achieves product inhibition.

Product inhibition occurs when the ratio of O 2
•− to MnSOD is high 33,34 .At concentrations of O   •− that are much greater than enzyme, catalysis proceeds through the reversible inhibition reactions k 3 ( rst-order) and k 4 (zero-order).The k 3 kinetics are attributed to forming a "productinhibited" complex that is unreactive to O 2 •− and only disassociates through the zero-order k 4 reaction that dictates the lifetime of the complex.Several kinetic models suggest that inhibition is initiated from the reaction of a Mn 2+ ion with O  ] or [Mn 3+ -− OOH] (k 3 ) 33,34 .The inhibited complex is then relieved by at least one protonation to yield Mn 3+ and H 2 O 2 (k 4 ).
Since k 2 and k 3 both use Mn 2+ and O 2 •− as reactants, they are competing reactions, and their ratios determine the propensity for MnSOD to become product-inhibited.Human MnSOD has equal k 2 and k 3 (Table 1), which means at high O 2 •− to enzyme ratios, ~ 50% of Mn 2+ reactions with O 2 •− form the inhibited complex 53,54 .The factors that govern k 3 and k 4 are unclear though the residue Tyr34 appears to contribute to the inhibition process 55,56 .
Tyr34 is a conserved residue in the active site of MnSOD 55,57 .Physiologically, the residue becomes nitrated in several human neurodegenerative diseases, leading to inactivated MnSOD 58-65 .
Mechanistically, Tyr34 is thought to protonate dioxygen species to produce H 2 O 2 during the Mn 2+ to Mn 3+ redox transition 32,34,57 .This is in line with our previous neutron structures, where Tyr34 is poised to donate a proton in Mn 2+ SOD (Fig. 1d) 13 .With mutation of the residue to phenylalanine, Tyr34Phe MnSOD is unable to proceed through the fast Mn 2+ to Mn 3+ redox transition (k 2 ), and catalysis proceeds exclusively through the product-inhibited pathway (k 3 Table 1).Since k 2 < < k 3 , ~ 99% of Mn 2+ reactions with O 2 •− form the inhibited complex.There is also higher retention of the inhibited complex, with half the disassociation rate compared to wildtype (k 4 , Table 1).The perturbed kinetics and enrichment of product inhibition from the Tyr34Phe variant could be rationalized by the loss of a hydroxyl group for proton transfer, especially since the active site of the Tyr34Phe variant from the X-ray structure is nearly identical to that of the wildtype (Fig. 1e) 57 .Interestingly, the Trp161Phe variant, which does not eliminate a hydroxyl group, has similar kinetics to the Tyr34Phe variant (Table 1) 35 .This suggests that the contribution of Tyr34 toward catalysis is not solely proton transfer.To study the effects that govern the reactions k 1 -k 4 of MnSOD, we performed a series of experiments with the Tyr34Phe variant.
Several studies have reported that mixing large H 2 O 2 concentrations with Mn 3+ SOD leads to the formation of the product-inhibited complex that decays to Mn 2+ SOD 35,[66][67][68] .Mutagenesis studies found the process to be correlated with the forward reactions k 2 -k 4 .These puzzling results may be explained by excessive concentrations of H 2 O 2 initiating a backward reaction with Mn 3+ SOD to produce O     55 Here, we sought to elucidate the role of Tyr34 in MnSOD reactivity by utilizing the Tyr34Phe variant in combination with neutron crystallography, X-ray absorption spectroscopy (XAS), and quantum mechanical (QM) chemistry calculations.With neutron crystallography, we captured the product-bound, reduced, and oxidized states of Tyr34Phe MnSOD free from radiation-induced perturbations.For each state, we used XAS to probe the electron orbitals of the Mn ion.Then, we used QM calculations to quantitatively interrogate the Mn ion orbitals that determine redox activity.Lastly, the Tyr34Phe data were compared with wildtype 13 and Trp161Phe 70 to piece together a MnSOD mechanism that describes the reactions k 1 -k 4 .With respect to oxidoreductases in general, our work presents a thorough characterization of how a single tyrosine modulates PCET catalysis.

Capture of MnSOD product inhibition with H 2 O 2 soaking
With XAS, we sought to determine whether product inhibition of MnSOD may be achieved with excess H 2 O 2 .In Mn K-edge XAS, the X-ray absorption near edge structure (XANES) contains information on the oxidation and coordination states, while the extended X-ray absorption ne structure (EXAFS) region provides information on Mn-ligand bond distances.For studying product inhibition, the Tyr34Phe MnSOD variant is advantageous because it accumulates and retains the product-inhibited complex (Table 1) 33,34,55,57,67,72 .First, we pursued the XANES spectral signatures of Tyr34Phe Mn 3+ SOD and Tyr34Phe Mn 2+ SOD to establish reference points of oxidation and coordination state.Then, we compared the XANES spectra for Tyr34Phe Mn 3+ SOD mixed with O 2 The energy of the rising edge of the XANES region increases with higher oxidation states while the intensity of the pre-edge contains information about the coordination of the Mn ion.The pre-edge corresponds to 1s to 3d orbital transitions, with gains in intensity from 4p character mixing into the 3d orbitals from symmetry distortions and/or loss of inversion symmetry [73][74][75] .Based on electron paramagnetic resonance (EPR) data, the Mn ion is expected to be high-spin in both Mn 3+ and Mn 2+ states 76 .For oxidized Tyr34Phe MnSOD, the rising edge is observed at higher energy compared to the reduced data, indicating a difference in the Mn ion valency.Given that these samples were mixed with stoichiometric excesses of redox reagents, we interpret oxidized Tyr34Phe MnSOD as Mn 3+ and reduced Tyr34Phe MnSOD as Mn 2+ .For both oxidized and reduced Tyr34Phe MnSOD, the pre-edge intensity (i.e., the area under the curve, inset Fig. 2a) corresponds to a distorted ve-coordinate trigonal bipyramidal complex that is also observed in the crystal structures 57,77 .Orbital assignment to the pre-edge intensities will be addressed later with the Kα high-energy resolution uorescence detected absorption (HERFD) method and excited state simulations.The spectral signatures of Tyr34Phe Mn 3+ SOD and Tyr34Phe Mn 2+ SOD were measured for comparison with the product-inhibited complex.
To isolate the product-inhibited complex with XAS, we either introduced O 2 MnSOD.O 2 •− was used to isolate the complex from reaction k 3 (Table 1), while H 2 O 2 was used to attain the complex with a back reaction 67,78 .Following the methods of previous studies, the superoxide-soaked inhibited complex was attained by mixing O 2 •− dissolved in dimethyl sulfoxide (DMSO) and di-benzo-18crown-6-ether with Tyr34Phe MnSOD dissolved in aqueous solution 66,67,79,80 .The peroxide-soaked inhibited complex was obtained by simply mixing aqueous H 2 O 2 with aqueous MnSOD.For both approaches, the inhibited complex has been reported to form within 44 ms of mixing with Tyr34Phe MnSOD 67 .Indeed, the approaches yield nearly identical XANES spectra and reveal that the same electronic structure is formed (Fig. 2a).The superoxide and peroxide-soaked spectra are most similar to Tyr34Phe Mn 2+ SOD, suggesting the presence of a divalent Mn ion.However, these spectra have unique features relative to Tyr34Phe Mn 2+ SOD, with lower intensities at ~ 6550-6555 eV and higher intensities at ~ 6565-6575 eV.Likewise, the pre-edge intensities are highly similar to Tyr34Phe Mn 2+ SOD to indicate that the resulting complex from either mixing O 2 •− or H 2 O 2 is ve-coordinate (Fig. 2a).Altogether, by using the Tyr34Phe variant that enriches for the product-inhibited complex, we show that an electronically distinct ve-coordinate Mn 2+ complex forms from either exposure to O 2 Next, we investigated the EXAFS region of the superoxide and peroxide-soaked Tyr34Phe MnSOD K-edge spectra.The Fourier transform of the EXAFS data, χ(k), yields χ(R) that provides information on the atomic radial distribution around the absorbing Mn ion (Fig. 2b, c).Overall, the spectra in both k space and R space are highly similar between the soaked Tyr34Phe MnSOD counterparts and yielded the same Mn bond distance solutions (Table 2).For both superoxide and peroxide-soaked samples, the rst shell of coordination observed at ~ 2. Neutron structure of the Tyr34Phe product-inhibited complex To visualize the product-inhibited complex and the corresponding active site protonation states, we solved a cryocooled neutron structure of perdeuterated Tyr34Phe MnSOD soaked with deuterium peroxide (D 2 O 2 ) at 2.30 Å resolution.Neutron crystallography is advantageous because it probes H/D atom positions and is absent of photoreduction effects that metal-oxygen species interactions are susceptible to from X-ray exposure 37 .For the neutron structures, proton positions can be assigned at 2.5 Å or better.We rst solve the all-atom structure of the entire enzyme, excluding the active site.Then, with these phases, we carefully interrogate the active site nuclear density maps for the active site coordination and protonation states. of the active sites for any visually distinct dioxygen species.For chain A, nuclear density next to the Mn ion is observed at 3.0 σ and is oblong (Fig. 3a).We interpreted the density as a dioxygen species with a single proton (denoted as LIG for ligand) that has taken the place of the WAT1 position observed in the resting states (Fig. 1b).The proton of LIG, D 2 , points toward Trp161 and appears to be interacting with the pi system (Fig. 3a).The O 2 (LIG) and D ε21 (Gln143) atoms are close in proximity (2.2 Å apart) though LIG and Gln143 are not in optimal geometry for hydrogen bonding (Fig. 3a, b).LIG re ned well at full occupancy and led to Mn bond distances that closely resemble those measured from the EXAFS spectra (Table 2).At physiological temperatures, optical absorption spectra also suggest a displacement of WAT1 and binding of a dioxygen species, which agrees with our cryocooled diffraction data 82 .For chain B, the omit |F o | -|F c | is instead a spherical shape at 3.0 σ (Supplementary Fig. 1b), indicating a − OD molecule that has been previously observed in wildtype neutron structures 13 .Contrasting structures between the two subunits of the asymmetric unit are often observed for the MnSOD P6 1 22 crystal form due to differences in solvent accessibility 13,71 .In chain B, the nuclear density near the Mn ion and − OD molecule is di cult to interpret and will not be discussed further (Supplementary Fig. 1b).Regardless, from the nuclear density at chain A, a singly protonated dioxygen species replaces WAT1 upon D 2 O 2 soaking and forms a complex with bond distances similar to those found from EXAFS spectra (Table 2).
The Mn bond distances of the neutron structure resemble those found from the XAS data and density function theory (DFT) calculations using a hydroperoxyl anion, − O 2 H (Table 2).Overall, our data veri es that the inhibited complex is a ve-coordinate Mn 2+ complex where the WAT1 position has been replaced a The second oxygen of the dioxygen ligand is not directly bound to the Mn ion.
b Broken-symmetry geometry optimization of Mn 2+ -• O 2 H with S = 2 collapses to a Mn 3+ -− O 2 H complex.
Tyr34 orients the Gln143-WAT1 SSHB of Mn 2+ SOD and limits product inhibition We next wondered whether enrichment of the product-inhibited complex in the Tyr34Phe variant was because of structural perturbations near the Mn ion or due to the absence of a hydroxyl group for proton transfer.To this end, we solved neutron structures of reduced and oxidized Tyr34Phe MnSOD at 2.50 and 2.30 Å resolution, respectively.Redox reagents were used that enacted their chemical effects without entering the active site 13,88 .For reduced Tyr34Phe MnSOD, the Mn bond distances are similar to vecoordinate wildtype Mn 2+ SOD (Supplementary Table 3).Furthermore, the protonation states resemble those of wildtype Mn 2+ SOD, where WAT1 is of the D 2 O form while Gln143 is deprotonated to the amide anion (Fig. 3c).Deprotonated amino acids are identi ed when attempts to model and re ne a proton result in negative |F o | -|F c | difference neutron scattering length density and all the other protons of the amino acid can be placed.Interestingly, the lack of the hydroxyl group in Tyr34Phe Mn 2+ SOD slightly perturbs the orientation of Gln143 and lengthens the WAT1-Gln143 hydrogen bond (Fig. 3d).The WAT1-Gln143 SSHB of the wildtype enzyme is critical for the back-and-forth proton transfers needed for redox cycling of the Mn ion 13 , and the Tyr34Phe Mn 2+ SOD neutron structure suggests one role Tyr34 plays in catalysis is correctly positioning Gln143 for rapid PCET catalysis.Indeed, the Mn 2+ to Mn 3+ redox transition is nearly ablated for Tyr34Phe MnSOD (k 2 , Table 1) 33,34 .Another consequence of the perturbation of the WAT1-Gln143 interaction is the potentially easier displacement of WAT1 by a dioxygen species to form the product-inhibited complex.Overall, the reduced Tyr34Phe MnSOD neutron structure suggests that Tyr34 orients Gln143 for a tight hydrogen bonding interaction with WAT1.
For oxidized Tyr34Phe MnSOD, the Mn ion is bound by an − OD molecule (Fig. 3e) and has covalent bond distances that resemble that of wildtype Mn 3+ SOD (Supplementary Table 3).Furthermore, the hydrogen bond distances among the interactions of O(WAT1)-D ε21 (Gln143) and D ε1 (Trp123)-O ε1 (Gln143) are identical to wildtype counterpart (Fig. 3f).Of potential consequence for the Tyr34Phe variant, however, is the absence of the anionic phenolate group that is seen in wildtype Mn 3+ SOD.As PCET mechanisms depend on the distribution of electrostatic vectors to propagate proton and electron transfers, the lack of an ionizable group may perturb enzyme kinetics.Such electronic effects are suggested by the lower kinetic rates of the Mn 3+ to Mn 2+ redox transition found for the Tyr34Phe variant (k 1 , Table 1).Overall, the interactions between WAT1, Gln143, and Trp123 are the same in Tyr34Phe and wildtype Mn 3+ SOD.
The neutron structures of reduced and oxidized Tyr34Phe Mn 2+ SOD suggest the roles of Tyr34 in catalysis are (1) orient Gln143 for e cient interaction and proton transfer with WAT1 during the Mn 2+ to Mn 3+ PCET reaction, (2) limit formation of the product-inhibited complex by strengthening the WAT1-Gln143 hydrogen bond, and (3) contribute subtle electronic effects as a phenolate anion during the Mn 3+ to Mn 2+ reaction.These interpretations are supported by the kinetic rates of Tyr34Phe MnSOD 33,34 .For Tyr34Phe MnSOD, the fast Mn 2+ to Mn 3+ redox reaction is ablated (k 2 , Table 1), formation of the productinhibited complex is enriched (k 3 > > k 2 , Table 1), and the Mn 3+ to Mn 2+ redox reaction is cut in third (k 1 , Table 1).A unifying theme among Tyr34Phe MnSOD and other variants studied kinetically is that the precise orientation of Gln143 is critical for catalysis.Mutating residues directly adjacent to Gln143, such as Trp161, Trp123, and Tyr34, lead to similar kinetic consequences, where the Mn 2+ → Mn 3+ half reaction is ablated, product inhibition is enriched, and the Mn 3+ → Mn 2+ half reaction is slower compared to wildtype 33,34 .These redundant effects of the Trp161Phe, Trp123Phe, and Tyr34Phe variants suggest that a crucial role of residues neighboring Gln143 is to enforce a SSHB between WAT1 and Gln143.In the case of Tyr34, our neutron structures indicate that Tyr34 is responsible for positioning Gln143 for a SSHB with WAT1 during the Mn 2+ resting state and that the strength of the bond between Gln143 and WAT1 correlates with the extent of product inhibition.
Retention of the product-inhibited complex is dependent on the Gln143 position As the XAS and neutron crystallographic data of Tyr34Phe MnSOD demonstrated the formation of a vecoordinated Mn 2+ SOD with a singly-protonated dioxygen species replacing the WAT1 position, we wondered if the formation and retention of the inhibited complex could be distinguished with other variants of MnSOD.In the context of Tyr34Phe MnSOD, we also sought to de ne whether the enrichment of product inhibition was due to the loss of the ionizable Tyr group only or also due to the perturbation of the Gln143-WAT1 SSHB interaction.For example, the Trp161Phe MnSOD variant alters a residue directly adjacent to Gln143 and has enriched product-inhibition kinetics like those of Tyr34Phe MnSOD (Table 1) 66 .For wildtype, Tyr34Phe, and Trp161Phe, we performed XANES in the HERFD mode of detection, which allows a large improvement in energy resolution and sensitivity compared to conventional XANES [89][90][91] .Here, we focused on comparing the reduced form of the variants to those of peroxide-soaked counterparts.As all these complexes are expected to be ve-coordinated d 5 with spin S = 5/2, we sought to distinguish ne details of the spectra offered by the HERFD mode of detection.
For HERFD-XANES data of Tyr34Phe MnSOD, the features of the reduced and peroxide-soaked forms seen in conventional XANES (Fig. 2a) are also observed.While both forms have similar rising edge energies to indicate the same oxidation state of the Mn ion, the peroxide-soaked form is seen with a lower intensity at ~ 6550-6555 eV and a higher intensity at ~ 6565-6580 eV (Fig. 4a).Unique to the HERFD data is the observation of a higher intensity shoulder of peroxide-soaked Tyr34Phe MnSOD at ~ 6565 eV and the ability to better resolve the pre-edge peaks for both samples.The peak centers of the pre-edge are within ~ 0.2 eV of each other, while greater intensity for the higher energy tail (~ 6542.5 eV) is distinguishable for peroxide-soaked Tyr34Phe MnSOD.Overall, reduced and peroxide-soaked Tyr34Phe MnSOD spectra have distinct features between each other, and the general shape trends are reproducible both with conventional XANES (Fig. 2a) and HERFD-XANES (Fig. 4a) and suggest two differing ve-coordinate Mn 2+ complexes are measured.
Trp161Phe MnSOD, like Tyr34Phe MnSOD, exhibits kinetics indicating a highly product-inhibited enzyme (k 3 > > k 2 , Table 1).HERFD-XANES data of reduced and peroxide-soaked Trp161Phe MnSOD demonstrates the same trends as Tyr34Phe MnSOD, with a lower intensity at ~ 6550 eV and a higher intensity at ~ 6562-6570 eV (Fig. 4b).Likewise, a lower intensity for the pre-edge maxima at ~ 6540 eV and a greater intensity for the pre-edge tail at ~ 6542.5 eV is seen for peroxide-soaked Trp161Phe MnSOD compared to the reduced counterpart.Overall, similar trends are observed between reduced and peroxide-soaked forms of Tyr34Phe and Trp161Phe MnSOD.
Next, we wondered how well the product-inhibited complex could be isolated in wildtype MnSOD, where k 3 = k 2 (Table 1).Compared to Tyr34Phe and Trp161Phe, wildtype has the least noticeable difference in intensity among the 6550-6560 eV region between its peroxide-soaked and reduced counterparts (Fig. 4c).However, a higher intensity at ~ 6570 eV is still observed for peroxide-soaked wildtype as well as the higher intensity for the pre-edge at ~ 6542.5 eV.Of the reduced and peroxide-soaked pairs, the spectra of the wildtype pair are the most similar in their overall shape.Since the wildtype enzyme exhibits physiological product inhibition levels (k 3 = k 2 , Table 1) compared to the Tyr34Phe and Trp161Phe variants that are enriched for product inhibition (k 3 > > k 2 and lower k 4 compared to wildtype, Table 1), a potential explanation for the less pronounced differences for the wildtype pair is that the peroxide-soaked data may re ect a mixture of species rather than a fully isolated product-bound complex.
When comparing the HERFD-XANES data of wildtype, Tyr34Phe, and Trp161Phe peroxide-soaked MnSOD variants, several commonalities and trends are observed.First, the edge of the three samples lay on top of each other (Fig. 4d), indicating that the oxidation and spin state is maintained across the variants.For the pre-edge, the apex of the pre-edge intensity for the peroxide-soaked samples is found to be consistently lower than the reduced counterparts in addition to an increase in the intensity of the highenergy pre-edge tail (inset, Fig. 4a-d).While the apex peak of the pre-edge shifts within 0.2 eV among the samples tested (inset, Fig. 4d), the signi cance of the shift is unclear due to the closeness of the peaks.For the overall spectral shape, peroxide-soaked Trp161Phe MnSOD has the most drastic shift compared to its reduced counterpart (6550-6580 eV, Fig. 4b).Furthermore, peroxide-soaked Trp161Phe MnSOD also has the lowest intensity at the 6550 eV region compared to other peroxide-soaked variants (Fig. 4d).This may be because Trp161Phe MnSOD is the most product-inhibited of the three MnSOD forms tested, (k 3 > > k 2 and lowest k 4 , Table 1).Wildtype is the least product-inhibited when compared to Trp161Phe and Tyr34Phe, (k 3 = k 2 and higher k 4 , Table 1) and has the least drastic shape shift between its reduced and peroxide-soaked counterparts.Tyr34Phe, while still highly product-inhibited (k 3 > > k 2 ), has a k 4 value between that of Trp161Phe and wildtype and may explain why the 6550 eV intensity is intermediate (Fig. 4d).While previous kinetic models ascribe k 4 as the zero-order decay of the inhibited complex to a trivalent Mn ion [33][34][35]66,92 , some studies have indicated that the product-inhibited complex decays to divalent Mn ion 66,67 . Ineed, exposing our samples to lesser amounts of peroxide before HERFD-XANES data collection leads to spectra that more closely resemble reduced spectra (Supplementary Fig. 2).These observations indicate that retention of the inhibited complex is not exclusively dependent on a proton transfer from Tyr34 but instead on other factors of the active site.
To provide insight into the mechanism of product inhibition, we compared our D 2 O 2 -soaked Tyr34Phe MnSOD neutron structure with that of our previous D 2 O 2 -soaked Trp161Phe MnSOD neutron structure (Fig. 4e, PDB ID 8VHW) 70 .The orientation of the singly-protonated dioxygen ligand differs between the two structures.The O 1 -O 2 (LIG) axis for Trp161Phe is nearly coaxial with that of the Mn-Asp159 bond.For Tyr34Phe, the O 1 -O 2 (LIG) axis is at an angle of ~ 55° to the Mn-Asp159 bond.These orientation differences are most likely attributed to the residue at position 161.LIG closely interacts with Trp161 in Tyr34Phe MnSOD (Fig. 3a), and when this contact is absent, LIG can assume a different orientation.Interestingly, the strong LIG-Gln143 hydrogen bond in Trp161Phe MnSOD coincides with a longer retention of the product inhibited-complex (k 4 , Table 1).However, since the 161 position is occupied by a tryptophan residue in wildtype, the LIG orientation in Tyr34Phe MnSOD is probably close to what would occur physiologically.Both Tyr34Phe and Trp161Phe MnSOD have nearly ablated catalysis for the Mn 2+ to Mn 3+ redox reaction (k 2 , Table 1) and therefore proceed predominately through the product-inhibited pathway (k 3 and k 4 , Table 1).To investigate whether de cient k 2 catalysis in Tyr34Phe is from a loss of the Tyr34 ionizable group or the distortion of the Gln143 position, we compared the neutron structure of Tyr34Phe Mn 2+ SOD with our previous Trp161Phe Mn 2+ SOD structure that preserves the number of ionizable groups (Fig. 4f, PDB ID 8VHY) 70 .Both variant structures have a lengthened WAT1-Gln143 interaction compared to wildtype which is a site of proton transfer (Fig. 1b) 13 .This suggests that the k 2 PCET reaction is not solely dependent on the ionization of Tyr34 but also on tight WAT1-Gln143 hydrogen bonding.Furthermore, the weakened interaction for the variants allows WAT1 to be more easily displaced by dioxygen species for the formation of the product-inhibited complex through k 3 (Table 1).Altogether, both the Tyr34Phe and Trp161Phe Mn 2+ SOD structures highlight the importance of the WAT1-Gln143 interaction for catalysis.
Previous studies have indicated that relief of inhibited complex (i.e., disassociation of the dioxygen species from the Mn ion) is proton transfer dependent, with Tyr34 being the most obvious proton donor 33,34 .Indeed, mutation of Tyr34 to phenylalanine leads to a slower disassociation of the inhibited complex (k 4 , Table 1).However, several other point mutations lead to the same effect, including Trp161Phe and Trp123Phe 33,34 .What these variants have in common with Tyr34Phe is that the affected residue position is directly adjacent to Gln143, which has been shown to change protonation states (Fig. 1b) 13 .From the D 2 O 2 -soaked neutron structure of Trp161Phe MnSOD, we previously postulated that the strong interaction between LIG and Gln143 could represent a possible proton transfer site (Fig. 4e) 70 .
However, this suggestion is at odds with the Tyr34Phe counterpart, which has a weak hydrogen bonding interaction with the already protonated O 2 (LIG) atom and a faster Mn-dioxo dissociation (k 4 , Table 1).This means Gln143 may not directly protonate LIG and that a stronger LIG-Gln143 interaction instead contributes to longer retention of the inhibited complex.An alternative proton donor for the protonation of LIG to form H 2 O 2 could be a solvent molecule.Proton donation from a solvent molecule would have to compete with the LIG-Gln143 interaction, and this also explains why a short LIG-Gln143 hydrogen correlates with longer retention of the inhibited complex.Overall, our analysis of D 2 O 2 -soaked MnSOD variants suggests that Gln143 plays a role in the retention of the inhibited complex.
The electronic con guration of the Mn ion For enzymes that use metal centers to catalyze redox reactions, the arrangement of the metal 3d orbitals determines how electrons are exchanged and how substrates orient for catalysis 93 .For MnSOD, the metal ion is in a distorted C 3v symmetry environment with either 4 or 5 occupied electrons in the αmanifold, depending on the oxidation state of the metal 76 .For Mn 3+ with S = 2, the e π (xz/yz) and e σ (xy/x 2 -y 2 ) α orbitals are occupied, while Mn 2+ with S = 5/2 also has the z 2 α orbital occupied (Fig. 5a).The z 2 α orbital exchanges electrons during redox reactions as it is the lowest unoccupied molecular orbital (LUMO) for Mn 3+ and the highest occupied molecular orbital (HOMO) for Mn 2+ .This means that monoor dioxygen species are most likely to bind the Mn ion along the z-axis for reactivity (Fig. 5b).For further insight into the MnSOD metal 3d orbitals, we compared the K-pre-edge spectra found through timedependent DFT (TD-DFT) simulations with spectra measured from HERFD-XANES.
In K-edge XAS, the pre-edge corresponds to 1s to 3d transitions that gain intensity through 4p character mixing into the 3d orbitals from symmetry distortions and/or loss of inversion symmetry [73][74][75] .The contribution of the 4p electric dipole is about one hundred times stronger than the 3d quadrupole contributions, so a small amount of 4p mixing can signi cantly in uence the pre-edge spectra 73,74,77 .
With TD-DFT simulations, the 3d/4p contributions can be assigned to the experimental pre-edge spectra (Fig. 5c-h).In the plots, the vertical stick heights represent the intensity of 1s to 3d/4p transitions from quadrupole and dipole contributions, and percentage of 3d and 4p character from ground-state DFT are listed.To identify the impact of the Tyr34Phe variant on the electronic con guration of the metal, we compared its Mn 3+ SOD, Mn 2+ SOD, and H 2 O 2 -soaked pre-edge spectra to those of wildtype and Trp161Phe.
The experimental pre-edge spectra are similar between wildtype and Tyr34Phe Mn 3+ SOD, indicating that the Tyr34Phe variant does not signi cantly alter the Mn 3+ ion orbital con guration (Fig. 5c, d).The TD-DFT spectra are also similar between the two complexes, though the simulated spectra underestimate the splitting of orbital transitions and overestimates intensity.This misestimation has been previously reported for other transition metal complexes of similar symmetry 77,94 .In brief, TD-DFT overestimates the exchange interaction between the 1s core hole and the valence 3d orbitals, which results in decreased energy splitting between transitions.Regardless, the e σ orbitals are the primary contributors to the intensities of the pre-edge as they have signi cant dipole character from 4p mixing.Indeed, ground-state DFT indicates that the e σ β orbitals have 1.5-1.6%4p character and have the highest 4p mixing compared to the other 3d orbitals (inset table, Fig. 5c, d).In the experimental spectra, a highenergy tail is observed at ~ 6542.5 eV, which is likely to be the z 2 β transition as indicated by TD-DFT.
Assignment of the lower energy z 2 α and e π β transitions in the experimental spectra is less clear, but they may contribute to the left shoulders of the pre-edge peaks.Despite the misestimation of the energy splitting with TD-DFT, the contributions of the e σ and z 2 β transitions can be assigned to the experimental spectra.
For wildtype and Tyr34Phe Mn 2+ SOD, the experimental pre-edge spectra are nearly identical (Fig. 5e, f).DFT calculations suggest that the e σ β orbitals contribute the majority of the intensity to the pre-edge for both wildtype and Tyr34Phe Mn 2+ SOD due to 4p mixing.The z 2 β transitions found at higher energies are of pure quadrupole character that contributes weak intensity.At lower energy, the e π β transitions likely contribute weak intensity to the left shoulders of the pre-edge peaks.For the resting state Mn 2+ SOD complexes, the experimental and simulated spectra agree with each other and allow the assignment of all the orbital transitions.
The H 2 O 2 -soaked Trp161Phe and Tyr34Phe complexes have similar experimental spectra though the calculated spectra suggest differences in orbital transitions (Fig. 5g, h).The calculated spectra used a Mn 2+ ion along with a HO 2 − as the dioxygen species.For Trp161Phe, the e σ and e π β transitions are close in energy and suggest further distortions of C 3v symmetry and mixing of the d orbitals (Fig. 5g).These e transitions are mostly of dipole character, with the e σ set bearing the most.The z 2 β orbital is the highest energy transition with weak intensity.For Tyr34Phe, the d orbitals also mix, and the e σ transitions begin to split (Fig. 5h).Between Tyr34Phe and Trp161Phe, the energy of the z 2 β transition differs, with that of Trp161Phe being ~ 0.4 eV higher than Tyr34Phe (Fig. 5g, h).This is re ective of different Mn ion interactions along the z-axis, like with HO 2 − .However, the calculated intensities of the z 2 β transitions are weak, and the energies that these transitions occur in the experimental spectra are not obvious.Overall, the pre-edge spectral shapes of H 2 O 2 -soaked Trp161Phe and Tyr34Phe complexes are similar, though they have different transition energies.
Analysis of the K-pre-edge for various MnSOD complexes indicates that the hydroxyl group of Tyr34 does not signi cantly affect the metal 3d transitions of the Mn 3+ SOD and Mn 2+ SOD resting states (Fig. 5c-f).
The pre-edge spectra for wildtype and Tyr34Phe resting states are dominated by e σ transitions that are anked by weaker intensity e π and z 2 transitions.The majority of the intensities from the e σ orbitals are from dipole contributions as a result of 4p mixing.Speci cally, the metal 4p x and 4p y mix with the 3d x 2y 2 and 3d xy orbitals.These orbitals have σ-overlap with the Mn ligands along the xy plane, namely with residues His74, His163, and Asp159 (Fig. 5b).The deviation of these trigonal ligands away from idealized 120° angles in C 3v symmetry results in 4p mixing and signi cant intensity contributions to the pre-edge peak 77 .
The TD-DFT calculations for the Trp161Phe and Tyr34Phe Mn 2+ SOD that are bound by HO 2 − have similar spectral shapes though different energies of for the e σ and z 2 transitions (Fig. 5g, h).This may, in part, be explained by the different orientations of HO 2 − bound to the Mn 2+ ion that leads to different orbital characteristics (Fig. 4e).The orbital transitions for these complexes have strong covalent mixing with the Mn-bound ligands, leading to less overall d character compared to the resting state complexes.The d orbitals also mix with each other and suggest the complexes are further distorting away from C 3v symmetry (Fig. 5g, h).TD-DFT analysis of dioxygen-bound Trp161Phe and Tyr34Phe Mn 2+ SOD suggest that different modes of HO 2 − binding may result in similar spectra.
Tyr34 contributes to the pK a s of Tyr166/His30 and in uences the proton shu e across the subunit interface Second-sphere residues His30 and Tyr166 have unusual pK a s in wildtype MnSOD and change protonation states (Fig. 1c) 13 .To identify whether the Tyr34 mutation to Phe affects the protonations of His30/Tyr166, we investigated the nuclear density maps of Tyr34Phe MnSOD in D 2 O 2 -soaked, reduced, and oxidized forms.For chain A of D 2 O 2 -soaked MnSOD that has dioxygen species bound (Fig. 3a, b), the omit |F o | -|F c | density indicates that Tyr166 is in the neutral, protonated form while His30 is singly protonated on the N δ1 atom (Fig. 6a).Interestingly, for chain B, Tyr166 is instead deprotonated and His30 is now protonated on N ε2 (Fig. 6b).The two residues form a 1.7 Å SSHB due to the negative charge of ionized Tyr166.Different chains of the same MnSOD neutron structure have been observed before to differ in protonation states, with the most explicable cause being differences in solvent accessibility 13 .However, the protonation con guration observed in chain B is unique and has not been seen before (Fig. 6b).It is not known if this proton con guration occurs in wildtype enzyme as neutron data for D 2 O 2soaked wildtype has yet to be collected.But we can compare reduced and oxidized Tyr34Phe MnSOD to wildtype 13 .
For reduced Tyr34Phe MnSOD, both chains have the same protonation states (Fig. 6c, d).Tyr166 is protonated and neutral while His30 is singly-protonated on the N δ1 atom.The omit density for D η (Tyr166) is elongated and spans to N ε2 (His30) (Fig. 6d).Elongated density has been observed between the two residues before in reduced wildtype MnSOD 13 .This may be indicative of a proton transfer occurring between Tyr166 and His30, in line with the different protonation states observed in the D 2 O 2 -soaked structure (Fig. 6a, b).
The protonation states of oxidized Tyr34Phe (Fig. 6e, f) are identical to that of reduced Tyr34Phe (Fig. 6c,  d), which is in contrast to wildtype MnSOD where the His30 protonation alters between oxidized and reduced states (Fig. 1c).This difference is probably due to the Phe mutation at position 34.In wildtype, Tyr34 is ionized when the Mn ion is oxidized (Fig. 1d).The negative charge on Tyr34 may exert electronic effects that alter the pK a of His30.Thus, Tyr34 may help modulate the pK a of nearby residues so e cient proton transfers occur for PCET catalysis.
Our three neutron structures of Tyr34Phe MnSOD highlight the importance of residues Tyr166, His30, and Tyr34 in maintaining the proton pool of the active site.Two protons are needed to protonate O 2 2− to H 2 O 2 during the fast Mn 2+ → Mn 3+ reaction (k 2 , Table 1).The D 2 O 2 -soaked structure unambiguously indicates that His30 and Tyr166 shu e protons, with proton transfers between O η (Tyr166) and N ε2 (His30) coinciding with changes in the N δ1 (His30) protonation state (Fig. 6a, d).In wildtype MnSOD, the proton between O η (Tyr166) and N ε2 (His30) was instead observed to be shared, perhaps because the wildtype structures were collected at room temperature (Fig. 1c, d).Another distinction between Tyr34Phe and wildtype MnSOD is that in the oxidized forms, the N δ1 (His30) have different protonation states.For Tyr34Phe, N δ1 (His30) is protonated (Fig. 6e, f) while in wildtype N δ1 (His30) is deprotonated (Fig. 1c).The pK a s of active site residues are a result of multiple effects, including residue composition and ionization states.An ionized Tyr34 coincides with a deprotonated N δ1 (His30) in wildtype MnSOD and suggests that Tyr34 contributes to modulating the pK a of nearby residues for oxidized MnSOD.Overall, the Tyr34Phe MnSOD neutron structures help elucidate the role of Tyr166, His30, and Tyr34 in MnSOD catalysis.

Summary and comparison of Tyr34Phe active site con gurations
Comparing the Tyr34Phe MnSOD neutron structures with our previous wildtype and Trp161Phe neutron structures helps our understanding of the role of Tyr34 in catalysis.For oxidized wildtype and Tyr34Phe MnSOD, the WAT1-Gln143 protonation states and hydrogen bond interaction are similar (Fig. 7a, b).However, in reduced Tyr34Phe, His30 is observed to have different protonation states compared to wildtype, and the proton between His30 and Tyr166 is not shared.For the reduced counterparts, the hydrogen bonding of Gln143 with WAT1 and Trp123 is weaker in Tyr34Phe MnSOD (Fig. 7c, d).The Gln143 hydrogen bonds in wildtype are SSHBs (< 1.8 Å), while those of Tyr34Phe are typical hydrogen bonds (> 1.8 Å).Reduced wildtype is observed to have a shared proton between His30 and Tyr166 while Tyr34Phe does not.However, chain B of reduced Tyr34Phe MnSOD is observed to have elongated nuclear density between His30 and Tyr166 (Fig. 6d), which indicates that a proton may be shared.For peroxide-bound Trp161Phe and Tyr34Phe, HO 2 − binds the Mn ion in different orientations (Fig. 7e, f).
Furthermore, in Trp161Phe, a H 2 O 2 molecule is bound with SSHBs between His30 and an anionic Tyr34.
The lack of H 2 O 2 at this site in Tyr34Phe may be due to the absence of the hydroxyl group.Overall, Tyr34 plays a role in several aspects of the active site, and several enzymatic details are revealed from this collection of neutron structures.
First, Tyr34 helps control the charge at the active site and provides an environment conducive to chargedependent proton and electron transfers.For example, Tyr34 and His30 both lose protons when the active site is oxidized and gain protons when the active site is reduced (Fig. 7a, 7c, 1c-d).As a result, we previously hypothesized that Tyr34 and His30 protonate O 2 2− during the fast Mn 2+ → Mn 3+ reaction (k 2 , Table 1) 13 .In the Tyr34Phe variant, the His30 protonation state remains consistent between resting states, which implicates Tyr34 in in uencing the pK a of His30 through electronic effects (Fig. 7b, 7d, 6cf).These charge effects of Tyr34 may be a partial contributor to the de cient catalysis observed for the Tyr34Phe variant (Table 1).
Second, Tyr34 plays a signi cant role in the fast Mn 2+ → Mn 3+ reaction as suggested by the nearly ablated k 2 for Tyr34Phe (Table 1).Comparison of the resting Mn 2+ SOD neutron structures of wildtype and Tyr34Phe indicates that Tyr34 enforces a strong WAT1-Gln143 interaction (Fig. 7c-d, 3d).This interaction is important for PCET, where the WAT1 → Gln143 proton transfer coincides with the Mn 2+ → Mn 3+ redox reaction (Fig. 1b) 13 .Another potential reason for ablated k 2 catalysis for the Tyr34Phe variant is the loss of an ionizable group that could protonate O 2 2− or HO 2 − .However, the Trp161Phe variant also has an ablated k 2 and maintains the number of ionizable groups (Table 1).The shared feature for these two variants is the weakened WAT1-Gln143 interaction (Fig. 4f).Since our previous structures indicate that Tyr34 loses a proton during the Mn 2+ → Mn 3+ reaction (Fig. 1d), it is possible that the WAT1 → Gln143 proton transfer precludes a Tyr34 → O 2 2− proton transfer.Overall, our structures indicate Tyr34 contributes to orienting WAT1 and Gln143 for a proton transfer event during the Mn 2+ → Mn 3+ redox reaction.
Third, the formation of the product-inhibited complex is dependent on Tyr34.The complex is characterized by an HO 2 − molecule replacing the WAT1 position in the Mn 2+ oxidation state (Fig. 7e-f,   4e).As such, the capacity to form the inhibited complex may correlate with the ease an HO 2 − molecule can displace WAT1.The Tyr34Phe and Trp161Phe variants have a higher propensity to accumulate the inhibited complex, and both have a weakened WAT1-Gln143 interaction in the Mn 2+ oxidation state that would permit easier displacement of WAT1 (Fig. 4f).This suggests that the Tyr34-Gln143-WAT1 hydrogen bond network suppresses product inhibition.
Lastly, retention of the product-inhibited complex correlates with the strength of hydrogen bonding between HO 2 − and Gln143.The inhibited complexes captured in the Tyr34Phe and Trp161Phe variants reveal two different HO 2 − binding orientations and hydrogen bond interactions (Fig. 7e-f, 4e).The hydrogen bonding between HO 2 − and Gln143 is stronger in Trp161Phe compared to Tyr34Phe, and this stronger interaction correlates with a slower disassociation of the inhibited complex for Trp161Phe (k 4 , Table 1).As protonation of HO 2 − to H 2 O 2 is required for relief of the inhibited complex, the hydrogen bonding of Gln143 with HO 2 − may compete with a H 2 O → HO 2 − proton transfer to form H 2 O 2 .
Comparison of the product-inhibited complex of Tyr34Phe and Trp161Phe provides clues into the relief of the inhibited complex.

Suggested mechanism
From our collection of neutron structures and XAS data, we have constructed a mechanism for the fast reaction pathways k 1 and k 2 and the reversible product inhibition reaction pathways k 3 and k 4 (Fig. 8).
Note that our data describe the inhibited complex as Mn 2+ -containing (Fig. 2a, 4a-c), in contrast with several mechanistic models that assign a Mn 3+ oxidation state to the complex [33][34][35]66,67 . Furthrmore, in the absence of data that describe how O 2 •− interacts with an oxidized active site, O 2 •− reacting with the Mn 3+ ion is represented only by Mn 3+ gaining an electron (Fig. 8).For a reduced active site, O 2 •− likely binds between His30 and Tyr34 for PCET to form H 2 O 2 13,43 .Overall, the mechanism delineates how MnSOD's reversible inhibition pathway may branch from the fast reaction pathway.
For the k 1 reaction that represents the fast Mn 3+ → Mn 2+ half-reaction, reduction of the Mn ion occurs alongside three proton transfers, Gln143 → WAT1 and protonation of His30 and Tyr34 by solvent (Fig. 8a).In the reduced state, anionic Gln143 is stabilized by SSHBs with Trp123, WAT1, and Tyr34 (Fig. 8b).For the k 2 reaction that represents the fast Mn 2+ → Mn 3+ half-reaction, a WAT1 → Gln143 proton transfer occurs alongside protonation and reduction of O 2 •− (Fig. 8c).Here, O 2 •− participates in a long-range electron transfer and is protonated by His30 and Tyr34 to form H 2 O 2 (Fig. 8d).If Mn 3+ gains an electron while the resultant H 2 O 2 is still present, H 2 O 2 donates a proton to His30 and subsequently replaces WAT1 to form the product inhibited complex (k 3 , Fig. 8e-f).The inhibited complex is characterized by the inability to perform the WAT1 → Gln143 proton transfer and prohibits fast catalysis (Fig. 8f).Relief of the inhibited complex is achieved by protonation of HO 2 − by a solvent molecule (k 4 , Fig. 8g).H 2 O 2 departs the active site and is replaced by WAT1 to form the Mn 2+ resting state (Fig. 8h).
From relief of inhibition, fast catalysis may proceed again through k 2 (Fig. 8c-d).
The key determinant of whether product inhibition is engaged is the His30 proton donor during Mn 3+ reduction.If H 2 O 2 is the proton donor, the inhibited complex is formed (k 3, Fig. 8e).If a solvent molecule is the proton donor, the enzyme proceeds through fast catalysis (k 1 , Fig. 8a).This explains why high concentrations of O 2 •− lead to product inhibition, as O 2 •− may enter the active site before H 2 O 2 departs.
Tyr34 may also serve as a potential proton acceptor for H 2 O 2 since Tyr34 also gains a proton during reduction to Mn 2+ .However, Tyr34 is not necessary to form the inhibited complex, as indicated by our Tyr34Phe neutron structure (Fig. 3a-b).Our work provides insights into the PCET mechanism of MnSOD, especially in the context of product inhibition.
Altogether, our investigation reveals how a single tyrosine residue has a profound effect on PCET catalysis.Tyr34 plays a part in every MnSOD kinetic step from its roles of (1) acting as a proton donor/acceptor, (2) orienting nearby molecules Gln143 and WAT1 for proton transfer, (3) limiting the formation of the inhibited complex, and (4) shortening the lifetime of the inhibited complex.These roles of Tyr34 place the residue as a central regulator of H 2 O 2 output in the mitochondria.H 2 O 2 produced from MnSOD has several cellular effects, including stimulating apoptotic signaling pathways 45,46 , coordinating protein localization and activity 47 , and mitochondrial biogenesis 44 .Inactivation of Tyr34 by nitration is observed in neurological disease and further highlights the physiological role of Tyr34 58-65 .In total, the work provides insight into how a PCET enzyme facilitates catalysis and how an oxidoreductase molecularly facilitates mitochondrial function.

Perdeuterated expression and puri cation
For deuterated protein expression of MnSOD, the pCOLADuet-1 expression vector harboring full-length cDNA of MnSOD was transformed into Escherichia coli BL21(DE3) cells.Transformed cells were grown in D 2 O minimal media within a bioreactor vessel using D 8 -glycerol as the carbon source 95 .Induction was performed with 1 mM isopropyl -D-thiogalactopyranoside, 8 mM MnCl 2 , and fed D 8 -glycerol until an OD 600 of 15.0.Expression was performed at 37°C for optimal Mn metal incorporation 96 .Harvested cell pastes were stored at -80°C until puri cation.For protein puri cation (with hydrogenated reagents), cells were resuspended in a solution of 5 mM MnCl 2 and 5 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.8.Clari ed lysate was incubated at 55°C to precipitate contaminant proteins that were subsequently removed by centrifugation.Next, soluble protein was diluted with an equal volume of 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5, yielding a nal concentration of 25 mM.Measurement of pH veri ed a value of 5.5 after dilution.Protein was applied onto a carboxymethyl sepharose fast ow column (GE Healthcare) and eluted with a sodium chloride gradient that contained 50 mM MES pH 6.5.

Crystallization
Perdeuterated Tyr34Phe MnSOD crystals were grown in a microgravity environment aboard the International Space Station (ISS) 97 .Crystals growth was achieved in Granada Crystallization Boxes (GCBs, Triana) through capillary counterdiffusion using fused quartz capillary tubes (VitroCom) that had inner diameters of 2.0 mm and outer diameters of 2.4 mm 98 .25 mg ml − 1 protein-lled capillaries were plugged with 40 mm of 2% agarose (w/w) and inserted into GCBs lled with precipitating agent composed of 4 M potassium phosphate, pH 7.8.The pH of the phosphate buffer was achieved through 91:9 ratios of K 2 HPO 4 :KH 2 PO 4 .The GCBs were delivered to the ISS by SpX-17 as part of the Perfect Crystals NASA payload and returned to earth 1 month later on SpX-18.The crystals within GCBs were observed to be resilient against travel damage and were placed within carry-on baggage during further aircraft travels to the UNMC Structural Biology Core Facility and ORNL.Perdeuterated Tyr34Phe MnSOD crystals were 0.3-0.6 mm 3 in size, and further details of microgravity crystallization have been published 97 .For X-ray diffraction, crystals were grown in 1.8 M potassium phosphate, pH 7.8 by hangingdrop vapor diffusion.Protein (23 mg ml − 1 ) and reservoir solution were mixed at a 1:1 ratio to give a 4.0 µL drop.Crystals for X-ray diffraction were less than 0.1 mm 3 in size and were fully grown after 14 d.

Crystal Manipulations
For deuterium exchange, microgravity-grown crystals were rst placed in 1 mL of hydrogenated 4 M potassium phosphate pH 7.8.Deuterium was introduced with 0.1 mL incremental additions every 2 min of 4 M deuterated potassium phosphate (K 2 DPO 4 :KD 2 PO 4 ) pD 7.8 (calculated by adding 0.4 to the measured pH reading) for a total of ve times and a net volume addition of 0.5 mL.After 10 min, 0.5 mL of the solution was removed leading to a 1 mL solution consisting of 33% deuterium.The process was repeated enough times to gradually increase the deuterium content to ~ 100%.The 4 M deuterated potassium phosphate also served as the cryoprotectant for the cryocooling process.Further details of the process were published 99 .
For redox manipulation, the deuterated potassium phosphate solutions were supplemented with either 6.4 mM potassium permanganate (KMnO 4 ) to achieve the Mn 3+ oxidation state or 300 mM sodium dithionite (Na 2 S 2 O 4 ) to achieve the Mn 2+ state.Crystals were either sealed in capillaries or in 9-well glass plates to ensure the desired oxidation state was maintained.For the Tyr34Phe structure soaked with D 2 O 2 , redox reagents were not used.The dioxygen-bound complex was achieved by supplementing the cryoprotectant that the crystal was immersed in with D 2 O 2 at a nal concentration of 1% w/v (~ 0.28 M) and soaking for 1 min before cryocooling.Flash-cooling was performed with an Oxford diffraction cryostream 100 .Further details of ligand cryotrapping were published 99 .

Crystallographic Data Collection
Time-of-ight, wavelength-resolved neutron Laue diffraction data were collected from perdeuterated crystals using the MaNDi instrument 101,102 at the Oak Ridge National Laboratory Spallation Neutron Source with wavelengths between 2 to 4 Å.Sample sizes ranged from 0.3 to 0.6 mm 3 and data were collected to 2.30 Å resolution for oxidized and D 2 O 2 -soaked structures, while the reduced structure was collected to 2.5 Å resolution (Supplementary Table 5).Crystals were held in stationary positions during diffraction, and successive diffraction frames were collected along rotations of the Φ axis.X-ray diffraction data were collected using a Rigaku FR-E SuperBright home source or the Stanford Synchrotron Radiation Lightsource (SSRL) beamline 14 − 1 (Supplementary Table 5).

Crystallographic Data Processing and Re nement
Neutron data integrated using the MANTID software package [103][104][105] and wavelength-normalized and scaled with LAUENORM from the Daresbury Laue Software Suite 106 .X-ray diffraction data were processed using HKL-3000 107 .Re nements of both neutron and X-ray models were completed separately with PHENIX.REFINE from the PHENIX suite 108 .The re nements were intentionally performed separately due to the known perturbations that X-rays have on the solvent structure, metal redox state, and metal coordination 36,109 .The X-ray model was rst re ned against its corresponding data set and subsequently used as the starting model for neutron re nement.Torsional backbone angle restraints were derived from the X-ray model and applied to neutron re nement using a geometric target function with PHENIX.REFINE 108 .Mn-ligand restraints for neutron re nement were derived from DFT calculations rather than the X-ray model to remove any in uence of photoreduction.The neutron re nement was performed by modeling the D atoms of the active site last to limit phase bias.wavelength of 250 nm with a molar extinction coe cient of 2686 M − 1 cm − 1 79,111 .Superoxide solutions were stored at 4°C under a layer of argon in glass vials sealed with rubber septum.
Mn K-edge HERFD-XANES spectra were recorded at beamline 15 − 2 of the Stanford Synchrotron Radiation Lightsource (SSRL), while Mn K-edge EXAFS spectra were collected at beamline 7 − 3.At both beamlines, data were collected at 10 K using a liquid He cryostat, and the incident energy was tuned to the rst derivative of an internal Mn foil at 6539 eV.X-ray irradiation was carefully monitored so that two subsequent scans of the same spot did not have photoreduction differences, and different spots along samples were scanned.When appropriate, aluminum foil was inserted into the beam path to attenuate the incident ux.For HERFD-XANES measurements, a Johann-type hard X-ray spectrometer with six Ge(333) analyzer crystals was used with a liquid-nitrogen cooled Si(311) double crystal monochromator, and energy was calibrated to a glitch with measurement of Mn Foil.For EXAFS, measurements were recorded with a 30-element Ge solid-state detector, and a Si(220) monochromator at Φ = 90° was used.

X-ray Absorption Spectroscopy Data Analysis
EXAFS data reduction, averaging, and re nement were carried out with the LARCH software package 112 .
Re nement of the k 2 χ(k) EXAFS data used phases and amplitudes obtained from FEFF 113    when the Mn ion changes oxidation states.d Tyr34 is deprotonated in Mn 3+ SOD and protonated in Mn 2+ SOD. e Superposition of wildtype MnSOD (yellow, PDB ID 5VF9) and Tyr34Phe MnSOD (red, PDB ID 9BWR) active sites with a root-mean-square deviation of 0.07 Å among C α atoms.Panel a was created from MnSOD X-ray structure (PDB ID 5VF9) 71 , and panels b-d were created from the neutron structures of Mn 3+ SOD (PDB ID 7KKS) and Mn 2+ SOD (PDB ID 7KKW) 13 .All hydrogen positions were experimentally determined except for solvent molecules in panel a that were randomly generated to accentuate the solvent in the active site funnel.All distances are in Å.        c-d The fast Mn 2+ → Mn 3+ half-reaction corresponding to k 2 .e-f Formation of the inhibited complex that is dependent on the presence of H 2 O 2 during the reduction of the Mn 3+ ion.In the absence of H 2 O 2 , the enzyme proceeds through the fast Mn 3+ → Mn 2+ half-reaction (k 1 ).g-h Relief of the inhibited complex.
Our data describe the inhibited complex as Mn 2+ -containing in contrast to other published mechanistic models [33][34][35]66,67 . In th absence of data that indicate how O 2 •− interacts with an oxidized active site, O 2 •− reacting with the Mn 3+ ion is represented only by Mn 3+ gaining an electron.For a reduced active site, O 2 •−

For D 2 O 2 -
soaked Tyr34Phe MnSOD, we rst analyzed the omit |F o | -|F c | nuclear scattering-length density

Figures
Figures

Figure 1 Structure
Figure 1

Figure 7 Comparison
Figure 7

Table 1
•− with MnSOD to form the inhibited complex67,68.Importantly, this would provide an avenue to structurally analyze product inhibition without relying on volatile O 2 Individual steady-state rate constants of MnSOD.

Table 2
Comparison of superoxide and peroxide-soaked Tyr34Phe MnSOD bond distances found through EXAFS tting, neutron crystallography, and DFT calculations.
the active site (e.g., hydroxyl group of serine/tyrosine) were manually inspected for obvious positive omit |F o | -|F c | neutron scattering length density at a contour of 2.5σ or greater and modeled as a fully occupied deuterium.If the density was not obvious, and there was no chemically sensible reason for the residue to be deprotonated (which is the case for residues outside the active site), the proton position was H/D occupancy re ned.D 2 O molecules outside the active site were then modeled and adjusted according to the nuclear density.Last, D atoms of the active site were modeled manually.At the active site, a residue is considered deprotonated when (1) attempts to model and re ne a proton result in negative |F o | -|F c | difference neutron scattering length density, (2) all the other protons of the residue can be placed, and (3) the heavy atom that is deprotonated acts as a hydrogen-bond acceptor.As chemically ideal covalent bond distances of D atoms were ensured during model building and re nement, small deviations from D atom positions and omit |F o | -|F c | neutron scattering length density centers were expected from the data resolution (2.3-2.5 Å).
79,tia110ounds of re nement to t protein structure included only non-exchangeable D atoms, which have stereochemical predictable positions.Afterward, H/D atoms were modeled onto the position of each amide proton, and occupancy was re ned.In general, the asymmetric units of the neutron crystal structures had a deuterium content of ~ 85% for the amide backbone, and areas with low deuterium exchange (< 50%) coincided with the presence of hydrogen bonds forming a secondary structure.Next, exchangeable proton positions of residues outside 2+ SOD resting state, and either 20 mM superoxide or 280 mM (1% w/v) hydrogen peroxide to isolate the product-inhibited state.Superoxide stock solutions were generated by mixing 1.3 M potassium superoxide (KO 2 , Sigma Aldridge) in a mixture of dry DMSO (Thermo Scienti c) and 0.30 M di-benzo-18crown-6-ether (Thermo Scienti c) following published protocols 66,67,79,80,110.The concentration of superoxide in the DMSO/18-crown solution was measured by a UV-Vis spectrophotometer at a . For each t, Laboratory, is supported by the US Department of Energy (DOE), O ce of Science, O ce of Basic Energy Sciences under Contract DE-AC02-76SF00515.The SSRL Structural Molecular Biology Program is supported by the DOE O ce of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (P30GM133894).The contents of this publication are solely the responsibility of the authors and do not necessarily represent the o cial views of NIGMS or NIH.Quantum chemical computations were completed using the Holland Computing Center of the University of Nebraska, which receives support from the Nebraska Research Initiative.