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Mechanism of Organophosphonate Catabolism by Diiron Oxygenase PhnZ: A Third Iron-Mediated O-O Activation Scenario in Nature Chongyang Zhao, and Hui Chen ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b00578 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 10, 2017

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Mechanism of Organophosphonate Catabolism by Diiron Oxygenase PhnZ: A Third Iron-Mediated O-O Activation Scenario in Nature Chongyang Zhao,†,‡ and Hui Chen*,† †

Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory of Photochemistry,

CAS Research/Education Center for Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100049, P. R. China

*Correspondence author, [email protected] ACS Paragon Plus Environment

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ABSTRACT: Diiron oxygenase PhnZ catalyzes the catabolism of organophosphonate (Pn) (R)-2-amino-1hydroxyethylphosphonic to glycine and inorganic phosphate (Pi). In this Pn catabolism way, PhnZ oxidatively cleaves the highly stable C-P bond in Pn to produce Pi. However, the mechanism of this enzyme that affords aquatic and marine bacteria in Pi-limited environments to utilize the most abundant environmental Pn (2-amino-ethylphosphonic acid) as the source of Pi, is still unclear. In this work, extensive QM/MM calculations reveal that the mechanism of PhnZ consists of four consecutive steps: (1) Rate-limiting α-H abstraction of Pn by FeIII-superoxo; (2) Formation of FeIIIOOCα peroxide; (3) Concerted O insertion into Cα-P bond of Pn initiated by “inverse” heterolytic O-O cleavage; (4) Phosphate hydrolysis to glycine and Pi. Intriguingly, the enzymatic reaction mechanism of PhnZ for the crucial breakage of the C-P bond is characterized by the “inverse” heterolytic O-O cleavage of FeIIIOOCα intermediate, which renders the distal O atom more oxidative to oxygenate Pn than the homolytic O-O cleavage. In this way, PhnZ adopts a mechanism quite different from the related diiron oxygenase MIOX, with His62 residue playing an important role. This unusual “inverse” heterolytic O-O cleavage mode, apart from the well-known homolytic and “normal” heterolytic ones, constitutes a third iron-mediated OO activation scenario in nature, which is expected to have its broad occurrence in oxidative transformation involving heteroatoms of sulfur and phosphorus.

Keywords:

C-P bond cleavage, QM/MM, Diiron enzyme, superoxo, heterolytic O-O cleavage, organophosphonate catabolism, ferric-peroxide, phosphorus oxidation

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1. INTRODUCTION Oxygen molecule activation is the central topic in numerous chemical and biochemical oxygenation transformations.1-7 Iron, as an earth abundant transition metal, and probably the most important trace metal for biological organisms, plays pivotal roles in oxygen molecule activation in both enzymes and catalyses.8-11 In iron-mediated O2 activations of many heme and non-heme oxygenases, O2-activating ferrous iron (FeII) combines O2 to commonly generate ferric or ferrous (hydro)peroxide species FeIII/IIOOR(H) with the assistance of co-substrate (such as in non-heme α-ketoglutarate-dependent dioxygenase TauD and AlkB12), substrate (such as in non-heme homoprotocatechuate 2,3-dioxygenase HPCD,13-15 2-hydroxyethylphosphonate dioxygenase HEPD,16-20 heme tryptophan 2,3-dioxygenase TDO and indoleamine 2,3-dioxygenase IDO21-24), or exogenous proton/electron source (such as in many heme enzymes of cytochrome P450s25,26 and heme oxygenase HO27,28). From FeIII/IIOOR(H) species, generally the subsequent key O-O bond cleavage step may proceed either homolytically or heterolytically. As shown in Scheme 1a, homolytic O-O cleavage from FeIII/IIOOR(H) leads to radicaloid •OR(H) plus highvalent ferryl FeIVO or ferric FeIIIO. While for heterolytic O-O cleavage, all the enzymes reported up to date adopt such a scenario as shown in Scheme 1b, that the pair of electrons shared by two O atoms in the heterolytically breaking O-O σ-bond go to the distal O atom. As a result, this type of O-O heterolytic cleavage, which we call “normal” O-O heterolytic cleavage, stores 2e oxidation equivalent in the proximal O and its bonding iron cofactor. Depending on the ferric or ferrous oxidation state of iron prior to the OO cleavage, “normal” O-O heterolytic cleavage produces the high-valent perferryl FeVO or ferryl FeIVO in non-heme enzymes, and FeIVO plus a cationic hole (1e oxidation equivalent) in the macrocyclic porphyrin ring in heme enzymes. These high-valent perferryl-oxo or ferryl-oxo species are the terminal oxidants from O2 activations, which are capable of oxygenating substrate with the stored 2e oxidation


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equivalent. On the contrary, the alternative scenario of heterolytic O-O cleavage from FeIII/IIOOR(H) as shown in Scheme 1c, in which the pair of electrons in the heterolytically breaking O-O bond unusually go to the proximal O atom to generate FeIIIO– or FeIIO–, while resultantly rendering the distal O atom 2eoxidative to oxygenate the substrate, was never found in any enzymes.29,30 Here in this work, through combined quantum mechanical/molecular mechanical (QM/MM) computational approach, we disclosed that this enzymatically unprecedented and extremely unusual heterolytic O-O cleavage scenario for the O2 activation, which we call “inverse” O-O heterolytic cleavage, is adopted by a non-heme diiron oxygenase PhnZ.

Scheme 1. Three possible enzymatic O-O cleavage mechanisms from FeIII/IIOOR(H) intermediates in Nature, including: (a) homolytic mode, (b) “normal” heterolytic mode, and (c) unknown “inverse” heterolytic mode, initiated from the common ferric/ferrous peroxide (hydroperoxo) intermediates

Scheme 2. PhnY-PhnZ metabolism relay pathway from 2-AEP to glycine and Pi


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PhnZ is a non-heme diiron oxygenase recently discovered in HD-domain protein superfamily, which catalyzes the conversion of organophosphonate (Pn) (R)-2-amino-1-hydroxyethylphosphonic acid ((R)OH-AEP) to glycine and inorganic phosphate (Pi), as shown in Scheme 2.31,32 Combined with PhnY, which is a non-heme mononuclear α-ketoglutarate-dependent (αKG) dioxygenase to effect typical hydroxylation transformation of 2-amino-ethylphosphonic acid (2-AEP) to (R)-OH-AEP, PhnY-PhnZ relay pathway affords aquatic and marine bacteria in Pi-limited environments to utilize 2-AEP, the most abundant environmental Pn, as the source of Pi. In this Pn catabolism pathway, PhnZ oxidatively cleaves the highly stable C-P bond in Pn to produce Pi, which constitutes a widely distributed one among the three known enzymatic C-P bond cleavage routes in marine ecosystems. Notably, this route in PhnZ is unlimited to the presence of β-carbonyl group in Pn as required in the alternative non-oxidative route for C-P bond cleavage in many phosphohydrolases.33

Figure 1. Active site of PhnZ (PDB code: 4MLN34) with its substrate (R)-OH-AEP bound, in comparison with MIOX (PDB code: 2HUO35). ACS Paragon Plus Environment

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Recently, it has been demonstrated experimentally that PhnZ employs a quite unusual mixed-valent FeII/FeIII diiron cofactor to activate O2 and to effect the degradation of substrate (R)-OH-AEP.36 This is same as the non-heme diiron enzyme of myo-insitol oxygenase (MIOX),37,38 which also belongs to HD proteins. MIOX functions by oxidative cleavage of C-C bond in substrate, which is comparable to the function of oxidative cleavage of C-P bond in PhnZ. Furthermore, in the recent X-ray crystal structures of PhnZ,34 the substrate binding mode was found to be similar to that in MIOX,35 both using one iron of two in diiron cofactor to bind the substrate, as depicted in Figure 1. These similarities in cofactor, in substrate binding mode, as well as the fact that PhnZ is most similar to MIOX in structural homology,34 all appear to point to one implication, i.e., that MIOX and PhnZ shall have similar reaction mechanism. In fact, this conjecture, while still unproved, has been utilized to propose the tentative mechanism of PhnZ by analogy to that of MIOX.31 However, as will be shown in this work, we found that PhnZ has a quite different reaction mechanism compared to MIOX. Most notably, MIOX adopts a usual homolytic O-O cleavage mechanism, while PhnZ has a very unusual “inverse” O-O heterolytic cleavage mechanism. This unusual O-O cleavage mode opens a completely new avenue to mechanistic scenarios of the O2 activations in diiron oxygenases, which would enrich the chemistry of enzymes containing iron cofactor, and shed light on the complicated mechanisms of oxygenation reactions in chemistry and biochemistry.

2. COMPUTATIONAL DETAILS Setup of system. The initial coordinates were taken from X-ray crystal structure of PhnZ (PDB code 4MLN).34 The subunit A of the X-ray crystal structure, which was proposed to be the active Michaelis complex,34 was adopted in our study. The protonation states of titratable residues were determined by combining the pKa values predicted by the PROPKA39 and visual inspection for nearby hydrogen bonding. After these procedures, we found that all glutamic and aspartic residues are not protonated, while the histidine residues are singly protonated. The missing hydrogen atoms were added through HBUILD ACS Paragon Plus Environment

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module40 and optimized by CHARMM22 force field41 using CHARMM program42. Then a 16Å thick solvent water layer was built around the enzyme. The inner 8Å of water layer was then relaxed through a procedure involving (1) MM optimization; (2) heating to 300K; (3) equilibration for 3ps; (4) a second optimization. This procedure for adding solvent water was repeated twice to ensure that less than 100 additional waters were added in one procedure. The QM/MM setup procedure had been recently reviewed for QM/MM modeling of enzymatic processes.43 QM/MM Methodology and Software. QM/MM calculations were carried out by ChemShell program,44 which interfaces Turbomole45 for QM calculation and DL_POLY46 for MM calculation. An electronic embedding scheme47 was applied to include the polarizing effect of the enzymatic environment on the QM region. Hydrogen link atoms with charge-shift model47 were applied for treating the QM/MM boundary. Density functional theory (DFT) method was employed for QM part, while CHARMM force field was utilized for MM part. Geometry optimization and potential energy surface scan along the reaction coordinate were performed by B3LYP48 functional in combination with 6-31G* basis set for hyper-valent phosphorus atom and 6-31G basis set for all the other main-group elements,49 while LACVP50 basis set for iron atoms (basis sets denoted as B1). The transition states were determined as the highest point in the energy profile along reaction coordinate. To refine the electronic energy, single point B3LYP/MM calculation with larger basis sets of triple-ζ polarized quality, i.e., LANL2TZ(f) for irons51 and 6-311G** for the rest atoms52 (basis sets denoted as B2), were conducted based on the geometry from B3LYP(B1)/MM calculations. QM Region. As the core region of the active site of the enzyme, the QM region in our calculations generally covers the diiron metals, and all groups/molecules that are directly coordinated with diiron (Fe1 and Fe2). These diiron-coordinating parts include the six residues directly coordinated to diiron (His34, His58, His104, His80, Asp59, Asp161), hydroxo/water bridge linking two irons, substrate ((R)-OH-AEP) ligating Fe2 site, as well as the O2 or H2O ligating Fe1 site. All these collected diiron and iron-coordinating parts were denoted as ODV. Besides ODV, the molecules which form important hydrogen bonds with ODV (Glu27, His62, Lys108, Thr129, Gln133, Arg158, Glu162, crystal waters Wat331, Wat348, Wat363) were also included in the QM region. All the residues in the QM region were cut off from main chain at the truncated carbon positions that simultaneously minimize QM fragments and keep integer MM


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charges, with the cut C-C bond saturated by a hydrogen link atom (for example, the glutamic acid was treated as CH3COO–, glutamine was modeled as NH2-COH). Optimized QM/MM Region. During the QM/MM geometry optimization, the active region to be optimized and relaxed consists of QM region and all other residues/molecules within 6Å of ODV (ODV, Tyr24, Ile25, Gly26, Glu27, Ile29, Asn30, Gln31, Leu32, Gly33, His34, Ser35, Leu36, Gln37, Cys38, Ala39, Phe41, Leu53, Ala54, Ala55, Leu56, Leu57, His58, Asp59, Leu60, Gly61, His62, Tyr63, Met72, Tyr75, Gly76, Val77, Trp78, Gln79, His80, Lys82, Val83, Gly84, Ala85, Leu100, Ile101, Glu102, Gly103, His104, Val105, Ala106, Ala107, Lys108, Tyr118, Leu122, Ser123, Ala125, Ser126, Arg127, Thr129, Leu130, Gln133, Cys154, Lys156, Ile157, Arg158, Ala159, Trp160, Asp161, Glu162, Lys163, Gly164, Lys165, Gln166, Wat302, Wat306, Wat307, Wat314, Wat317, Wat320, Wat321, Wat325, Wat326, Wat331, Wat332, Wat336, Wat343, Wat348, Wat355, Wat360, Wat363, solvent waters Sol1442, Sol1481, Sol1638, Sol1698, Sol1902, Sol2129). The vibrational analyses for the optimized stationary points were also done, as shown in Table S1 in the SI.

3. RESULTS AND DISCUSSION 3.1. Protonation States of Substrate and Hydroxo Bridge for Diiron Cofactor Before any meaningful theoretical modeling for PhnZ, it is advisable to determine the protonation states of active site as accurate as possible. In PhnZ, except the titratable residues with their protonation states already determined by combining their calculated pKa values with the visual inspection for nearby hydrogen bonding interaction, there are still two protonation issues to be clarified. As depicted in Figure 2a, the first issue is about the currently ambiguous protonation state assignment for the hydroxo bridge linking two irons. In X-ray crystal structures, visual inspection only implies that as a H-bond donor to Asp161, μ-oxo bridge is not a possible form (we also tested the possibility of μ-oxo as a H-bond acceptor with the H-bond donor of protonated Asp161, but the QM/MM geometry optimization starting from such an initial protonation setting turned out to be the form of hydroxo plus deprotonated Asp161). However, ACS Paragon Plus Environment

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whether it is hydroxo or water bridge, is still not unambiguously distinguishable only from the X-ray crystal structures.34 To check this protonation issue, we did QM/MM geometry optimizations assuming both hydroxo and water bridges. The results indicate that hydroxo bridge reproduces the X-ray crystal structure slightly better,34 especially for the most relevant Fe1-O(hydroxo) and Fe2-O(hydroxo) bond distances, as depicted in Figure 2a. In addition, the calculated free energy of the hydroxo bridge protonation by the water outside the enzyme at the biologically relevant pH favors the bridge form of hydroxo rather than water. Thus, we now know that it is hydroxo bridge that exists in PhnZ to link the two irons. Notably, the protonation state of hydroxo bridge is not a trivial issue in reaction mechanism of PhnZ, since previously it has been proposed for PhnZ that hydroxo bridge can act as a base to be directly involved in the O2 activation,31 in a similar way as for MIOX,53 whereas water bridge apparently cannot play such a role.

Figure 2. Deviations (D) of QM/MM-calculated key structural parameters (in Å, with protonation state denoted by color) from the X-ray crystal structure, for PhnZ active site with different protonation states of (a) hydroxo bridge, (b) substrate (R)-OH-AEP.

The second uncertainty in protonation state is about the phosphonic moiety in the substrate (R)-OHAEP. The amino group in (R)-OH-AEP can easily be determined to be protonated by its much higher pKa


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above 7 (~ 11 for 2-AEP),54 as also implied from the solvation by two water molecules and electrostatic interaction with Glu27 in the X-ray crystal structure.34 However, the protonation state of phosphonic group is much more ambiguous due to its second pKa slightly below 7 (~ 6.3 for 2-AEP).54 Apparently, the protonation state of substrate (R)-OH-AEP, which is directly coordinated to iron, could severely affect the electron transfer property between iron and (R)-OH-AEP during the reaction, and hence being crucial to the mechanism of PhnZ. To determine the protonation state of iron-binding (R)-OH-AEP in PhnZ, we compared the QM/MM-optimized geometries of protonated and unprotonated phosphonic group in (R)OH-AEP. As depicted in Figure 2b, the (R)-OH-AEP binding geometry with unprotonated phosphonic group matches the X-ray crystal structure better than the ones with protonated (R)-OH-AEP (especially the one with protonation on O1 site). In addition, the calculated QM/MM energies also demonstrate that protonations on O2 and O3 sites are even disfavored than O1 protonation by about 7 and 14 kcal/mol, respectively, indicating also that these protonations are not likely to be relevant in PhnZ. Hence we conclude that the (R)-OH-AEP substrate is not protonated at the phosphonic position in PhnZ.

3.2. Electronic Structure of Resting State and Superoxo Intermediate Among various diiron oxygenases, PhnZ and MIOX are very unusual in that they use the mixedvalent FeII/FeIII cofactor to activate O2,36-38 unlike all the others that employ the di-ferrous FeII/FeII cofactor. Compared with MIOX,55 PhnZ is unique in modulation of oxidation state of mixed-valent FeII/FeIII cofactor upon substrate coordination. Thus, as proposed by Zechel, Jia, and their coworkers, an intriguing induced-fit mechanism triggered by binding of (R)-OH-AEP is possibly involved to avoid premature O2 activation in the absence of substrate.34 As shown in Scheme 3, the binding of (R)-OH-AEP can induce the flip of the loop containing Tyr24-Glu27 region by electrostatic interaction between Glu27


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and protonated 2-amino group of (R)-OH-AEP, which hence liberates the binding site of Fe1 for water and then finally for O2 coordination. There exists experimental evidence that Fe1 is in the oxidation state of III when Tyr24 still occupies the coordinate site of Fe1.34 However, as shown in Scheme 3, it is still unclear at what stage the single electron transfer (SET) from Fe2 to Fe1, which is necessary for the final O2 coordination and activation, occurs in this interesting induced-fit mechanism.

Scheme 3. The induced-fit mechanism triggered by binding of (R)-OH-AEP proposed in ref 34, and the corresponding unclear issue concerning the single electron transfer (SET) between Fe1 and Fe2

To clarify this issue related to the electronic structure of the resting state of PhnZ with its substrate binding but without O2 loaded, we did QM/MM calculations. As shown in Scheme 4, we found that interestingly, with the water coordination as well as the induced-fit flip of the loop containing Tyr24Glu27 region triggered by the binding of (R)-OH-AEP, the diiron cofactor is still in the oxidation state of III/II pattern for Fe1/Fe2. Thus Fe1 is still a ferric iron, which indicates that only the substrate binding ACS Paragon Plus Environment

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and the resulting induced-fit removal of Tyr24 from Fe1 for water coordination as observed in X-ray crystal structure34 are not enough to induce the SET from Fe2 to Fe1. Oxygen molecule binding may be prerequisite and indispensable for this electron transfer event to occur, which implies that O2 may “selfcatalyze” its own iron-binding and activation. Thus, it is now necessary to further explore the geometric and electronic structures of superoxo species of PhnZ.

Scheme 4. Electronic structure and iron oxidation states of the resting state of mixed-valent FeII/FeIII cofactor

Generally, concerning the possible superoxo species, there are two well-known coordination modes of O2 at single iron site. One is the end-on mode, the other is the side-on one. After extensive computational search for the electronic and geometric structures of superoxo species, we found the following results as summarized in Scheme 5. First, among all our calculated diiron-superoxo species, side-on Fe-O2 geometry with electronic structure labeled as LF (Stotal=1/2), which has a low-spin (S=0, L) diiron unit and ferromagnetic (F) coupling between O2•– and Fe1, is the most stable one. Two end-on electromers, labeled as LA (Stotal=1/2) and HA (Stotal=9/2), which respectively have low-spin (S=0, L) and high spin (S=5, H) diiron units, and with Fe1 anti-ferromagnetically (A) coupled with O2•–, lie 2.8 and 4.4 ACS Paragon Plus Environment

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kcal/mol higher in energy. The side-on electromer HF (Stotal=11/2), which has high-spin (S=5, H) diiron unit ferromagnetically (F) coupled with O2•–, follows in energy by lying 5.8 kcal/mol higher than the most stable side-on diiron-superoxo species LF. In general, the low-spin (S=0, L) diiron unit with two Fe (S=5/2) anti-ferromagnetically (A) coupled has energetic advantage over the high-spin (S=5, H) one with two Fe (S=5/2) ferromagnetically (F) coupled, which is in line with the Mössbauer experimental observation for PhnZ.36 To explore alternative electronic structure coupled from Fe(III) electronic configuration other than high-spin (S=5/2) one, we also tried to get Fe1 with medium spin (S=3/2, M), which is coupled with high-spin (S=5/2) Fe2 and O2•– to form Stotal of 1/2 and 3/2. These two electromers, denoted as MF and MA, lie above the most stable side-on LF diiron-superoxo species by 11.4 and 3.2 kcal/mol in energy, respectively. These results echo the experimental discovery of high-spin (S=5/2) Fe(III) subsites,36 and confirm the preference of such an high-spin electronic configurations adopted by the single iron in PhnZ. It is notable that the calculated energies for many Fe-O2 species, such as LF, LA, HA, and HF, are quite close, which means their transformation may be energetically feasible.


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Scheme 5. Various geometric and electronic structures calculated in this work for FeIII/FeIII-superoxo species. The relative energies (in kcal/mol) of the more stable geometry for each electromer (taking the most stable side-on LF state as zero point) and side-on/end-on gaps (for cases wherein both geometries exist) are labeled in parentheses and bracket, respectively

When both end-on and side-on diiron-superoxo species exist, an interesting trend for their relative stability is that, for those electronic states with Fe1 and O2•– (superoxo linked by Fe1) ferromagnetically coupled, side-on geometry is more stable than the corresponding end-on one, while the opposite is true if Fe1 and Fe1-linked O2•– are anti-ferromagnetically coupled. This general trend is notably in agreement with the previous theoretical finding in a bioinorganic synthetic mononuclear iron(III)-superoxo system with N-tetramethylated cyclam N,N,N,N-tetradentate ligand (TMC).56 After the electronic structures of iron-superoxo species being clarified, we go on to explore the reaction mechanism of Pn catabolism initiating from this active species.


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3.3. Mechanism of organophosphonate catabolism by PhnZ The calculated reaction profiles of oxidative catabolism of (R)-OH-AEP to glycine and Pi by PhnZ initiated from four low-lying electronic states (LF, LA, HA, and HF) of FeIII/FeIII-superoxo species, are depicted in Figure 3. The reaction mechanism can be divided into four stages: (1) H-abstraction; (2) Peroxide formation; (3) Concerted or stepwise O atom insertion into C-P bond of Pn substrate initiated by unusual “inverse” heterolytic or usual homolytic O-O cleavage; (4) Hydrolysis of the generated phosphate to finally produce glycine and Pi. Below we separately present and discuss our findings concerning these processes.

Figure 3. QM/MM-calculated reaction profile of oxidative catabolism of (R)-OH-AEP to glycine and Pi by PhnZ initiated from four low-lying electronic states (LF, LA, HA, and HF) of FeIII/FeIII-superoxo species. The relative energies (in kcal/mol) are labeled. ACS Paragon Plus Environment

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As shown in Figure 3, the Pn catabolism reaction in PhnZ is found to start with H-abstraction of αH in (R)-OH-AEP by Fe1-superoxo, which supports the mechanistic proposal by Zechel et al.31,34 Interestingly, towards this H-abstraction, the four low-lying states (LF, LA, HA, and HF) of FeIII/FeIIIsuperoxo species exhibit quite different reactivities that can be classified as two types. Among the four states, LF and HA states have similar and relatively high reactivity by bearing relatively low barriers of about 24-25 kcal/mol through H-abstraction transition state TSA-B (LF structure depicted in Figure 4) and quite exothermic reaction energies of about 14-16 kcal/mol from AFeO2 to BFeOOH. On the contrary, LA and HF states are relatively much disfavored in reactivity by bearing either quite endothermic reaction energies of at least 16 kcal/mol, or extraordinary high barrier of more than 40 kcal/mol. The origin of such distinguishing reactivities can be rationalized and attributed to their electronic structure difference, as exemplified for LF and LA states shown in Scheme 6. It can be seen that due to the parallel spin alignment of unpaired electrons on Fe2 and O2•–, Fe2 in LA state cannot be reduced from the ferric to ferrous form by the single electron of Cα radical resulting from the H-abstraction. Hence the radical in LA state unstably remains on Cα in (R)-OH-AEP, unlike LF state for which due to the anti-parallel spin alignment of Fe2 and O2•–, this reduction occurs without inhibition. The same difference can be identified to account for the poor reactivity of HF state in comparison to HA state (see Scheme 5 for their electronic configurations). Of note is that the proton of the hydroxyl group in (R)-OH-AEP has been transferred to His62 residue in intermediate BFeOOH after H-abstraction. As a result, the hydroxyl group in (R)-OH-AEP is transformed to carbonyl group (Cα=O) in BFeOOH. This is the first place along the reaction profile that His62 plays a role as a proton acceptor/donor. We will see later in this study that it is not the only place for His62 to play such a role.


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Figure 4. The structure of H-abstraction transition state TSA-B for LF state.

Scheme 6. The electronic configuration difference between LF and LA states accounting for their distinguishing H-abstraction reactivities

All the above H-abstraction reactivities are from the four lowest-lying electronic states, which all consist of high-spin (S=5/2) iron(III) building blocks. For energetically even higher-lying states coupled from medium-spin (S=3/2) iron(III) like MF, the reactivity is uniformly lower than that of LF or HA states, the details of which is relegated to the supporting information (SI) document (see Figure S1 in the


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SI). It is notable that MA state should be even less favored in H-abstraction than MF state due to the same electronic state difference as between LA and LF that hinders the reduction of Fe2 from the ferric to ferrous form by the single electron left on the Cα after Cα-H abstraction. Hence we would not further discuss the reaction mechanisms based on MF and MA states hereafter in this work. The next stage after H-abstraction is the peroxide formation from the iron(III)-hydroperoxo intermediate BFeOOH. Thus, as shown in Figure 3, through nucleophilic O-attack (via TSB-C) to Cα atom of newly formed carbonyl group Cα=O in (R)-OH-AEP after H-abstraction, two types of peroxide intermediates CFeOOC and C′FeOOC are formed with the assistance of hydroxo bridge acting as a base to simultaneously accept the proton from the hydroperoxo moiety. Importantly, CFeOOC and C′FeOOC differ in structure by the position of proton in the His62-H-OCα fragment. In CFeOOC, His62 keeps protonated with Cα=O in carbonyl form as the preceding intermediate BFeOOH, while in C′FeOOC, His62 gives the proton back to Cα=O to form Cα-OH moiety. Notably, this structural difference between CFeOOC and C′FeOOC, although generated from LF and HA states, is not limited to these two states. Hence, both LF and HA states have their CFeOOC and C′FeOOC intermediates. As seen below, this structural difference between CFeOOC and C′FeOOC would bring significant disparity in the subsequent mechanisms. After CFeOOC/C′FeOOC is formed, the next step is O-O cleavage. As shown in Figure 3, surprisingly, from CFeOOC intermediate, we found that an “inverse” heterolytic O-O cleavage via TSC-D (structure shown in Figure 5a, with the displacement vector of the imaginary frequency given in Figure S5 in the SI, which indicates the correct mode of concerted O-O/C-P cleavage for transition state TSC-D) spontaneously brings about three non-synchronous concerted structural evolvements: (1) Cα-P breaking; (2) Ob-P forming involving the distal oxygen atom (Ob); (3) Oa-Cα forming involving the proximal oxygen atom (Oa), finally reaching the intermediate DFeOCO. During this concerted process, His62 also plays the role as


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proton acceptor/donor again to give the proton back to Cα=O. Thus, in only one single step, three covalent bonds are broken and three others are formed. To our best knowledge, this is the most complicated oxidative rearrangement done in one single reaction step ever disclosed in nature to date. More surprising is that this extremely complicated single reaction step involving a sequence of multiple bond breakages/formations only needs to overcome small barriers of 5.4 and 6.3 kcal/mol for LF and HA states, respectively. From C′FeOOC, however, the O-O cleavage is much simpler. As found in MIOX,53 homolytic O-O cleavage starting from Fe(III)-peroxide CFeOOC via TSC′-E (structure shown in Figure 5b) leads to Fe(IV)-oxo on Fe1 site plus the moiety containing the •ObR radical (EFeO/OC), with barrier of around 10 kcal/mol to overcome for both LF and HA states. This distal •ObR radical then rearranges to insert into the C-P bond via TSE-F to FFeO/CO, during which the single unpaired electron of the •ObR radical reduces the Fe(IV)-oxo on Fe1 site to Fe(III)-O–. This O insertion into C-P through TSE-F needs to overcome a considerable barrier of at least 18.4 kcal/mol. More importantly, TSC-D, as the highest-lying transition state for the “inverse” heterolytic O-O cleavage pathway after the first H-abstraction step until the end of Pn catabolism pathway, is more stable than the corresponding highest-lying transition state TSE-F for the homolytic O-O cleavage pathway by at least 8.8 kcal/mol. With these results, we conclude that the reaction pathway characterized by the unusual “inverse” heterolytic O-O cleavage, is more preferable than that characterized by usual homolytic O-O cleavage. More detailed analyses of the heterolytic/homolytic O-O cleavage are described in the SI document (see Figure S2 and the corresponding discussions).


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Figure 5. The structures of (a) the “inverse” heterolytic O-O cleavage transition state TSC-D for LF state, (b) the usual homolytic O-O cleavage transition state TSC′-E for HA state. The bond distance between the proximal (Oa) and distal (Ob) oxygen atoms is labeled.

The final stage of the Pn catabolism mechanism in PhnZ is the hydrolysis of the generated phosphate. Usually, this type of hydrolysis is considered to proceed via the nucleophilic acyl substitution mechanism, which consists of a first nucleophilic addition to the carbonyl group, followed by an elimination of the leaving group (Pi) for regenerating the carbonyl C=O bond. For DFeOCO as the immediate intermediate from the “inverse” heterolytic O-O cleavage, however, the Oa-Cα bond has been formed between the nucleophilic Oa in Fe(III)-Oa– and the carbonyl Cα of the phosphate, which means that the first addition step of the hydrolysis is already done. From DFeOCO, TSD-G serves as the Cα-Ob breaking transition state of the second Pi elimination step for hydrolysis, with a barrier of 14.5/14.3 kcal/mol for LF/HA states. Interestingly, during this hydrolysis process, His62 plays its role as proton acceptor/donor for a third time to get the proton from the Cα-OH fragment. Different from DFeOCO, from FFeO/CO generated in the homolytic O-O cleavage pathway, hydrolysis of the phosphate occurs not in a stepwise manner but in a SN2-like concerted manner via TSF-H, with a barrier of 15.9/13.8 kcal/mol for LF/HA states. Notably, the hydrolysis reaction profiles from DFeOCO lie below those from FFeO/CO by about 10 kcal/mol, indicating ACS Paragon Plus Environment

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the preference of the former in the “inverse” heterolytic pathway for the phosphate hydrolysis in PhnZ. Importantly, our mechanism based on QM/MM calculations is consistent with the experimental finding in that only one but not two O atoms in O2 molecule goes into the Pn catabolism product glycine,36 which lends more credence to the mechanism uncovered herein. In summary, the most favorable whole Pn catabolism mechanism of (R)-OH-AEP in PhnZ consists of four steps: (1) H-abstraction of α-H atom by Fe1(III)-superoxo species; (2) Formation of Fe1(III)OOCα peroxide intermediate; (3) Concerted Fe1-distal O atom insertion into C-P bond of Pn substrate initiated by “inverse” heterolytic O-O cleavage, which accomplishes breaking/forming four chemical bonds in one step; (4) Hydrolysis of the generated phosphate by the Fe1-proximal O atom, producing the final products of glycine and Pi. The rate-limiting step is the H-abstraction step. The enzymatic reaction mechanism is characterized uniquely by the “inverse” heterolytic O-O cleavage of Fe(III)OOCα peroxide intermediate, which generates Fe(III)-O– and renders the distal O atom more oxidative to oxygenate the substrate than the homolytic O-O cleavage. This unusual O-O cleavage mode, apart from the well-known homolytic and “normal” heterolytic O-O cleavages, constitutes a third iron-mediated O-O activation scenario in nature.

3.4. Comparison with MIOX, HEPD, and synthetic analogue system PhnZ is not the only enzyme that can oxidatively cleave the C-P bond. Mononuclear non-heme enzyme

HEPD

also

has

this

function

specifically

towards

the

substrate

(S)-1-HEP

(HEP=hydroxyethylphosphonate),17 as shown in Scheme 7. As such, it would be interesting to see the mechanism difference between PhnZ and HEPD in the C-P cleavage process. In Scheme 7 we display their mechanistic difference for the initial H-abstraction step. It can be seen that in PhnZ, the unpaired electron left on Cα from the H-abstraction of Cα-H bond transfers to Fe2 and hence reduces this ferric iron


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that binds the substrate, which makes the ferric Fe1-OOH as the H-abstraction product to remain in ferric state. While in HEPD case, because there is only one iron, this single electron transfer reduces ferric FeOOH to ferrous one.19 It is notable that MIOX and PhnZ, which share very similar active site structure, have similar electron transfer pathway in this H-abstraction step as shown also in Scheme 7.53

Scheme 7. The iron-superoxo H-abstraction mechanism comparison between PhnZ containing diiron cofactor and HEPD containing monoiron cofactor


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Scheme 8. The comparison of O-O cleavage mechanisms in PhnZ, MIOX, and HEPD

Thus, do the similar active site structures between MIOX and PhnZ generally guarantee a similar reaction mechanism? Concerning the key O-O cleavage step, the answer to this question is definitely negative. As shown in Scheme 8, the most favorable pathway in PhnZ involves an extremely unusual “inverse” heterolytic O-O cleavage, which initiates complicated concerted multiple bond breakages/formations and leads directly to the insertion of O into the C-P bond. Of note is that high-valent iron-oxo species is avoided in this oxidation pathway. On the contrary, the O-O cleavage in MIOX (and also in HEPD) is homolytic one, which commonly leads to the high-valent Fe(IV)-oxo species plus a radical on the distal O atom. Then it is followed by a series of complicated stepwise rearrangement processes involving H-abstractions by distal O radical and Fe(IV)-oxo, C-C cleavage, electron transfer,


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and proton transfer processes, during which the FeIV/FeIII cofactor generated from O-O homolytic cleavage in MIOX is finally reduced to FeIII/FeII by the substrate.53 A key residue inducing such an unusual “inverse” O-O heterolytic cleavage in PhnZ is His62, which is H-bonded to the hydroxyl group of the bound substrate. As shown in Figure 3, for peroxide intermediate CFeOOC immediately preceding the “inverse” O-O heterolytic cleavage process, this His residue acts as a proton acceptor. While for DFeOCO immediately after this “inverse” O-O heterolytic cleavage, His62 changes to act as a proton donor. In addition, comparing PhnZ and MIOX in Scheme 8, it is intriguing to see that in MIOX, the Lys127 residue corresponding to His62 in PhnZ is not able to act as proton donor for the deprotonated hydroxyl group in myo-inositol substrate. All these results strongly imply that His62 specifically plays pivotal role for PhnZ to adopt the “inverse” heterolytic cleavage reaction pathway, by acting as temporal proton acceptor/donor to the hydroxyl group. Interestingly, during the initial Habstraction step generating the ferric hydroperoxo species BFeOOH, His62 accepts the proton from the hydroxyl group of the substrate. However, His62 returns this proton back to the hydroxyl group after ferric peroxide species C′FeOOC is reached, whereas for CFeOOC, His62 keeps this proton. This result, as well as the back-and-forth proton transfer between His62 and hydroxyl group binding to iron, indicate that His62 is the sensitive harnessing factor for reaction mechanism in PhnZ. In summary, His62 is key to render the unusual mechanism of PhnZ. Our current discovery of the “inverse” O-O heterolytic cleavage in PhnZ is reminiscent of the recent study of a synthetic mononuclear non-heme iron(III)-hydroperoxo complex containing TMC ligand by Nam, Shaik and their coworkers, in which similar “inverse” O-O heterolytic cleavage was proposed to account for the mechanism of sulfoxidation reaction thereof.57 Interestingly, it was found in that synthetic mononuclear complex that both high-spin (S=5/2) and medium-spin (S=3/2) iron(III)-hydroperoxo can


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induce the “inverse” O-O heterolytic cleavage,57 while here in diiron enzyme PhnZ, only high-spin iron(III)-hydroperoxo unit (LF, HA) shows this character and the corresponding medium spin iron(III)hydroperoxo (MF) fails to induce this “inverse” O-O heterolytic cleavage, but only exhibits usual O-O homolytic cleavage behavior (Figure S1 in the SI), which highlight the difference between the diiron enzyme and mononuclear synthetic systems. All in all, the current results on the dinuclear non-heme enzyme for oxidative C-P cleavage, associated with the previous ones on a synthetic mononuclear nonheme system for sulfoxidation reaction,57 strongly suggest that the “inverse” O-O heterolytic cleavage mode is not only restricted to enzyme or diiron system, but also could have broader potential implication on mononuclear iron synthetic and enzymatic systems for oxidative process involving the heteroatoms such as sulfur and phosphorus. Generally, this “inverse” O-O heterolytic cleavage mode is a promising alternative O-O activation scenario to avoid the involvement of high-valent iron-oxo species, as featured in some recently emerging iron-mediated oxidizing reactions.57-59

4. CONCLUSIONS In this work, from QM/MM calculations we reveal that the mechanism of organophosphonate catabolism by diiron oxygenase PhnZ consists of four consecutive steps: (1) H-abstraction of α-H atom in organophosphonate substrate (R)-OH-AEP by Fe(III)-superoxo; (2) Formation of Fe(III)OOCα peroxide; (3) Concerted O atom insertion into C-P bond of organophosphonate substrate initiated by “inverse” heterolytic O-O cleavage; (4) Hydrolysis of the generated phosphate to produce the final products of glycine and Pi. The rate-limiting step of the whole pathway is the first H-abstraction step. Concerning the key breakage of the highly stable C-P bond, the enzymatic reaction mechanism is characterized uniquely by the “inverse” heterolytic O-O cleavage of Fe(III)OOCα peroxide intermediate, ACS Paragon Plus Environment

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which generates Fe(III)-O– and renders the distal O atom more oxidative to oxygenate the substrate. In this way, PhnZ adopts a quite different mechanism compared with the closely related and structurally most similar non-heme diiron oxygenase MIOX, for which His62 residue in PhnZ is found to play a key role. This unusual “inverse” heterolytic O-O cleavage mode, apart from the well-known homolytic cleavage and usual “normal” heterolytic O-O cleavage, constitutes a third iron-mediated O-O activation scenario in nature. Based on this work for PhnZ and previous work on non-heme mononuclear synthetic complex, this extremely unusual “inverse” heterolytic O-O cleavage is expected to find its broad occurrence in oxidative transformation involving heteroatoms such as sulfur and phosphorus. Finally, it is disclosed that by inducing the electron transfer between the mixed-valent FeII/FeIII diiron cofactor, oxygen molecule may interestingly “self-catalyze” its own iron-binding and activation in PhnZ.

ASSOCIATED CONTENT Supporting Information. Tables S1-S4 and Figures S1-S5 of computational results, Cartesian coordinates of the optimized structures. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments. This work was supported by the National Natural Science Foundation of China (21290194, 21521062, and 21473215), and Institute of Chemistry, Chinese Academy of Sciences.

5. REFERENCES ACS Paragon Plus Environment

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6. TOC


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