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From Alkane to Alkene: The Inert Aliphatic C-H Bond Activation Presented by Binuclear Iron Stearoyl-CoA Desaturase with a Long di-Fe Distance of 6 Å Ming-Jia Yu, and Shi-Lu Chen ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00456 • Publication Date (Web): 04 Apr 2019 Downloaded from http://pubs.acs.org on April 4, 2019

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From Alkane to Alkene: The Inert Aliphatic C−H Bond Activation Presented by Binuclear Iron Stearoyl-CoA Desaturase with a Long diFe Distance of 6 Å Ming-Jia Yu and Shi-Lu Chen* Key Laboratory of Cluster Science of Ministry of Education, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing 100081, People’s Republic of China ABSTRACT: The desaturation of inert aliphatic C–H bonds from alkane to alkene, the initial step in biological fatty acid metabolism, can be catalyzed by a non-heme di-iron (NHFe2) stearoyl-CoA desaturase (SCD1) with the longest di-Fe distance of > 6 Å known in NHFe2 enzymes. The SCD1 di-iron core is devised by nine histidines with penta- (FeA) and tetra-coordinations (FeB) mixed. Utilizing density functional theory calculations, we demonstrate that SCD1 employs a mechanism unknown previously in other NHFe2 enzymes, which involves the binding of O2/water leading to FeA(II)-●OH + FeB(II)-●OOH, the addition of H+/e forming FeA(II)-water, the O-O bond dissociation assisted by a hydrogen transfer from FeA(II)-water to FeB-bound oxygen to form a unique triple-hydroxyl intermediate of FeA(II)-●OH + FeB(II)-(●OH)2, a hydrogen transfer inside FeB(II)-(●OH)2 resulting in a FeA(II)-●OH and a high-valent FeB(IV)=O, the respective abstraction of the C9- and C10-hydrogens of substrates by FeB(IV)=O and FeA(II)-●OH producing alkene, and the regeneration of FeB(II)-●OH to FeB(II)-(OH2)2 with another H+/e added. The remote di-iron with penta- and tetra-coordinations mixed cooperates closely and achieves a good reactivity balance between O-O dissociation and hydrogen abstraction. The activity order of various Fe-containing species in the aliphatic C-H bond activation was obtained. Other important mechanistic characteristics and chemistry were revealed also. Our investigation lays a foundation for the design of lowcost and easily-synthesized biomimetic catalysts for the aliphatic C−H bond activation, such as homogeneous di-Fe(II) complexes with pure N-containing ligands and heterogeneous porous Fe(II)-N metal-organic frameworks.

KEYWORDS: Stearoyl-CoA desaturase • di-iron center • density functional calculations • O-O bond dissociation • C-H bond activation

1. INTRODUCTION C-H bond activation/functionalization is one of the most attractive topics in chemistry1, and is often achieved in the C-H bonds adjacent to nitrogen, oxygen, benzyl, and allyl groups. The direct activation of unreactive aliphatic C−H bond is rare and highly challenging. It has been disclosed that stearoyl-CoA desaturase (SCD1), a non-heme di-iron enzyme (NHFe2 enzyme), can catalyze the insertion of one double bond into the alkyl chains of stearic acids to form mono-unsaturated fatty acids (MUFAs), like oleate and palmitoleate (Figure 1).2-4 Such kind of desaturation of inert aliphatic C–H bonds from alkane to alkene usually serves as the initial step of fatty acid metabolism in life, which is of biological and chemical significance and generally divided into catabolism generating energy and anabolism creating biologically important molecules5 such as triglycerides, phospholipids,6 second messengers, local hormones, and ketone bodies. Therefore, it is highly significant to scrutinize the mechanism of SCD1, as it will advance the understanding of fatty acid metabolism in life and fundamental chemistry in aliphatic C-H bond functionalization, facilitating the discovery of new methods of C-H bond activation using non-precious metal catalysts.

Figure 1. The desaturation of fatty acids catalyzed by stearoylCoA desaturase (SCD1). It is proposed in this work that a water molecule should be included in the SCD1 reaction as co-reactant.

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Figure 2. Overall view of SCD1 and close-up view of its active site. Coordinates from PDB code 4YMK8 were used to generate the pictures. In the right picture, the atom colors of C, N, Fe, and substrate, are shown in yellow, blue, red, and grey, respectively. The FeA ion is penta-coordinated with five histidines, while FeB is tetra-coordinated with four histidines.

Scheme 1. Proposed mechanism (mechanism A-α) for the SCD1catalyzed desaturation of stearoyl acids. This mechanism is the green pathway in Figure 5 shown later.

With a dioxygen (O2), two protons (H+), and two electrons (e-) included, SCD1 is capable of catalyzing the biosynthesis

of a double bond between the ninth (C9) and tenth (C10) carbons in the long alkyl chains of stearoyl and palmitoyl acids conjugated by coenzyme A (CoA) (Figure 1).2-4 The required electrons are derived from a NADPH coenzyme (the reduced form of nicotinamide adenine dinucleotide phosphate) and transferred via flavocytochrome b2 and cytochrome b5 and finally to the di-iron center in SCD1.7 The crystallographic analysis shows that SCD1 consists of four transmembrane domains with both NH2 and COOH terminals oriented toward the cytosol (Figure 2).8 The long, thin, and hydrophobic substrate binding site is able to kink the substrate chain to force the C9 and C10 carbons upon the di-iron catalytic center, providing the structural basis for the regioselectivity and stereospecificity of the SCD1 desaturation reaction.3 The di-iron center is bound by a unique nine histidine regions with four and five histidines coordinating to the two irons respectively (Figure 2), separating the two iron by ~ 6.4 Å.8 This is quite different from another binuclear iron desaturase (Δ9 desaturase) where the two irons are bridged by a glutamate and located in a much closer distance of 4.2 Å (PDB code: 1AFR).9 Such a di-Fe distance in SCD1 is also much longer than those in other binuclear iron enzymes, such as monooxygenase hydroxylase (3.4 Å in 1MMO10 and 3.0 Å 1FZ711) and ribonucleotide reductase (3.3 Å in 1XIK12 and 3.9 Å in 1KGN).13 The catalytic mechanism of Δ9 desaturase has been previously proposed to mainly involve a peroxy-bridging diferric intermediate (P), a hydroperoxy-bridging diferric intermediate (prot-2), and the subsequent H-atom abstraction from the substrate C10 site by the prot-2 species.14 Considering that the two irons in SCD1 are separated in a much longer distance than in Δ9 desaturase and even in any previously solved soluble diiron enzymes,15,16 it is interesting and critical to inspect whether SCD1 employs a different mechanism from Δ9 desaturase, which may act as a perfect example to explore the chemical nature how the di-metal distance in binuclear metal enzymes affects the reaction mechanisms. In this work, using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP,17,18 we have investigated the reaction mechanism of binuclear iron SCD1 on the basis of a chemical model constructed from an X-ray crystal structure (PDB code: 4YMK).8 We here present the energetics for the SCD1-catalyzed desaturation of the saturated fatty acid (stearic acid) and provide the characterization of the stationary points involved. The calculations indicate that the SCD1 reaction proceeds through a mechanism (i.e., mechanism A summarized in Scheme 1) completely different from the one in Δ9

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desaturase, where the two irons separated by a long distance cooperate closely. It is found that only the FeB ion having a tetra-coordination at the beginning is able to bear the O-O bond dissociation and only Fe-oxyl (Fe(IV)=O/Fe(III)-O●) is the active species to activate the C-H bond of stearic acids. Other various species have been shown to be inactive in the Habstraction, including Fe(II)-●OH/Fe(III)-OH- hydroxyls, Fe(III)-O-O● radicals, and Fe(II)-●OOH/Fe(III)-O-OH- peroxyls. The order of their activities in abstracting the hydrogen of a saturated C-H bond has been established. The binuclear ferrous core with pure-histidine penta- and tetra-coordinations mixed is further demonstrated to be an ingenious architecture that achieves a good reactivity balance between the O-O bond dissociation and the hydrogen abstraction, providing a perfect prototype for the low-cost and easily-synthesized biomimetic catalysts for the aliphatic C−H bond activation (e.g. the chemical conversion from alkane to alkene).

2. COMPUTATIONAL METHODS The calculations were performed using unrestricted density functional theory (UDFT) with the hybrid functional B3LYP14,15 as implemented in the Jaguar 8.8 package19. Geometry optimizations were carried out with the LACVP basis set20 for the Fe atom, the 6-31G(d,p)+ for the alkyl substrate, the 6-31G(d,p) for the functional groups of residues, and the 631G for the alkyl chains of residues. The diffuse functionincluded 6-31G(d,p)+ was also used for the O2, water, and protons participating the reaction. Based on the optimized geometries, more accurate energies were obtained by performing single-point calculations with larger basis sets, i.e., LACV3P+ for Fe and 6-311G-3df-3pd+ for other elements. To estimate the effects of the protein environment on the calculated energies, solvation effects were calculated at the same theory level as the optimizations by performing single-point calculations on the optimized structures using the self-consistent reaction field (SCRF) method with a Poisson-Boltzmann solver21,22 and a dielectric constant (ε) of 4 which is a standard value that has been used in many previous studies23,24. Using the Gaussian 09 program package25, frequency calculations were performed at the same theory level as the optimizations to obtain zero-point energies (ZPE) and to confirm the nature of the stationary points. Dispersion effects were considered using the empirical formula by Grimme et al. (i.e., DFTD3)226,27. It is worth stressing that dispersion corrections were included in both geometry optimizations and energy evaluation in the present work. Unless otherwise indicated, the energies reported here have been corrected for solvation, zeropoint vibrational, and dispersion effects. The present procedure has been carefully benchmarked24,28,29 and successfully applied to a large number of enzymes by different research groups,23,30,31 including some binuclear iron enzymes32,33.

ies.13,14To reduce computational consumption and conformational uncertainty, the stearoyl-CoA substrate was cut by keeping extra two carbons adjacent to the pivotal C9 and C10 carbons, i.e., finally represented by a hexane. The main chains of residues were truncated but retaining the full side chains. To preserve the spatial arrangement of the residues and substrate, the atoms where the truncations have been done were fixed to their X-ray crystal positions. The atom number of the model with a hexane included is 157. Without O2/H2O included, this active-site model has been optimized in diverse electronic states (see Table S1 in the Supporting Information, SI), where the broken-symmetry (BS) approach and Noodleman correction were applied.35-39 It is found that the di-iron core with high-spin ferrous ions is the ground state and has almost the same energies between ferromagnetic and antiferromagnetic couplings, actually indicating little coupling between the two long-distance irons in SCD1. Moreover, the Noodleman correction was observed to have little effects on the energetics, which is also found in the following calculations of enzymesubstrate complexes and reaction pathways.

3.2. Enzyme-Substrate Complex. We first considered whether the reaction mechanism used by Δ9 desaturase is available in the case of SCD1. To check this, we included an O2 molecule in the present model of SCD1 to optimize the peroxy-bridging diferric intermediate, the key intermediate P involved in the mechanism of Δ9 desaturase14. The optimization of the hydroperoxy-bridging diferric intermediate (intermediate prot-2 in Δ9 desaturase)14 was also tried. However, all attempts failed and always led to minima with O2/OOH terminally coordinating to either of irons. This may be attributed to the significant elongation of di-iron distance in SCD1 compared with Δ9 desaturase and excitingly implies that SCD1 must employ a different mechanism from Δ9 desaturase. Considering the long di-iron distance of ~ 6.4 Å in SCD1 and the potential cooperation of the two irons during the catalysis, another molecule (water most likely) besides O2 is supposed to be included in the initial enzyme-substrate complex of SCD1. Therefore, a water molecule along with an O2 were added in the model to optimize the enzyme-substrate complex. Because the two irons are not identical with respect to coordination number, we obtained two kinds of enzyme-substrate complexes with different and approximately symmetrical binding forms (see Reactα and Reactβ in Figure 3). One has water binding to penta-coordinated FeA and O2 to tetracoordinated FeB (referred to as Reactα), while another has opposite bindings (i.e., Reactβ). Both Reactβ and Reactα have been optimized in various electronic states (see Tables S2 and S3 in the SI).

3. RESULTS AND DISCUSSION 3.1. Chemical Model. A chemical model of the SCD1 active site (see Figure 3) was built on the basis of the X-ray crystal structure of SCD1 with a stearoyl-CoA substrate bound (PDB code: 4YMK).8 It contains two iron atoms, nine first-shell histidine ligands (His297, His121, His116, His153, His157, His156, His265, His294, His298), and a stearoyl-CoA substrate. The two irons in this model are set to be divalent, since the initial state of di-iron in Δ9 desaturase has been well established to be divalent by experimental24,34 and theoretical stud-

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Figure 3. Optimized structure of the SCD1 active site with a water, a dioxygen, and a hexane (the model of stearoyl-CoA substrate) bound. The complex with water and dioxygen coordinating to FeA and FeB respectively is referred to as Reactα, while the one having the opposite bindings is named by Reactβ. To show Reactα more clearly, a schematic 2D and a simplified 3D structures are given. In the simplified Reactα, all histidines are simplified to nitrogen atoms and the terminal methyls of hexane are omitted, which is also applied to Reactβ in this figure and Figures 4 and 6 below. Asterisks indicate the atoms which are fixed to their X-ray positions. All distances are in angstrom (Å). The unpaired spin populations are indicated by "S".

Figure 4. Optimized structure of transition states (TS1β and TS1α) and intermediates (Int1β and Int1α) for hydrogen transfer from Fe(II)water to Fe(III)-O-O● radical.

The ground state of Reactβ was found to be in the openshell singlet state with the two high-spin irons antiferromagnetically coupling (see entry 1 in Table S2). In this singletstate Reactβ, the unpaired spin populations at FeA and FeB are

4.04 and -3.75 with a few spins delocalized at the first-shell ligands (see Figure 3 and entry 1 in Table S2). Interestingly, the O2 molecule coordinating to FeA is calculated to have spins of -0.11 and -0.44 at the oxygen binding to FeA (named by OA)

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and the terminal oxygen (OT) respectively, indicating that, with the binding of O2 to FeA, an electron has been transferred from the FeA(II) ion to O2 and the nature of the FeA site of Reactβ is a FeA(III)-O-O● radical species. In this state of Reactβ, the FeB ion retains divalent. This is consistent with a shorter Fe-O distance at the FeA site (FeA-OA = 2.01 Å) than that at FeB (FeB-OB = 2.16 Å) (Figure 3). Although the other two states consisting of high-spin irons (entries 2 and 3 in Table S2) have quite close energies compared with the singletstate Reactβ (entry 1 in Table S2), their long FeA-OA distances and almost equivalent high spins at the OA and OT atoms point out that the O2 moiety is still an inactivated triplet-state dioxygen. Therefore, the singlet-state Reactβ discussed here (shown in Figure 3 and entry 1 in Table S2) was used as the initial enzyme-substrate complex for the following calculations of reaction mechanisms (if not otherwise indicated, the name of Reactβ hereinafter means the ground state discussed here). In addition, it was found that although the singlet-state Reactα is not the ground state (see entry 1 in Table S3), it is easily reachable and lies only 5.2 kcal/mol higher than the ground state of Reactα with the highest multiplicity (entry 2 in Table S3). Considering that the O2 moiety in the latter is also almost inactivated as a triplet-state dioxygen (the spins of the two oxygens are 0.73 and 0.65, entry 2 in Table S3), the former (the singlet-state Reactα) was used in the following calculations (if not otherwise indicated, the name of Reactα hereinafter means the singlet state discussed here). It should be noted that the FeB site of Reactα is also shown to be a FeB(III)-O-O● radical species (see Figure 3 and entry 1 in Table S3). The diiron distances in Reactβ and Reactα are 6.43 and 6.61 Å respectively, which are quite close to the corresponding crystallographic distance of ~ 6.4 Å.8 Due to the approximately symmetrical bindings of O2/H2O in Reactα and Reactβ, the reaction pathways starting from the Reactα state may have the mirror pathways happening from Reactβ. Considering that the two irons differ in coordination number, they may render the different favorability of a pathway initiating from Reactα or Reactβ. Therefore, in this work various reaction pathways starting from Reactα and their mirror pathways from Reactβ are both taken into account. The singlet-state Reactβ (entry 1 in Table S2) is computed to be the ground state among all examined states of Reactα and Reactβ, and thus chosen as the energy reference utilized in the subsequent calculations of reaction mechanisms. The result that Reactβ, a complex with a water bound to FeB, has a lower energy than Reactα is consistent with the crystal structure where a crystal water is bound to FeB.8

3.3. Hydrogen Transfer from Fe(II)-water to Fe(III)-OO● Radical. From the ground-state enzyme-substrate complex of Reactβ, a transition state for a hydrogen transfer from FeB(II)-water to FeA(III)-O-O● radical (TS1β, Figure 4) has been optimized and confirmed to be a first-order saddle point with an imaginary frequency of 1893i cm−1. The key distances of the transferred hydrogen (HW) to the FeB-bound oxygen (OB) and the terminal oxygen of FeA(III)-O-O● (OT) are 1.19 and 1.21 Å, respectively. This hydrogen transfer leads to an intermediate involving FeB-OH and FeA-O-OH species (Int1β, Figure 4). In Int1β, the nonnegligible spin densities at the OH (OB = -0.35) and OOH moieties (OA = 0.49 and OT = 0.14), as well as the increasing and decreasing of spins at FeB (-4.07) and FeA (-3.97) respectively relative to Reactβ, indicate that this is a hydrogen-atom transfer process. The resultant species are a

FeB(II)-●OH hydroxyl radical (most likely a resonance state between FeB(II)-●OH and FeB(III)-OH- hydroxide) and a FeA(II)-●OOH peroxyl radical (most likely a resonance state between FeA(II)-●OOH and FeA(III)-OOH- peroxide). The energy barrier for this step is quite low (5.5 kcal/mol), while Int1β has an energy (-0.9 kcal/mol) very close to Reactβ (see Figure 5). This implies that the conversion between Reactβ and Int1β, i.e. the hydrogen transfer between FeB(II)-water and FeA(III)-O-O●, is rapid and reversible. From another enzyme-substrate complex of Reactα, we also obtained an intermediate resulted from the hydrogen-atom transfer from FeA(II)-water to FeB(III)-O-O● radical, which includes a FeA(II)-●OH hydroxyl radical and a FeB(II)-●OOH peroxyl radical (Int1α, Figure 4). Different from the case of Reactβ, this hydrogen transfer starting from Reactα shows a big exothermicity of 12.4 kcal/mol (6.6 kcal/mol compared with Reactβ) (Figure 5). However, we failed in the location of the transition state for this hydrogen transfer, which always led to Reactα or Int1α. This may be attributed to the nature of very early transition state derived from the relatively large exothermicity. It is conceivable that this missing transition state may have close geometry and energy to be compared to Reactα, implying a low barrier for this hydrogen transfer, that is, the hydrogen transfer from Reactα to Int1α is also fast. Considering that Reactα lies only 5.8 kcal/mol higher than Reactβ (Figure 5), the interconversion among the four minima (Reactβ, Int1β, Reactα, and Int1α) is facile but with Int1α being the favorite state (i.e., FeA(II)-●OH + FeB(II)-●OOH). Such a preference for Int1α rather than Int1β (FeB(II)-●OH + FeA(II)● OOH) reveals a bigger hydrogen affinity of FeB(III)-O-O● than FeA(III)-O-O●, a feature that implies that the tetracoordinated FeB site may has a stronger reactivity than the penta-coordinated FeA, which will be investigated in more detail later.

3.4. Mechanism A: Adding One Pair of H+/e to Int1 (Fe(II)-●OH + Fe(II)-●OOH). 3.4.1. Addition of One Pair of H+/e. From the Int1α and Int1β states (i.e., Fe(II)-●OH + Fe(II)-●OOH), diverse mechanisms can be initiated by introducing one pair of proton and electron (H+/e), two pairs of H+/e, or none of H+/e. We first considered the mechanism with one pair of H+/e added to the Int1 states (referred to as mechanism A hereinafter). The pair of electron and proton are originated from a NADPH coenzyme and/or the solution (Figure 1) and supposed to finally end in the Fe(II)-●OH/Fe(III)-OH site of Int1, converting the Fe(II)-●OH to Fe(II)-water (named by Int2A). Because a NADPH is able to provide two electrons, this step, with Int1α as the example, is formulated as 1/2 NADPH + 1/2 H+ + FeA(II)-●OH/FeA(III)-OH- (Int1α) → 1/2 NADP+ + FeA(II)-OH2 (Int2A-α)

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Figure 5. Schematic 3D (upper) and 2D (lower) potential energy profiles for mechanism A of the SCD1 reaction. The 2D profile is obtained by spreading every face of the 3D.

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Figure 6. Optimized structures of stationary points in the pathway α of mechanism A (the green curve in Figure 5).

The Int2A-α (Figure 6) has been optimized to have the spins of -3.79 and 0.00 at the FeA and OA atoms respectively with the FeA-OA bond elongated to 2.28 Å from 1.95 Å at Int1α, showing that its FeA site is a FeA(II)-water species. Its FeB site is maintained to be a FeB(II)-●OOH/FeB(III)-OOH- resonance state with the spins of FeB and OB being 4.07 and 0.30 respectively. The redox potential for the oxidation of NADPH to NADP+ has been determined to be 0.324 V at pH = 7,40 i.e., NADPH + H+ → NADP+ + 2e- + 2H+ E = 0.324 V

a value that is corresponding to a free energy change of ∆GNADPH = -14.9 kcal/mol. Coupled with the experimental solvation energy of one proton (-264.0 kcal/mol)41, the formation of Int2A-α from Int1α is estimated to be thermodynamically feasible with a rough exothermicity of around 123.9 kcal/mol (i.e., 130.5 kcal/mol with Reactβ as the energy reference) (see the green curve in Figure 5). When Int1β serves as the H+/e accepter, the addition of one pair of H+/e is also estimated to be thermodynamically achievable with a large exothermicity of 131.4 kcal/mol (132.3 kcal/mol compared with

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Reactβ) (see the red curve in Figure 5), where the resultant intermediate (Int2A-β, given in Figure S1 in the SI) is composed of a FeB(II)-water and a FeA(II)-●OOH/FeA(III)-OOHresonance state.

3.4.2. O-O Bond Dissociation. O-O bond dissociation is usually a crucial step and receiving particular attention in the O2-consumption chemistry. From Int2A-α (i.e., FeA(II)-OH2 + FeB(II)-●OOH/FeB(III)-OOH-), a transition state for the OB-OT bond cleavage (TS2A-α, Figure 6) has been located with an OBOT distance of 1.85 Å. It is calculated to have an imaginary frequency of 648i cm-1, which turns out to correspond to a vibrational mode that, simultaneously with the O-O bond dissociation, one of hydrogens in FeA(II)-OH2 is transferred to the OB atom. This step leads to an interesting and unique tripleOH intermediate (Int3A-α, Figure 6) including three OH species with one located at the FeA site and the other two at FeB, making both of irons being hexa-coordinated. In Int3A-α, the spins at the FeA (-4.13), FeB (4.09), OA (-0.28), OB (0.78), and OT (0.67) point out that the hydrogen transfer from FeA(II)OH2 to OB involved in this step is a hydrogen-atom transfer and three hydroxyl radicals are formed, i.e., FeA(III)-OH/FeA(II)-●OH + FeB(II)-(●OH)2. The barrier for this step is predicted to be reachable (13.4 kcal/mol) with an exothermicity of 13.9 kcal/mol (Figure 5). The two irons cooperate closely in this O-O bond dissociation step from Int2A-α, although they are most separated in a distance longer than 6 Å during the whole catalysis (Figure 7). The FeB, being tetra-coordinated at the beginning, plays the main catalytic power by inducing the peroxyl O-O bond cleavage, which can be reflected by the significant shortening of the FeB-OT distance from 2.27 Å at Int2A-α to 2.00 Å at Int3A-α (2.10 Å via TS2A-α). The FeA, penta-coordinated originally, assists in the O-O bond dissociation by activating the water and providing a hydrogen atom. We could not obtain a transition state of O-O cleavage without the hydrogen transfer from FeA(II)-OH2 to OB, or an intermediate with an oxyl and a hydroxyl located at FeB together, demonstrating that, without the collaboration of FeA(II)-OH2 (providing a hydrogen atom), the single tetra-coordinated FeB ion could not achieve the peroxyl O-O bond cleavage. It can further be speculated that the O-O bond dissociation at the penta-coordinated FeA site, initiating from the Int2A-β state (i.e., FeA(II)-●OOH/FeA(III)-OOH- + FeB(II)-OH2), may not follow the same pattern as the case of FeB since the FeA could not offer an extra binding position for the leaving hydroxyl. As expected, from Int2A-β, we obtained another kind of transition state (TS2A-β, Figure S1) where, accompanying with the OA-OT bond cleavage, one of hydrogens in FeB(II)-OH2 is transferred to the terminal OT atom instead of the OA ligating to FeA, resulting in a FeA(IV)=O, a FeB(II)-●OH/FeB(III)-OH-, and a free water (see the Int3A-β intermediate in Figure S1). However, its barrier is estimated to be somewhat high (23.8 kcal/mol, see the red curve in Figure 5), showing that the O-O bond dissociation at the penta-coordinated FeA site is likely difficult or at least slow. This, from another perspective, reveals that the O-O bond dissociation happening at the tetracoordinated FeB site (from Int2A-α) is a favorable pathway in SCD1, rendering a stronger reactivity of tetra-coordinated iron than penta-coordinated and the evolution necessity of a tetracoordinated iron in the SCD1 active site. It is worth noting that the β-pathway with one pair of H+/e added to Int1β (the red curve in Figure 5) could not be entirely ruled out. In such case, the TS2A-β will be the highest obstacle with a barrier of 23.8

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kcal/mol. To explain this, it may be speculated that the protoncoupled electron transfer (the addition of H+/e) may not be fast. Coupled with the facile interconversion among the four minima (Reactβ, Int1β, Reactα, and Int1α) and the lowest energy of Int1α discussed in Section 3.3, Int1α may have the highest concentration among the four minima, causing most of SCD1 reactions proceed through the α-pathway (the green curve in Figure 5). It could not be excluded that minor SCD1 reactions may take place via the β-pathway at a very low reaction rate but probably not result in the complete inactivation of enzyme. A slow proton/electron transfer is not rare in enzymatic reactions, which, for example, was also suggested in the oxidative C-N bond dissociation of dimethylamine to methylamine and formaldehyde catalyzed by a heme-dependent Ndemethylase.42 React

6.8 6.6

Prod

6.4

Int1α

6.2 6 5.8

5.6

Int6A-α

5.4

Int5A-α

Int2A-α

Int3A-α

Int4A-α Figure 7. The di-Fe distances of all minima along mechanism A-α of the SCD1 reaction (the green pathway in Figure 5). All distances are in angstrom (Å).

3.4.3. Formation of High-valent Fe(IV)=O. From the triple-hydroxyl intermediate (Int3A-α, i.e., FeA(III)-OH-/FeA(II)● OH + FeB(II)-(●OH)2), a hydrogen-atom transfer from OT to OB between the two FeB-bound hydroxyls (i.e., FeB(II)-(●OH)2) was found to occur (see the TS3A-α transition state in Figure 6) with a barrier of 7.8 kcal/mol (Figure 5). It leads to a FeAhydroxyl and a water-co-bound FeB-oxyl species (see the Int4A-α intermediate in Figure 6) which is confirmed to be a high-valent non-heme FeB(IV)=O by the spins at the FeA (4.14), FeB (-3.03), OA (0.28), OB (-0.02), and OT (-0.76) atoms. In this case, the ferryl and oxyl species (FeB-oxyl) is in a resonance state between FeB(IV)=O and FeB(III)-O●. With the water excluded from the FeB site of Int4A-α, the intermediate of FeA(II)-●OH + FeB(IV)=O (Int4-dhA-α, Figure S2) has been reoptimized and estimated to have an energy 6.8 kcal/mol higher than Int4A-α (Figures 5 and S3). Such FeB(IV)=O species with the water removed (Int4-dhA-α) shows a higher overall barrier for abstracting the substrate hydrogen than the one with water (Int4A-α), which will be discussed in more detail in Section 3.4.4. From the triple-hydroxyl intermediate of Int3A-α, another pathway via a hydrogen-atom transfer from FeA(III)-OH- to one of hydroxyls at FeB (i.e., the OB atom) was found to be feasible (see the TS3A-αβ transition state in Figure S4) with a little barrier of 0.9 kcal/mol (Figure 5). It results in a complex of FeA(IV)=O and water-co-bound FeB(II)-●OH/FeB(III)-OHhydroxyl (see Int4A-β in Figure S4) with the spins of -3.05,

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4.10, -0.65, 0.01, and 0.43 at the FeA, FeB, OA, OB, and OT atoms. In this case, the ferryl and oxyl species (FeA-oxyl) is in a resonance state between FeA(IV)=O and FeA(III)-O●. The exclusion of the water from the FeB site of Int4A-β, leading to an intermediate of FeA(IV)=O + penta-coordinated FeB(II)-●OH (Int4-dhA-β, Figure S5), has been estimated to be endothermic by 4.1 kcal/mol (Figure S3). The subsequent Habstraction by Int4-dhA-β is also shown to have a higher overall barrier than the one with water bound (Int4A-β), which will be discussed in the next section. Interestingly, it was found that the FeA(IV)=O and FeB(II)-●OH species in Int4-dhA-β are not interconvertible via a transition state of hydrogen-atom transfer between them (TS4-dhA-αβ, given in Figure S2). Accumulated from Int4A-β (taking into account the energy of the water exclusion), the barrier for the H-transfer from FeB(II)● OH to FeA(IV)=O, leading to Int4-dhA-α (FeA(II)-●OH + FeB(IV)=O), is 20.5 kcal/mol (Figure S3). This means that the Int4-dhA-β and Int4-dhA-α intermediates could hardly transform into each other, and that the Int4-dhA-α intermediate should be formed by the direct dehydration from Int4A-α described above.

3.4.4. Hydrogen abstraction of hexane (stearic acids). It is conceivable that the resulting high-valent FeB(IV)=O/FeB(III)-O● in Int4A-α or FeA(IV)=O/FeA(III)-O● in Int4A-β may be the active species to abstract the hydrogen of hexane (a substrate model of stearic acids). From Int4A-α (FeB(IV)=O + FeA(III)-OH-), a transition state for the hydrogen abstraction from the substrate C9 site by FeB(IV)=O (TS4A-α, Figure 6) has been optimized and calculated to have an imaginary frequency of 708i cm−1. In TS4A-α, the distances of the transferred hydrogen (H9) to the OB and C9 are 1.42 and 1.23 Å respectively. This is a hydrogen-atom transfer leading to a complex composed of a C9 radical, a FeB(III)-OH-/FeB(II)-●OH hydroxyl, as well as a FeA(III)-OH-/FeA(II)-●OH (Int5A-α, Figure 6). The barrier for this step is predicted to be accessible (10.0 kcal/mol, Figure 5), a value that is slightly lower than the one (13.4 kcal/mol) for the O-O bond dissociation resulting in the triple-hydroxyl intermediate discussed earlier (i.e., from Int2A-α to Int3A-α via TS2A-α) (see the green curve in Figure 5). The two barriers are such that it is not safe to say which step is rate-limiting. To probe whether a Fe-OH species can be an active species to abstract the hydrogen of hexane, we took Int4-dhA-β as an example to obtain a transition state for the C9-hydrogen abstraction by FeB(III)-OH-/FeB(II)-●OH (TS5b-dhA-β, displayed in Figure S6) and the following complex of a C9 radical + FeB(II)-water (Int5b-dhA-β, Figure S6). However, its barrier (25.5 kcal/mol) is much higher than the C9-hydrogen abstraction by FeB(IV)=O (10.0 kcal/mol). Other attempts to use various Fe-OH species to abstract the substrate hydrogen also lead to the barriers at least larger than 26 kcal/mol (discussed in Sections 3.5 and 3.6 below). This indicates that it is the high-valent Fe(IV)=O that acts as the initial active species to abstract the substrate hydrogen, instead of the Fe-OH hydroxyl. The resultant C9 radical in Int5A-α must be unstable and ready to donate its adjacent C10-hydrogen to the FeA(III)-OH/FeA(II)-●OH hydroxyl. This kind of transition state (TS5A-α, Figure 6) has been located and found to be very early with the key C10-H and H-O distances being 1.21 and 1.67 Å respectively. This step results in a C9=C10 alkene product (i.e., palmic acids), a FeA(II)-water, and a water-co-bound FeB(III)-OH/FeB(II)-●OH hydroxyl (Int6A-α, Figure 6). The barrier is predicted to be very low (-2.3 kcal/mol) with a quite large exo-

thermicity of 35.1 kcal/mol, which is consistent with the character of an early transition state. A negative barrier is an error derived from the incorporation of the corrections obtained with a medium-size basis set into the energies calculated with a larger basis set. This type of error was often found in a hydrogen transfer reaction with a low barrier28, and should not alter the conclusion about the mechanism that the hydrogen transfer from Int5A-α to Int6A-α is fast. Finally, the FeB(III)OH-/FeB(II)-●OH hydroxyl can be regenerated to a FeB(II)water (Prod, Figure 6) by the addition of another pair of H+/e, which is estimated to have an exothermicity of 138.5 kcal/mol (Figure 5) by taking account into the redox potential of NADPH (0.324 V)40 and the experimental solvation energy of one proton (-264.0 kcal/mol).41 The overall reaction exothermicity from the enzyme-substrate complex of Reactβ is estimated to be 326.8 kcal/mol, which is close to the driving force (-311.7 kcal/mol) predicted by the uncatalyzed reaction of hexane + O2 + NADPH + H+ → hexene + 2H2O + NADP+ (Figure S7). Using Int4A-β (FeA(IV)=O/FeA(III)-O● + FeB(II)● OH/FeB(III)-OH-) as the starting state, the hydrogen abstraction of hexane follows the same pattern as the case of Int4A-α, and similar stationary points have been optimized including the transition state for the C10-hydrogen abstraction by FeA(IV)=O (TS4A-β, Figure S4), the resulting intermediate consisting of a C10 radical and a FeA(II)-●OH/FeA(III)-OHhydroxyl (Int5A-β, Figure S4), the transition state for the C9hydrogen abstraction by FeB(II)-●OH (TS6A-β, Figure S4), the following complex of a C9=C10 alkene product + a FeA(II)● OH/FeA(III)-OH- hydroxyl + a FeB(II)-(OH2)2 (Int6A-β, Figure S4). The FeA(II)-●OH/FeA(III)-OH- in Int6A-β is also able to be renewed to FeA(II)-water by the addition of another pair of H+/e from NADPH and solution. The rate-limiting step in this pathway is determined to be the C10-hydrogen abstraction by FeA(IV)=O (i.e., from Int4A-β to Int5A-β via TS4A-β) with a barrier of 11.9 kcal/mol (see the blue curve in Figure 5), which is slightly higher than that (10.0 kcal/mol) for the C9-hydrogen abstraction by FeB(IV)=O via TS4A-α (Figure 5), showing that the latter pathway involving the C9-hydrogen abstraction by FeB(IV)=O via TS4A-α (i.e., pathway α shown by the green curve in Figure 5) is somewhat more favorable. The pathway β displayed by the blue curve in Figure 5 is accessible, too. Using Int4-dhA-α and Int4-dhA-β, i.e., with the water excluded from the FeB site of Int4A-α and Int4A-β respectively, the hydrogen-abstraction by Fe(IV)=O follows the same form as the cases with water bound to FeB (i.e. the pathways α and β described above). The optimized structures starting from Int4dhA-α and Int4-dhA-β are given in Figures S2 and S5 respectively and the energetics are summarized in Figure S3. It is found that, with the water removed from FeB, the abstractions of the substrate hydrogen have higher overall barriers (accumulated with the energies of water exclusion) than the pathways α (10.0 kcal/mol) and β (11.9 kcal/mol) with water bound, i.e., 15.6 and 18.5 kcal/mol for the pathways from Int4A-α to Int5-dhA-α via TS5-dhA-α (the green curve in Figure S3) and from Int4A-β to Int5-dhA-β via TS5-dhA-β (the blue curve in Figure S3), respectively. This implies that the pathways shown in Figure S3 with water excluded are unfavorable. Interestingly, both ground-state and excited-state Fe(IV)=O species, as well as the corresponding transition states for hydrogen abstraction, were found. Their energetics are presented in Figure S3 as the inserted pictures. It should be mentioned that all Fe(IV)=O species appearing in the pathways of Figures

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5 and S3 are in the ground state. Although the hydrogen-abstraction pathways in Figure S3 are unfavorable, their comparison with the pathways α and β in Figure 5 give an interesting result that the penta-coordinated FeB(IV)=O has the lowest hydrogen-abstraction barrier (8.8 kcal/mol from Int4-dhA-α to Int5-dhA-α via TS5-dhA-α, Figure S3) compared with other three hexa-coordinated Fe(IV)=O species, which have the barriers of 10.0 kcal/mol from Int4A-α to Int5A-α via TS4A-α, Figure 5), 11.9 kcal/mol from Int4A-β to Int5A-β via TS4A-β, Figure 5), and 14.4 kcal/mol from Int4dhA-β to Int5-dhA-β via TS5-dhA-β, Figure S3). Since the C9 and C10 carbons are basically identical hydrogen donors, this probably implies that the penta-coordinated high-valent Fe(IV)=O is a stronger hydrogen acceptor than the hexacoordinated Fe(IV)=O. Furthermore, when considering a stronger activity of tetra-coordinated FeB in terms of the O-O bond dissociation discussed above, an exciting finding is emerging, that is, the di-iron core with different penta- and tetra-coordination at the two cations, assembled by the SCD1 enzyme using nine histidines, is an ingenious architecture that achieves a quite good activity balance between the O-O bond dissociation and the hydrogen abstraction from stearic acids. It can be inferred that the construction of double pentacoordination in the di-iron core may decelerate the O-O bond dissociation with the barrier increased to more than 23 kcal/mol, while double tetra-coordination may reserve the catalytic efficiency of the native SCD1 enzyme.

3.5. Mechanism B: Adding Two Pairs of H+/e to Int1 (Fe(II)-●OH + Fe(II)-●OOH). The addition of two pairs of H+/e to the Int1α or Int1β state (i.e., Fe(II)-●OH + Fe(II)● OOH), named by mechanism B, is supposed to result in a Fe(II)-water, a Fe(IV)=O, and a free water. With Int1α as the example, it is summarized as: NADPH + H+ + FeA(II)-●OH + FeB(II)-●OOH (Int1α) → NADP+ + FeA(II)-OH2 + FeB(IV)=O (Int2B-α) + H2O The Int2B-α intermediate composed of a FeA(II)-OH2 and a high-valent penta-coordinated FeB(IV)=O has been optimized and depicted in Figure S8 in the SI. The FeB site is verified to be a high-spin quintet-state Fe(IV)=O/FeB(III)-O● diradicaloid by the very short FeB-OB distance (1.64 Å) and the spins at the FeB (3.04) and OB (0.77) atoms. With the consideration of the NADPH redox potential (0.324 V)40 and the experimental solvation energy of two protons (-528.0 kcal/mol),38 this step of proton-coupled electron transfer is estimated to be largely exothermic by 281.0 kcal/mol (see the blue curve in Figure 8). It is interesting to find that, once Int2B-α is formed, it will immediately transit to a much deeper minimum (Int3B-αβ, Figure S8) including two Fe(II)/Fe(III)-bound hydroxyls/hydroxides (i.e., hexa-coordinated FeA(II)-●OH/FeA(III)-OH- and pentacoordinated FeB(II)-●OH/FeB(III)-OH-) via an H-atom transfer from FeA(II)-OH2 to FeB(IV)=O, almost without barrier (failed in the transition state optimizations which always resulted in Int3B-αβ). Int3B-αβ (the red state in Figure 8) lies 24.2 kcal/mol lower than Int2B-α. The addition of two pairs of H+/e to the Int1β intermediate (FeA(II)-●OOH/FeA(III)-O-OH- + FeB(II)-●OH/FeB(III)-OH-) leads to a complex of hexa-coordinated FeA(IV)=O/FeA(III)O● and FeB(II)-OH2 (Int2B-β, Figure S8) with an exothermicity of 284.6 kcal/mol (the purple curve in Figure 8). The latter is also quickly, almost without barrier, converted to the doublehydroxyl intermediate of Int3B-αβ through an H-transfer transition state from FeB(II)-OH2 to FeA(IV)=O (TS2B-β, Figure S8).

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Int3B-αβ is 26.3 kcal/mol lower than Int2B-β (Figure 8). This, coupled with the remarkable lowering of 24.2 kcal/mol relative to Int2B-α, demonstrates that such a double-hydroxyl intermediate of Int3B-αβ is sitting in a very deep minimum, making it difficult to revert to the Int2B-α or Int2B-β state (i.e., Fe(IV)=O + Fe(II)-OH2). Even if Int3B-αβ could return to Int2B-α or Int2B-β with a low probability, the subsequent Habstraction of steric acids by high-valent FeB(IV)=O in Int2B-α or FeA(IV)=O in Int2B-β should require a sizable barrier, thus deepening the Int3B-αβ potential well. Now that Int3B-αβ, involving two Fe-bound hydroxyls, is a very stable intermediate in mechanism B, it is important to inspect its activity with respect to the hydrogen abstraction from steric acids. From Int3B-αβ, we have obtained two transition states for hydrogen abstraction (TS3B-α and TS3B-β) and their subsequent intermediates (Int4B-α and Int4B-β), which are shown in Figure S8. TS3B-α is a H-transfer transition state from the substrate C9 carbon to the penta-coordinated FeB(II)● OH/FeB(III)-OH- resulting in a C9 radical (Int4B-α), while TS3B-β is a C10-H abstraction transition state by hexacoordinated FeA(II)-●OH/FeA(III)-OH- forming a C10 radical (Int4B-β). However, both barriers are calculated to be very high, i.e., 31.0 and 35.5 kcal/mol for TS3B-α and TS3B-β respectively (Figure 8). This means that the double-hydroxyl intermediate of Int3B-αβ is an inert state that is not the active species to activate the C-H bonds of steric acids. With this, it can be concluded that mechanism B is inaccessible with an ending at the deep minimum of Int3B-αβ (double-Fe-bound-hydroxyl intermediate). Combined with the feasible mechanism A discussed earlier, the speculation mentioned in Section 3.4.2, that the proton-coupled electron transfer (the addition of H+/e) may not be fast, may ensure that only mechanism A, instead of mechanism B, is triggered by the slow addition of every pair of H+/e.

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Figure 8. Schematic 3D (upper) and 2D (lower) potential energy profile for mechanism B of the SCD1 reaction. Each face in schematic 3D has a corresponding face state in 2D.

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Figure 9. Potential energy profile for mechanism C of the SCD1 reaction.

3.6. Mechanism C: Not Adding H+/e to Int1 (Fe(II)● OH + Fe(II)-●OOH). In case that the slow addition of H+/e could not immediately consume the Int1α or Int1β state (i.e., Fe(II)-●OH + Fe(II)-●OOH), it is necessary to examine the reactivities of some potential active species contained by the initial four minima (Reactα, Reactβ, Int1α, and Int1β,), including the FeB(III)-O-O● radical in Reactα, the FeA(III)-O-O● in Reactβ, the FeA(II)-●OH hydroxyl and FeB(II)-●OOH peroxyl in Int1α, and the FeA(II)-●OOH and FeB(II)-●OH in Int1β. Four transition states for the corresponding H-abstraction from the hexane are located (given in Figure S9 in the SI), involving the C9-H abstraction by penta-coordinated FeB(III)-O-O● in Reactα (TS2C-α), the C10-H abstraction by hexa-coordinated FeA(III)-O-O● in Reactβ (TS2C-β), C10-H abstraction by hexacoordinated FeA(II)-●OH in Int1α (TS3C-α), and the C9-H abstraction by penta-coordinated FeB(II)-●OH in Int1β (TS3C-β). All of them are calculated to have high barriers, i.e., 23.4, 20.1, 39.6, and 26.9 kcal/mol for TS2C-α TS2C-β, TS3C-α, and TS3C-β respectively (Figure 9). Although the barrier of 20.1 kcal/mol via TS2C-β seems not to be entirely unreachable, it may be high enough to gain time for the addition of one pair of H+/e, ensuring that the SCD1 reaction proceeds through a more favorable mechanism A shown in Figure 5. We also obtained a transition state (TS4C-β, Figure S9) for the H-abstraction from

C10 to OA in FeA(II)-OOH (Int1β), which has a high barrier of 28.8 kcal/mol (Figure 9). In addition, all attempts to find the H-abstraction transition states by the terminal oxygens of Fe(II)-●OOH peroxyls failed and always led to the Int1α or Int1β state. These results indicate that all states mentioned in this section (various Fe(III)-O-O● radicals, Fe(II)-●OH hydroxyls, and Fe(II)-●OOH peroxyls) are not the active species to abstract substrate hydrogens and that the mechanism C without H+/e added is unreachable. Furthermore, we also considered the mechanistic possibility of a direct O-O cleavage in Int1α. To check this, we have optimized two kind of intermediates resulted from the direct O-O cleavage in Int1α. One is the complex of FeA(IV)=O + FeB(IV)=O + a free water, which is derived from the hydrogen transfer from FeA-bound oxygen (OA) to the terminal oxygen of OOH (OT). The other is composed of FeA(IV)=O and FeB(II)-(●OH)2, which originates from the hydrogen transfer from the OA to the FeB-bound oxygen (OB). These two intermediates are calculated to have the energies 17.9 and 23.8 kcal/mol higher than Int1α respectively, thus showing the unfavorability of the pathway via the direct O-O cleavage in Int1α.

4. CONCLUSIONS In this paper, using an active-site model of about 160 atoms and the DFT method, a full account has been given of the reac-

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tion mechanisms of stearoyl-CoA desaturase (SCD1), a nonheme di-iron enzyme (NHFe2 enzyme) with nine histidines as ligands, where the penta- (FeA) and tetra-coordinated irons (FeB) are not connected covalently. Although DFT is usually considered as the practical method of choice for the NHFe2 systems, this has been the first application of DFT in modeling a NHFe2 enzyme with the longest di-iron distance (> 6 Å). Diverse mechanisms have been inspected here and finally it is found that SCD1 employs a novel mechanism (i.e., mechanism A-α summarized in Scheme 1 and the green curve in Figure 5) that is completely different from the one for Δ9 desaturase (another NHFe2 enzyme capable of desaturating stearic acids), mainly including the binding of O2/water leading to the complex of FeA(II)-●OH/FeA(III)-OH- + FeB(II)● OOH/FeB(III)-OOH-, the addition of one pair of H+/e forming FeA(II)-water, the O-O bond dissociation assisted by a hydrogen transfer from FeA(II)-water to FeB-bound oxygen to form a unique triple-hydroxyl intermediate of FeA(II)-●OH/FeA(III)OH- + FeB(II)-(●OH)2, a hydrogen transfer inside FeB(II)(●OH)2 resulting in a FeA(II)-●OH/FeA(III)-OH- hydroxyl and a high-valent FeB(IV)=O/FeB(III)-O●, the respective and successive abstraction of the C9- and C10-hydrogens of stearic acids by FeB(IV)=O and FeA(II)-●OH to form a alkene product (palmic acids), and the regeneration of FeB(II)-●OH/FeB(III)OH- to FeB(II)-(OH2)2 with another pair of H+/e added. Both O-O bond dissociation and C9-hydrogen abstraction by FeB(IV)=O may contribute to the rate limiting with a barrier of 13.4 kcal/mol for the former. A water molecule is required as a co-reactant in this mechanism. The following important mechanistic characteristics and chemistry can be refined from the calculations: (i) Although the two irons in SCD1 are separated in a long distance (Figure 7), much longer than Δ9 desaturase9 and recently characterized methane monooxygenase,43 and could not construct the key peroxy-bridging (intermediate P) and hydroperoxy-bridging (intermediate prot-2) diferric intermediates involved in the mechanism of Δ9 desaturase,14 they cooperate closely during the catalysis. In particular, without the hydrogen transfer from the hexa-coordinated FeA(II)-water, the O-O bond dissociation in the penta-coordinated FeB(II)-●OOH peroxyl could not be launched to form the unique triplehydroxyl intermediate of FeA(II)-●OH + FeB(II)-(●OH)2, a crucial precursor for the active Fe(IV)=O in the present mechanism of SCD1. Such kind of O-O bond dissociation is currently rare. (ii) Only the FeB ion having a tetra-coordination at the beginning is able to bear the O-O bond dissociation. The FeA having one more histidine ligand would trigger the O-O bond cleavage through a different kind of pathway which has a much higher barrier. (iii) Only Fe-oxyl (Fe(IV)=O/FeA(III)-O●) is the active species to activate the C-H bond of stearic acids (i.e., hydrogen abstraction). Other various species have been demonstrated to be inactive, including Fe(II)-●OH/Fe(III)-OH- hydroxyls, Fe(III)-O-O● radicals, and Fe(II)-●OOH/Fe(III)-O-OH- peroxyls. Based on the barriers, their activities in abstracting the hydrogen of a saturated aliphatic C-H bond can be summarized to follow an order of penta-coordinated Fe(IV)=O > hexa-coordinated Fe(IV)=O > hexa-coordinated FeA(III)-OO● > penta-coordinated FeB(III)-O-O● > penta-coordinated FeB(II)-●OH/FeB(III)-OH- > hexa-coordinated FeA(II)● OH/FeA(III)-OH- > hexa-coordinated FeA(II)-●OOH/FeA(III)OOH- ≈ penta-coordinated FeB(II)-●OOH/FeB(III)-OOH-.

(iv) The binuclear iron core with penta- and tetracoordinations mixed is an ingenious architecture that achieves a quite good reactivity balance between the O-O bond dissociation and the hydrogen abstraction from stearic acids. Double penta-coordination in the di-iron core would decelerate the OO bond dissociation increasing the reaction barrier to an amount larger than 23 kcal/mol, while double tetracoordination may reserve the catalytic efficiency of the native SCD1 enzyme. (v) Many heme-dependent enzymatic reactions involving Fe(IV)=O have been found to occur through low-spin pathways (e.g., chlorite dismutase, peroxidase, and cytochrome P450, catalase),44-52 while non-heme-dependent enzymatic reactions through high-spin pathways (e.g., fumitremorgin B endoperoxidase, mononuclear iron tetrahydrobiopterindependent hydroxylase, α-ketoglutarate-dependent oxygenase, apocarotenoid oxygenase, phosphonate dioxygenase, cysteine dioxygenase, NHFe2 methane monooxygenase, and ribonucleotide reductase).23,32,53,54 The present case of SCD1 reaction including the Fe(IV)=O species forming by pure histidine ligands, whose imidazole groups are isoelectronic with the pyrroles in heme, also proceeds via high-spin pathways. This may imply that the difference between heme and non-heme reactions is probably attributed to the more complicated conjugated system in the porphyrin, instead of the non-nitrogenous ligands in non-heme Fe-dependent enzymes, which is worth studying further. The results here advance the understanding of the significant chemical conversion from alkane to alkene in the fatty acid metabolism, providing new insights into several important chemical fields including the di-iron catalysis, the dioxygen activation, the O-O bond dissociation, and the direct activation of unreactive aliphatic C−H bond. In particular, coupled with the chemistry learned here, the architecture of pure divalent Fe cations, pure histidine ligands, and long di-Fe distance in the di-iron center of SCD1, presents a perfect prototype for the low-cost and easily-synthesized biomimetic catalysts utilized for the aliphatic C−H bond activation. Typically, the homogeneous binuclear Fe(II) complexes with pure N-containing ligands and/or the heterogeneous porous Fe(II)N metal-organic frameworks may be potential artificial catalysts for the chemical conversion from alkane to alkene.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Diverse electronic states of enzyme and enzyme-substrate complexes, optimized structures in various competing mechanisms, potential energy profile for the pathway with water excluded for FeB, the computation of uncatalyzed reaction, and Cartesian coordinates of optimized structures.

AUTHOR INFORMATION Corresponding Author *[email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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We thank Margareta R. A. Blomberg for valuable discussion. This work was financially supported by the National Natural Science Foundation of China (21673019).

REFERENCES (1) (2) (3) (4)

(5) (6) (7) (8) (9)

(10)

(11)

(12)

(13)

(14)

(15)

(16) (17) (18) (19) (20)

Shang, R.; llies, L.; Nakamura, E. Iron-Catalyzed C-H Bond Activation. Chem. Rev. 2017, 117, 9086-9139. Paton, C. M.; Ntambi, J. M. Biochemical and physiological function of stearoyl-CoA desaturase. Am. J. Physiol. Endocrinol. Metab. 2009, 297, E28-E37. Wang, H.; Klein, M. G.; Zou, H. Crystal structure of human stearoyl-coenzyme A desaturase in complex with substrate. Nat. Struct. Mol. Biol. 2015, 22, 581-585. Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S.-K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y.-S.; Zhou, J. Geometric and electronic structure/function correlations in non-heme iron enzymes. Chem. Rev. 2000, 100, 235349. Stryer, L. Biochemistry (4th ed.). New York: W. H. Freeman and Company. 1995, 603-628. Nelson, D. L.; Cox, M. M. Lehninger, Principles of Biochemistry (3rd ed.). New York: Worth Publishing. 2000. Broadwater, J. A.; Laundre, B. J.; Fox, B. G. Desaturation of trans-octadecenoyl-acyl carrier protein by stearoyl-acyl carrier protein delta9 desaturase. J. Inorg. Biochem. 2000, 78, 7-14. Yonghong, B.; Jason, G. M.; Elena, J. L.; Pablo, S.; Kanagalaghatta, R. R.; Brian, G. F.; Ming, Z. X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 2015, 524, 252-256. Lindqvist, Y.; Huang, W.; Schneider, G.; Shanklin, J. Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins. EMBO J. 1996, 15, 4081-4092. Rosenzweig, A. C.; Frederick, C. A.; Lippard, S. J.; Nordlund, P. Crystal structure of a bacterial non-haem iron hydroxylase that catalyses the biological oxidation of methane. Nature 1993, 366, 537-543. Whittington, D. A.; Sazinsky, M. H.; Lippard, S. J. X-ray Crystal Structure of Alcohol Products Bound at the Active Site of Soluble Methane Monooxygenase Hydroxylase. J. Am. Chem. Soc. 2001, 123, 1794-1795. Logan, D. T.; Su, X., Aberg, A.; Regnstrom, K.; Hajdu, J.; Eklund, H.; Nordlund, P. Crystal structure of reduced protein R2 of ribonucleotide reductase: the structural basis for oxygen activation at a dinuclear iron site. Structure 1996, 4, 1053-1064. Hogbom, M.; Huque, Y.; Sjoberg, B.; Nordlund, P. Crystal Structure of the Di-iron/Radical Protein of Ribonucleotide Reductase from Corynebacterium ammoniagenes. Biochemistry 2002, 41, 1381-1389. Jakub, C.; Tibor, A. R.; Yuki, K.; Takeshi, Y.; Edward, I. S.; Lubomir, R.; Martin, S. Reactivity of the binuclear non-heme iron active site of Δ⁹ desaturase studied by large-scale multireference ab initio calculations. J. Am. Chem. Soc. 2014, 136, 15977-15991. Hӧgbom, M.; Huque, Y.; Sjoberg, B. M.; Nordlund, P. Crystal structure of the di-iron/radical protein of ribonucleotide reductase from Corynebacterium ammoniagenes. Biochemistry. 2002, 41, 1381-1389. Sazinsky, M. H.; Lippard, S. J. Correlating structure with function in bacterial multicomponent monooxygenases and related diiron proteins. Acc. Chem. Res. 2006, 39, 558-566. Becke, A. D. Perspective on “Density functional thermochemistry. III. The role of exact exchange”. J. Chem. Phys. 1993, 98, 1372-1377. Lee, C.; Yang, W.; Parr, R. G. Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density. Phys. Rev. B. 1988, 37, 785-789. Jaguar, version 8.8, Schrodinger, LLC, New York, NY. 2015. Hay, P. J.; Wadt, W. R. Ab initio effective core potentials for molecular calculations. Potentials for K to Au including the outermost core orbitals. J. Chem. Phys. 1985, 82, 299-310.

Page 14 of 16

(21) Tannor, D. J.; Marten, B.; Murphy, R.; Friesner, R. A.; Sitkoff, D.; Nicholls, A.; Ringnalda, M.; Goddard III, W. A.; Honig, B. Accurate First Principles Calculation of Molecular Charge Distributions and Solvation Energies from Ab Initio Quantum Mechanics and Continuum Dielectric Theory. J. Am. Chem. Soc. 1994, 116, 11875-11882. (22) Marten, B.; Kim, K.; Cortis, C.; Friesner, R. A.; Murphy, R. B.; Ringnalda, M. N.; Sitkoff, D.; Honig, B. New Model for Calculation of Solvation Free Energies:  Correction of Self-Consistent Reaction Field Continuum Dielectric Theory for Short-Range Hydrogen-Bonding Effects. J. Phys. Chem. 1996, 100, 1177511788. (23) Blomberg, M. R.; Borowski, T.; Himo, F.; Liao, R. -Z.; Siegbahn, P. E. M. Quantum chemical studies of mechanisms for metalloenzymes. Chem. Rev. 2014, 114, 3601-3658. (24) Siegbahn, P. E. M. Mechanisms of metalloenzymes studied by quantum chemical methods. Q. Rev. Biophys. 2003, 36, 91-145. (25) Gaussian 09, Revision D.01, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Petersson, G. A.; Nakatsuji, H.; Li, X.; Caricato, M.; Marenich, A. V.; Bloino, J.; Janesko, B. G.; Gomperts, R.; Mennucci, B.; Hratchian, H. P.; Ortiz, J. V.; Izmaylov, A. F.; Sonnenberg, J. L.; Williams-Young, D.; Ding, F.; Lipparini, F.; Egidi, F.; Goings, J.; Peng, B.; Petrone, A.; Henderson, T.; Ranasinghe, D.; Zakrzewski, V. G.; Gao, J.; Rega, N.; Zheng, G.; Liang, W.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Throssell, K.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M. J.; Heyd, J. J.; Brothers, E. N.; Kudin, K. N.; Staroverov, V. N.; Keith, T. A.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A. P.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Millam, J. M.; Klene, M.; Adamo, C.; Cammi, R.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Farkas, O.; Foresman, J. B.; Fox, D. J. Gaussian, Inc., Wallingford CT. 2013. (26) Grimme, S. Accurate description of van der Waals complexes by density functional theory including empirical corrections. J. Comput. Chem. 2004, 25, 1463-1473 (27) Goerigk, L.; Grimme, S. A thorough benchmark of density functional methods for general main group thermochemistry, kinetics, and noncovalent interactions. Phys. Chem. Chem. Phys. 2011, 13, 6670-6688. (28) Zhang, H. -M.; Chen, S. -L. Include dispersion in quantum chemical modeling of enzymatic reactions: the case of isoaspartyl dipeptidase. J. Chem. Theory Comput. 2015, 11, 2525-2535. (29) Chen, S. -L.; Fang, W. -H.; Himo, F. Technical aspects of quantum chemical modeling of enzymatic reactions: the case of phosphotriesterase. Theor. Chem. Acc. 2008, 120, 515-522. (30) Siegbahn, P. E. M.; Blomberg, M. R. Quantum chemical studies of proton-coupled electron transfer in metalloenzymes. Chem. Rev. 2010, 110, 7040-7061. (31) Huang, W. J.; Llano, J.; J. Gauld, W. Redox mechanism of glycosidic bond hydrolysis catalyzed by 6-phospho-alphaglucosidase: a DFT study. J. Phys. Chem. B. 2010, 114, 1119611206. (32) Siegbahn, P. E. M. A comparison of dioxygen bond-cleavage in ribonucleotide reductase (RNR) and methane monooxygenase (MMO). Chemical. Phys. Lett. 2002, 351, 311-318. (33) Fox, B. G.; Shanklin, J.; Somerville, C.; Munck, E. Stearoylacyl carrier protein delta 9 desaturase from Ricinus communis is a diiron-oxo protein. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 2486-2490. (34) Yang, Y. -S.; Broadwater, J. A.; Pulver, S. C.; Fox, B. G.; Solomon, E. I. Circular Dichroism and Magnetic Circular Dichroism Studies of the Reduced Binuclear Non-Heme Iron Site of Stearoyl-ACP Δ9-Desaturase:  Substrate Binding and Comparison to Ribonucleotide Reductase. J. Am. Chem. Soc. 1999, 121, 27702783. (35) Noodleman, L.; Case, D. A. Density Functional Theory of Spin Polarization and Spin Coupling in Iron Sulfur Clusters. Adv. In-

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org. Chem. 1992, 38, 423-470. (36) Mouesca, J. -M.; Chen, J. -L.; Noodleman, L.; Bashford, D.; Case, D. A. Density Functional/Poisson-Boltzmann Calculations of Redox Potentials for Iron-Sulfur Clusters. J. Am. Chem. Soc. 1994, 116, 11898-11914. (37) Han, W. -G.; Lovell, T.; Liu, T.; Noodleman, L. A density functional evaluation of an Fe(III)-Fe(IV) model diiron cluster: comparisons with ribonucleotide reductase intermediate X. Inorg. Chem. 2003, 42, 2751-2758. (38) Han, W. -G.; Giammona, D. A.; Bashford, D.; Noodleman, L. Density functional theory analysis of structure, energetics, and spectroscopy for the Mn-Fe active site of Chlamydia trachomatis ribonucleotide reductase in four oxidation states. Inorg. Chem. 2010, 49, 7266-7281. (39) Blachly, P.; Sandala, G. M.; Giammona, D. A.; Liu, T.; Bashford, D.; McCammon, J. A.; Noodleman, L. Use of BrokenSymmetry Density Functional Theory To Characterize the IspH Oxidized State: Implications for IspH Mechanism and Inhibition. J. Chem. Theory. Comput. 2014, 10, 3871-3884. (40) Karp, G. Cell and Molecular Biology: Concepts and Experiments. 5th Ed; Wiley: 2008. (41) Camaioni, D. M.; Schwerdtfeger, C. A. Comment on "Accurate experimental values for the free energies of hydration of H+, OH-, and H3O+". J. Phys. Chem. A. 2005, 109, 10795-10797. (42) Ortmayer, M.; Lafite, P.; Menon, B. R.; Tralau, T.; Fisher, K.; Denkhaus, L.; Scrutton, N. S.; Rigby, S. E.; Munro, A.W.; Hay, S.; Leys, D. An oxidative N-demethylase reveals PAS transition from ubiquitous sensor to enzyme. Nature 2016, 539, 593-597. (43) Cutsail, G. E.; Banerjee, R.; Zhou, A.; Que, L.; Lipscomb, J. D.; DeBeer, S. High-Resolution Extended X-ray Absorption Fine Structure Analysis Provides Evidence for a Longer Fe···Fe Distance in the Q Intermediate of Methane Monooxygenase. J. Am. Chem. Soc. 2018, 140, 16807-16820. (44) Sun, S.; Li, Z. -S.; Chen, S. -L. A dominant homolytic O-Cl bond cleavage with low-spin triplet-state Fe(IV)=O formed is revealed in the mechanism of heme-dependent chlorite dismutase. Dalton Trans. 2014, 43, 973-981. (45) Su, J. -X.; Chen, S. -L. Significant electron transfer in heme catalysis: The case of chlorite dismutase. J. Catal. 2017, 348, 40–46. (46) Poulos, T. L. Heme enzyme structure and function. Chem. Rev. 2014, 114, 3919-3962. (47) Alfonso-Prieto, M.; Oberhofer, H.; Klein, M. L.; Rovira, C.; Blumberger, J. Proton Transfer Drives Protein Radical Formation in Helicobacter pylori Catalase but Not in Penicillium vitale Catalase. J. Am. Chem. Soc. 2011, 133, 4285-4298. (48) de Visser, S. P.; Ogliaro, F.; Harris, N.; Shaik, S. Multi-State Epoxidation of Ethene by Cytochrome P450:  A Quantum Chemical Study. J. Am. Chem. Soc. 2001, 123, 3037-3047. (49) Hirao, H.; Kumar, D.; Thiel, W.; Shaik, S. Two States and Two More in the Mechanisms of Hydroxylation and Epoxidation by Cytochrome P450. J. Am. Chem. Soc. 2005, 127, 13007-13018. (50) Schöneboom, J. C.; Neese, F.; Thiel, W. Toward Identification of the Compound I Reactive Intermediate in Cytochrome P450 Chemistry:  A QM/MM Study of Its EPR and Mössbauer Parameters. J. Am. Chem. Soc. 2005, 127, 5840-5853. (51) Vidossich, P.; Alfonso-Prieto, M.; Carpena, X.; Loewen, P. C.; Fita, I.; Rovira, C. Versatility of the Electronic Structure of Compound I in Catalase-Peroxidases. J. Am. Chem. Soc. 2007, 129, 13436-13446. (52) de Visser, S. P.; Ogliaro, F.; P. Sharma, K.; Shaik, S. Hydrogen bonding modulates the selectivity of enzymatic oxidation by P450: chameleon oxidant behavior by compound I. Angew. Chem., Int. Ed. 2002, 41, 1947-1951. (53) Ji, J. -N. Chen, S. -L. Asymmetric abstraction of two chemically-equivalent methylene hydrogens: significant enantioselectivity of endoperoxide presented by fumitremorgin B endoperoxidase. Phys. Chem. Chem. Phys. 2018, 20, 26500-26505.

(54) Martinez, S.; Hausinger, R. P. Catalytic Mechanisms of Fe(II)and 2-Oxoglutarate-dependent Oxygenases. J. Biol. Chem. 2015, 290, 20702-20711.

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