Breaking Benzene Aromaticity â Computational ... - ACS Publications

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Cite This: J. Am. Chem. Soc. 2017, 139, 14488-14500

Breaking Benzene AromaticityComputational Insights into the Mechanism of the Tungsten-Containing Benzoyl-CoA Reductase Martin Culka,† Simona G. Huwiler,‡ Matthias Boll,‡ and G. Matthias Ullmann*,† †

Computational Biochemistry, University of Bayreuth, Universitätsstrasse 30, NW I, 95447 Bayreuth, Germany Microbiology, Faculty of Biology, University of Freiburg, Schänzlestrasse 1, 79104 Freiburg, Germany

‡

S Supporting Information *

ABSTRACT: Aromatic compounds are environmental pollutants with toxic and carcinogenic properties. Despite the stability of aromatic rings, bacteria are able to degrade the aromatic compounds into simple metabolites and use them as growth substrates under oxic or even under anoxic conditions. In anaerobic microorganisms, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-coenzyme A, which is enzymatically reduced to cyclohexa-1,5-dienoyl-CoA. The strictly anaerobic bacterium Geobacter metallireducens uses the class II benzoyl-CoA reductase complex for this reaction. The catalytic BamB subunit of this complex harbors an active site tungsten-bispyranopterin cofactor with the metal being coordinated by five protein/cofactor-derived sulfur atoms and a sixth, so far unknown, ligand. Although BamB has been biochemically and structurally characterized, its mechanism still remains elusive. Here we use continuum electrostatic and QM/MM calculations to model benzoyl-CoA reduction by BamB. We aim to elucidate the identity of the sixth ligand of the active-site tungsten ion together with the interplay of the electron and proton transfer events during the aromatic ring reduction. On the basis of our calculations, we propose that benzoyl-CoA reduction is initiated by a hydrogen atom transfer from a W(IV) species with an aqua ligand, yielding W(V)−[OH−] and a substrate radical intermediate. In the next step, a proton-assisted second electron transfer takes place with a conserved active-site histidine serving as the second proton donor. Interestingly, our calculations suggest that the electron for the second reduction step is taken from the pyranopterin cofactors rather than from the tungsten ion. The resulting cationic radical, which is distributed over both pyranopterins, is stabilized by conserved anionic amino acid residues. The stepwise mechanism of the reduction shows similarities to the Birch reduction known from organic chemistry. However, the strict coupling of protons and electrons allows the reaction to proceed under milder conditions.

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V).5,6 Facultative anaerobic microorganisms use ATP-dependent class I benzoyl-CoA reductase (BCR) to overcome the high energy barrier for the reduction of benzoyl-CoA to cyclohexa1,5-diene-1-carboxyl-CoA (dienoyl-CoA).2 Strict anaerobes use a different strategy. When grown on benzoate, the anaerobic bacterium Geobacter metallireducens initiates production of a class II BCRan eight-subunit complex BamBCDEFGHI (Bam stands for benzoic acid metabolism).7 This BamB-I complex drives the endergonic benzoyl-CoA reduction to dienoyl-CoA (E°′ = −622 mV),8 presumably by flavin-based electron bifurcation9 instead of coupling to ATP hydrolysis. The BamBC part with BamB harboring the active site has been further characterized biochemically and structurally.8,10,11 The Bam(BC)2 heterotetramer contains iron−sulfur clusters, tungsten, and zinc10,11 (Figure 1A). The BamC subunits, which presumably connect the BamBC to the rest of the BamB-I complex, bind three [4Fe−4S] clusters each. The BamB

INTRODUCTION Both natural and man-made homocyclic aromatic compounds are persistent in the environment due to the high stability of the aromatic rings. Toxic aromates originating from industrial waste pollute the groundwater and therefore pose a serious threat to human health. Only microorganisms are able to degrade the aromatic compounds to simple metabolites and thus ultimately serve as bioremediators of the environment. While under aerobic conditions, molecular oxygen is used to hydroxylate and cleave aromatic rings; anaerobic bacteria developed a reductive strategy to break the aromaticity.1 The thioester of benzoic acid and coenzyme A (benzoyl-CoA) is a central intermediate in the anaerobic degradation of aromatic compounds.2,3 Breaking the aromaticity of the benzene ring by reduction is a mechanistically difficult reaction. In organic chemistry, this reaction is facilitated by very strong reductants, such as sodium, lithium, or potassium in protic organic solvents, and is called Birch reduction.4 The reaction occurs in a stepwise manner, forming a high-energy anionic radical intermediate having a redox potential that is out of reach for biological systems (E° < −3 © 2017 American Chemical Society

Received: July 6, 2017 Published: September 18, 2017 14488

DOI: 10.1021/jacs.7b07012 J. Am. Chem. Soc. 2017, 139, 14488−14500

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Journal of the American Chemical Society

Figure 1. (A) X-ray structure of the Bam(BC)2 heterotetramer with the redox cofactors ([4Fe−4S] clusters and bis-WPT) and the substrate (benzoyl-CoA) highlighted. (B) Detail of the active site of the catalytic BamB subunit. Two pyranopterin cofactors along with C322 and an uncertain inorganic ligand X are coordinating the tungsten. Polar amino acids that are responsible for substrate binding and proton transfer during the reaction cycle are shown in the protonation state used as a starting point for the mechanistic investigation in QM/MM environment.

Despite the sequence and structural similarity of the BamB subunit to the aldehyde oxidoreductase (AOR) family of the tungsten enzymes, its catalytic mechanism is unlikely to be similar.15 In most of the Mo/W enzymes, the substrate or product is directly coordinated to the metal in the course of catalysis and a net oxygen atom transfer is performed.16,17 For BamB, an unusual second-shell mechanism is anticipated, since no oxygen atom is abstracted from or added to the benzoylCoA and the tungsten ion is shielded by its ligands. In addition, the surrounding residues hinder the bulky aromatic ring to approach the metal center directly. As with the majority of the Mo/W enzymes, BamB catalyzes a net two-electron transfer. This reaction is facilitated by the ability of tungsten to cycle between fully reduced W(IV) and fully oxidized W(VI) states with the possibility of a W(V) intermediate that may be involved in single-electron transfer/hydrogen atom transfer steps. EPR spectroscopy provided evidence of a stable W(V) state in the active site of the as-isolated BamB in the absence of substrate.10 The reduction of benzoyl-CoA was shown to be a reversible two-electron process occurring at the negative limit of biological electron transfer.8 The catalytic turnover number of the backward process, i.e., dienoyl-CoA oxidation, is 52 s−1.10 The presence of zinc ions was reported to inhibit the reaction.11 Moreover, zinc is only present in a presumably inactive substrate-free X-ray structure, suggesting a regulatory role of the zinc ion.11 Despite the above-mentioned experimental hints, the detailed mechanism of a rather difficult disruption of the aromaticity is far from clear. In this paper, we use a combination of computational approaches to get more insight into the mechanism of the enzymatic benzoyl-CoA reduction by BamB. Since the identity of the unknown ligand X is still unclear, one of the goals of this study is to address the impact of the identity of ligand X on the mechanism of catalysis. We consider common inorganic ligands found in the Mo/W enzymes (water and sulfide in its various protonation forms), as well as unusual diatomic ligands (cyanide and carbon monoxide) proposed by the EXAFS study.11 Continuum-electrostatics, in combination with Monte Carlo titrations, is used to determine the protonation behavior with respect to the oxidation state of the tungsten center.

subunit contains one additional [4Fe−4S] cluster that complements the electron transfer chain of BamC in order to supply electrons to the active site. The main element of the active site is the tungsten ion coordinated by four sulfur atoms of the dithiolene moieties of two pyranopterin cofactors (together designated as bis-WPT), a sulfur atom from a conserved cysteine residue (C322), and an additional ligand (denoted as X in further text), which could not be reliably resolved in the Xray structure (see Figure 1B). The electron density maps seemingly speak against smaller ligands (C/N/O) and suggest an electron-rich ligand such as sulfur.11 However, the best available BamBC structure was solved at 1.85 Å resolution. But to solve unambiguously the position of the ligands around an electron-rich element such as tungsten, one needs a highresolution structure below 1 Å,12 a goal that might not be easily achievable. Extended X-ray absorption fine-structure (EXAFS) data11 indicated the presence of a tungsten-bound atom at 2.0 Å and probably one at 3.2 Å, suggesting a diatomic ligand (cyanide or carbon monoxide) and excluding a sulfur species as the sixth ligand. The Fourier transform peak at 3.2 Å drastically decreases in the 1,5-dienoyl-CoA-reduced protein.13 Moreover, FTIR spectroscopy did not show a signature of cyanide or carbon monoxide,11 which raises doubts if the ligand is diatomic. The bam cluster of Geobacter metallireducens contains an FdhD-like protein. In Escherichia coli and some other organisms, FdhD is responsible for sulfuration of the molybdenum center of formate dehydrogenase during the cofactor maturation process and for the insertion of the cofactor into formate dehydrogenase. In the FdhD-like protein of G. metallireducens, however, one of the two active-site cysteines responsible for sulfuration14 is not conserved, which could indicate that this FdhD-like protein has lost the sulfuration activity and is only responsible for the cofactor insertion into BamB. A tungsten site with five sulfur ligands (four from the dithiolenes and one from the cysteine) is also inline with the EXAFS data.11 Nevertheless, the nature of the sixth ligand of the tungsten has not been resolved until now, but since the aromatic ring of the benzoyl-CoA is bound only 4 Å from ligand X (see Figure 1B), its role in the catalysis is expected to be crucial. 14489


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ranging from −4 to 20 in steps of 0.2 pH units. For every pH step, the MC calculation considered 300 equilibration and 20 000 production scans. For reprotonation energy calculations in the intermediate state, two conformations of the active site titratable residues derived from QM/MM calculations were considered in one Monte Carlo titration.30 The electrostatic interaction energy difference (calculated by CHARMM) and solvation energy difference (calculated by APBS32) were added in order to obtain the energy needed for conformational change.33 Continuum-electrostatic calculations were performed on both conformations, and Monte Carlo titrations were used to predict the most probable protonation states within the set of the two conformations. The free energy difference between the proton configuration in the QM/MM simulation after the first reaction step and the closest state with one more proton was taken as the reprotonation energy. Electrostatic potential maps were calculated using APBS.32 QM/MM Model Setup. The BamB subunit was divided into a core quantum-mechanical (QM) region surrounded by a molecularmechanical (MM) region. The QM region comprised of the bisWPT cofactor (including side chain of the coordinating cysteine residue C322 and the ligand X in several variants), the benzoylthioester part of the benzoyl-CoA, and side chains of H260, E251, E257, and K325 that form a proton transfer chain (Figure 1B). All side chains were truncated between Cα and Cβ. The rest of the protein, the crystal water, and an additional 6 Å surface water layer formed the classically treated MM region. For the active site, the following protonation states were used in the initial state based on the continuum-electrostatic calculations: the neutral form of H260 (protonated at Nε2); the protonated form of E251, E257, and E461; and the protonated form of K325 (see also Figure 1B). pDynamo,34 in combination with ORCA,18 was used for all QM/MM calculations. Unrestricted DFT (BP86 functional35,36 with the def2-SVP37 basis set) was used for the QM part. Stuttgart−Dresden ECPs were used for 60 core electrons of W.20,21 The CHARMM2725 force field was used for the MM part. A link-atom scheme and electrostatic embedding as implemented in pDynamo were used to model the QM/MM boundary. Harmonic restraints were applied on the MM atoms beyond 8 Å from any QM atom, i.e., the QM region was surrounded by 8 Å of flexible MM layer. Between 8 and 16 Å from the QM region, the force constants of the MM atoms restraints linearly increased from 0 to 12 kcal/(mol Å). Beyond 16 Å, the restraints were set to the maximal force constant 12 kcal/(mol Å). Reaction Path Search. The crystal-structure-derived initial coordinates were minimized by a conjugate gradient minimizer. Potential energy surface scans with a step size of 0.1−0.4 Å were preformed to find first estimates for transition states and intermediate states. Intermediate states were subsequently minimized by a conjugate gradient minimizer. The RMS gradient threshold for all minimizations and surface scans was set to 0.02 kcal/(mol Å). The stable minimized intermediates and transition state estimates from surface scans were further refined using the PyCPR38 implementation of the conjugated peak refinement (CPR) method39 in order to obtain estimates of the transition states. In addition, PyCPR was also used to derive a path between two stable intermediates de novo, or a roughly optimized path obtained from the growing string method40 was used as an input. The transition state energies presented in this paper correspond to the structures marked as saddle points by PyCPR.

Combined quantum mechanical/molecular mechanical methods (QM/MM) are employed to find the reaction path starting from a given protonation and oxidation state. In some cases, the QM/MM energy profiles are connected using the energy differences between protonation states derived from continuum-electrostatics. On the basis of our results, we are able to suggest the most probable reaction mechanism and exclude several alternative scenarios for enzymatic benzene ring reduction.

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METHODS

QM Calculations on Small Models. Molecular models of the tungsten center were minimized in the gas phase by the QM program ORCA18 using the B3LYP functional19 and the def2-TZVP basis set.20 Stuttgart−Dresden effective core potentials (ECPs) were employed for 60 core electrons of W.20,21 Each of these models consisted of a tungsten ion, two pyranopterin cofactors (truncated before the phosphate groups), methylthiolate as a model for cysteine, and a small inorganic ligand X, for which we tested several options. The pyranopterins were both considered in the tetrahydropterin form based on pyranopterin dihedral angle analysis in the available BamB crystal structures11 as described in ref 22. A large number of combinations of tungsten oxidation states [W(IV), W(V), and W(VI) states] and ligand X candidates (S2−/SH−/SH2, O2−/OH−/OH2, CN−/CNH, and CO) was considered. Furthermore, the diatomic ligands (CN/CNH and CO) were considered in two orientations: with the carbon atom pointing either toward or away from the tungsten ion. CHELPG charge fits23 were performed on the optimized structures to get partial atomic charges for MM and continuumelectrostatic calculations. Enzyme Structure Preparation and Analysis. The structure of the catalytic BamB subunit was taken from the X-ray structure of Bam(BC)2 heterotetramer11 with bound inhibitor cyclohex-1-ene-1carboxyl-CoA (monoenoyl-CoA) that has the best resolution of all available crystal structures (1.85 Å, PDB ID 4Z3X). Monoenoyl-CoA was replaced by benzoyl-CoA, the substrate of BamB. The “as-isolated structure” (PDB ID 4Z40, resolution 2.35 Å) was taken as a model for the calculations on the substrate-free structure. Structures with the tungsten center in three different oxidation states (IV, V, and VI) and with different ligands X in several protonation states (S2−/SH−/SH2, O2−/OH−/OH2, CN−/CNH, and CO) were prepared. The starting model was set up using the program CHARMM,24 and the force field parameters were taken from the CHARMM2725 force field. Charges of the tungsten center in different oxidation states with different inorganic ligands X were obtained from the QM-optimized model compounds in the gas phase. For the iron−sulfur cluster, we used charges for the reduced state taken from the literature.26 For benzoylCoA, force field parameters were taken from analogous atoms in the CHARMM force field. The charges for the benzoyl part were obtained from gas-phase QM optimization and CHELPG charge fits. The charges for the zinc site of the substrate-free structure were obtained from the cluster model containing the side chains of E251, E257, H260, and H255 (truncated at the Cβ) using gas-phase QM optimization and CHELPG charge fit. Missing hydrogen atoms were added by the HBUILD routine of CHARMM, and their positions were minimized while the non-hydrogen atoms were kept fixed. For the QM/MM calculations, the protonation state of the titratable side chains was set according to the results obtained from titration calculations as described below. Solvent channels were analyzed and filled with water molecules using McVol.27 Continuum-Electrostatic Calculations. The protonation probabilities were evaluated using the Poisson−Boltzmann continuumelectrostatic model with Monte Carlo titration.28 For these calculations, MEAD29 and GMCT30 were used. In the Poisson− Boltzmann continuum electrostatic model, the dielectric constant of the protein was set to 4 and that of the solvent to 80. The ionic strength was set to 0.15 M. The protonation probability was calculated using a Metropolis Monte Carlo algorithm31 as a function of pH

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RESULTS The central tungsten ion in the active site of BamB (Figure 1) is coordinated by two pyranopterin cofactors, a cysteine (C322), and an unidentified ligand X, which could not be reliably resolved from the crystal structure. 11 In our calculations, we considered several monatomic and diatomic ligands in different protonation states as candidates for X. Namely, we considered S2−/SH−/SH2, O2−/OH−/OH2, CN−/ CNH/NCH, and CO. 14490


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Figure 2. Electrostatic potential map of the active site of BamB with benzoyl-CoA bound to the enzyme (tungsten atom is in the center) and its changes depending on the identity and protonation of the ligand X and the oxidation state of the tungsten atom. Positive potential is shown in blue, neutral in white, and negative in red. The numbers represent an overall charge of the tungsten center.

calculated protonation free energy at pH 743 of E461 shifts from an avarage value of 5.5 to −2.1 kcal/mol, leading to the protonation of this residue. Using the program McVol, we identified a water channel enabling E461 protonation with the substrate bound in the cavity (Figure S1, SI). The side chains of K325, E257, H260, and E251 form a proton transfer chain spanning from two solvent channels (Figure S2, SI) to the active site where E251 and H260 neighbor the substrate (see Figure 1B). Note that E251, E257, and H260 together with solvent-exposed H255 are coordinating the zinc ion in the substrate-free structure, and thus, the proton transfer is hindered in the substrate-free state.11 The protonation of this proton transfer chain varies slightly with the oxidation state of the tungsten center and the identity of ligand X. The states, in which the total charge of the proton transfer chain is +1, are more accessible when the tungsten center is reduced [W(IV)], while upon tungsten oxidation to W(V) and W(VI), the states with a neutral proton transfer chain and the proton of H260 pointing away from the substrate are dominating (see Tables S6−S32, SI). Since several protonation states have very similar energies (differences within 5 kcal/mol) in combination with the W(IV) and W(V) oxidation states, we have chosen among the energetically accessible states the one where the protons are favorably oriented for catalysis and the proton transfer chain contains enough protons to allow reduction of the aromatic ring (Figure 1B). In particular, we have set K325 as protonated, E257 as protonated, H260 as singly protonated on Nε2, and E251 as protonated as a starting point for all reaction path searches. The protonation state of the tungsten center cannot be investigated by continuum electrostatic methods, since no pKa values of appropriate model compounds exist. Instead, we compared here the dependence of electrostatic potential maps of the active site on the protonation state of ligand X and the

Orientation of the Diatomic Candidates of Ligand X. Before starting the investigation of reaction paths, the orientation of the diatomic ligands relative to the tungsten ion had to be resolved. In the majority of the structurally known transition-metal complexes, the carbon atom is coordinating the metal, in the case of both cyanide and carbon monoxide.41,42 When we compared the calculated energy of the diatomic ligands when bound to the tungsten center in an isolated BamB bis-WPT-center model in the gas phase in the two different orientations, we find a clear trend. Namely, the energy of all relevant complexes with the carbon atom orientated toward the tungsten ion (W−[CN−], W−[CNH], and W−[CO]) were consistently lower compared to the alternative orientation {W−[NC−], W−[NCH], and W− [OC]; Table S1, Supporting Information (SI)}. These data indicate that the carbon atom of the diatomic ligand is oriented toward the tungsten ion. Thus, we considered only the variants with a W−C bond in further calculations with CN−, CNH, and CO. In total, nine variants of ligands X (S2−/SH−/SH2, O2−/ OH−/OH2, CN−/CNH, and CO) were considered in this study. Active Site Protonation State. As shown in Figure 1B, the aromatic ring of the benzoyl-CoA is bound in the vicinity of the tungsten center. But also several protonatable amino acid side chains are near this aromatic ring, which are likely to be involved in the proton transfer steps during catalysis. To probe the involvement of these residues, we analyzed the protonation behavior of BamB using continuum electrostatics. E461 forms a hydrogen bond to the carbonyl oxygen of the thioester of CoA and is therefore suggested to play a role in stabilizing negatively charged intermediates by partial protonation of this thioester carbonyl. This scenario requires that E461 is protonated. According to our calculations, E461 is deprotonated in the substrate-free state. However, upon substrate binding, the 14491


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the BamB subunit, including the phosphate groups of the pyranopterins, was treated by MM. The reduction of benzoyl-CoA to dienoyl-CoA involves the net transfer of two electrons and two protons. The starting state of the reaction can be either a fully reduced W(IV) oxidation state or a semireduced W(V) state. In the later case, the overall reduction would involve two W(V)−W(VI) transitions, and the tungsten center would have to be reduced by the iron−sulfur clusters of the protein between the first and the second reduction step. The reduction of the aromatic ring is accompanied by the transfer of protons to the para (C4) and meta (C3) position of the aromatic ring of benzoyl-CoA, resulting in a cyclic diene. In the vicinity of the aromatic ring, there are three potential proton donors (see Figure 1): H260, E251, and the protonated variants of ligand X. On the basis of the above-discussed electrostatic considerations, we analyzed SH−, SH2, OH−, OH2, CN−, CNH, and CO as potential candidates for ligand X in the W(IV) state and SH−, OH−, and CN− as potential candidates for ligand X in the W(V) state. In total, we considered 10 starting configurations of combinations of the tungsten oxidation state and various candidates of ligand X. For each of these configurations, the energetics of the first reaction step from one of the available proton donors was investigated with QM/MM calculations. A schematic representation of the different scenarios is given in Figure 4. We performed the simulation of the second step only for the variants that had promising energetics in the first step. In total, 27 alternative mechanistic possibilities were considered for the first reaction step and 12 possibilities for the second step. The reaction energies and barriers of all the tested alternative reaction steps will be discussed in the next paragraphs and are summarized in Tables 1 and 2. First Reaction Step. There are two potential situations for starting the reaction. The electron transfer reaction may start either from a W(IV) state or from a W(V) state. In addition, the reaction is accompanied by a first protonation of the reduced aromatic ring. The two alternative mechanisms are discussed in the following sections. First Reaction StepScenario I: Starting from W(IV). First, we analyze how the first electron and proton will be transferred starting from the fully reduced tungsten center [W(IV)]. If the neutral candidates for ligand X0 are considered (the superscript indicates the charge of ligand X; SH2, OH2, CNH, and CO), almost no unpaired spin density is observed in the optimized structure (Tables S2 and S3, SI). For the negatively charged candidates for ligand X− (SH−, OH−, and CN−), however, a significant amount of unpaired spin is observed on the tungsten center and on the substrate (Table S2, Figure S3, SI). This spin distribution indicates that electron transfer from the tungsten center to the substrate can happen at least partially prior to the proton transfer if the first coordination sphere of the tungsten ion is very negatively charged. The reaction energies and barriers for all scenarios starting from W(IV) are summarized in Table 1. The ligand X donates its proton to C4 of the substrate (Figure 4A,A′) with a reasonable barrier (4−13 kcal/mol) in the case of singly or doubly protonated sulfur or oxygen ligands (Figure 5A,C). In contrast, the barrier for protonated cyanide is much higher (18.1 kcal/mol, Figure 5B). In the case of neutral ligands (SH2, OH2, and CNH), an intermediate state InA with a benzoylCoA radical is formed [Figure 6 and Table S2 (SI)]. The intermediate InA is on roughly the same energy level as the initial state in the case of SH2 and OH2 (a difference of about

oxidation state of the tungsten ion. Since the active site is buried in the center of the protein, an overall balance of positive and negative electrostatic potential regions is expected. This balance can be seen in potential plots when red and blue regions take about the same areas. As seen from the electrostatic maps in Figure 2, in order to keep the electrostatic environment balanced in a rather hydrophobic and deeply buried active site cavity, it is advantageous to couple protonation and reduction. States of the tungsten center that seem to be stable from an electrostatic point of view have a charge of −1. The qualitative analysis of the electrostatic maps suggests that deprotonation of ligand X accompanying tungsten oxidation is favored during the catalytic process, indicating that ligand X is one potential proton donor. The finding that a charge of −1 is preferred at the deeply buried tungsten center is surprising at first sight. However, two α-helices are pointing with their N-terminal end toward the bisWPT-cofactor. Thus, the electrostatic dipoles of the α-helices cause a positive contribution to the electrostatic potential in this region (see Figure 3). In fact, helix dipoles can play an

Figure 3. Tertiary structure of BamB. The tungsten center (bis-WPT), the [4Fe−4S] cluster, and the benzoyl-CoA are highlighted. The two helices shown in orange (residues 323−333 and residues 355−369) are pointing with their N-terminal end toward the active site, thus making a positive contribution to the overall electrostatic potential in this region.

important role in tuning the electrostatic potential inside proteins without the placement of an explicit charge if they are not crossing the whole protein but end in the middle of the protein.44 Investigated Variants of the Mechanism. In order to investigate possible catalytic mechanisms, we constructed a QM/MM model of the catalytic BamB subunit in the abovedescribed protonation state. The QM region representing the active site consisted of the benzoyl moiety of benzoyl-CoA, including the thioester, the tungsten center with all the ligands (side chain of C322, two pyranopterins without the phosphate ions, and the ligand X), and the side chains of the discussed amino acids above (K325, E257, E251, and H260). The rest of 14492


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Figure 4. Schematic representation of the possible first reaction steps. Red arrows represents electron transfer steps, and blue arrows represent protonation steps (direction of the arrows in organic chemistry conventions). The intermediate is represented by the substrate molecule together with the relevant amino acid side chains. (A) Ligand X donates the proton to the substrate and a radical intermediate is formed. (A′) Ligand X donates the proton to the substrate and an anionic intermediate is formed [occurs starting from the W(IV) state with SH− or OH− as X]. (B) H260 donates the proton to the substrate. (C) E251 donates the proton to the substrate.

Table 1. Transition State and Reaction Energies (in kcal/mol) for the First (index 1) and Second (index 2) Proton−Electron Transfer Steps Starting from the W(IV) Statea D1 = ligand X (A and A′) 1st step X

ΔE⧧1

SH− SH2 OH− OH2 CN− CNH CO

3.8 5.0 13.0 11.0 −c 18.1 −c

D1 = H260 (B)

2nd step ΔE1

ΔE⧧2

−7.4 −2.0 −12.2 −1.9 −c 16.5 −c

0.5 4.7 0.3 9.3 −c 0.8 −c

1st step

ΔE2

ΔE⧧1

−13.0 −0.6 −14.8 −0.2 −c 0.5 −c

4.2 21.9 3.1 20.7 5.9 25.7 15.9

D1 = E251 (C) 2nd step

ΔE1

ΔE⧧2

−1.6 21.7 −6.3 20.5 −0.8 25.6 15.1

6.8 −b 4.6 −b 3.1 −b 10.2

1st step ΔE2

ΔE⧧1

−8.2 −b −4.5 −b −4.3 −b 7.5

16.5 29.7 13.0 29.0 23.9 28.6 32.5

2nd step ΔE1

ΔE⧧2

ΔE2

7.1 29.1 3.3 28.4 9.7 28.6 28.8

14.7 −b 12.7 −b 15.9 −b −b

13.2 −b 8.5 −b 13.9 −b −b

D1 represents the first proton donor; the second one is always H260. bNot determined, since the first step is already energetically unfeasible. Ligand X carries no proton to donate in the first step; thus, the second step is also not meaningful.

a c

In an alternative scenario, the first proton, which neutralizes the electron on the substrate, does not originate from ligand X but from H260 (Figure 4B). In our calculations, we moved the proton from H260 to the atom C4 of benzoyl-CoA. The spin population analysis (Table S3, SI) shows that proton transfer leads to a electron transfer from W(IV) to the substrate. H260 can donate a proton to C4 of the substrate with a low barrier (3−6 kcal/mol) for negatively charged X− ligands (SH−, OH−, and CN−) (Table 1). In the case of neutral ligands X0 (SH2, OH2, CNH and CO), however, the barrier is much higher (16−26 kcal/mol). The energy of intermediate InB drops to a lower level (−1 to −6 kcal/mol) with respect to the initial state in the case of negatively charged ligands X−, but it remains on the same level as the transition state (15−26 kcal/mol) in the case of neutral ligands X0. Note that in the case of a CN− ligand, it is possible to transfer a proton steeply downhill from H260 via the substrate to CN−. The proton transfer is accompanied by an electron transfer from the radical intermediate leading to W(IV)−[CNH]. Such an unwanted reaction can also occur for SH− and OH−. In these cases, the reaction has a reversible energetics (energy change 0.5 and −0.4 kcal/mol for SH− and OH−, respectively). In the case of CN−, however, this transition is associated with a large drop in

Table 2. Transition State and Reaction Energies (in kcal/ mol) for the First Proton−Electron Transfer Starting from W(V) State proton donor ligand X

a

H260

E251

X

ΔE⧧

ΔE

ΔE⧧

ΔE

ΔE⧧

ΔE

SH− OH− CN−

11.3 18.5 −a

5.1 3.3 −a

16.0 15.8 19.8

16.0 12.9 19.3

29.2 27.7 31.7

26.5 23.4 29.6

Ligand X carries no proton to donate.

−2 kcal/mol). For CNH, however, the energy of the radical intermediate remains high (16.5 kcal/mol) after the first reduction step. In the case of the negatively charged protonated ligands (SH− and OH−), no unpaired spin is seen (intermediate state InA′) (Table S2, SI), which indicates that the second electron has already been transferred and an anionic intermediate has been formed. The energy of the intermediate InA′ drops significantly compared to the initial state (−7 to −12 kcal/mol, Figure 5A,C). 14493


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Figure 5. Energy profiles of the most energetically feasible mechanism scenarios of BamB mechanism for ligand X = SH−/SH2, OH−/OH2, CN−/ CNH, and CO. All reaction profiles start from the W(IV) state. For the first protonation, different donors were tested. For the second protonation, H260 was always assumed to be the proton donor.

donors (Figure 4B,C) for all the ligand X candidates considered here. In addition, the energy of the intermediate remains at a high level (13−30 kcal/mol). In all the cases, the unpaired spin on the aromatic ring of the substrate indicates a radical intermediate (Table S5, SI). The electrostatic environment around the tungsten ion allows formation of W(VI) only when ligand X donates the proton. In other cases, the pyranopterin cofactors provide the electron for reducing the substrate instead and the W(V) state is retained (see Mulliken spin population in Table S5, SI). In summary, the reduction of the benzoyl-CoA is difficult when starting from W(V); the only feasible proton donor would be the SH− variant of the ligand X. To complete the overall reduction of the aromatic ring to a stable product, the tungsten center has to be reduced to the W(V) state for the second reduction step. Second Reaction Step. To finish the benzoyl-CoA reduction, a second reduction has to occur in the case of a radical intermediate (intermediates InA, InB, and InC in Figure 4) or the doubly reduced ring has to be protonated (intermediate InA′). Since the conjugated product (1,5dienoyl-CoA) is formed exclusively,45 the second proton needs to be donated to the C3 (meta) carbon of the intermediate. H260 is the only proton donor well positioned near the meta carbon (C3) of the substrate. The relative energy barriers and reaction energies of the second step are summarized in Table 1 directly next to the corresponding first step. Note that some cases were not considered for the second step due to the high energy barriers of the corresponding first step. Energy profiles of all mechanisms for which both steps were considered are depicted in Figure 5. In the case of a radical intermediate after ligand X0 donated the first proton to the substrate (InA in Figure 4), the barrier of the second proton transfer from H260 to the substrate (1−9

energy (−21.5 kcal/mol), making CNH a thermodynamic trap. The reaction barriers for this step are 6.8, 10.0, and 2.2 kcal/ mol for SH−, OH−, and CN−, respectively, implying a fast kinetics particularly in the case of CN−. In principle, also E251 could act as a first proton donor [Figure 4C and Table S4 (SI)]. However, our calculations show that E251 is a much worse proton donor than H260 (Table 1). In addition, the resulting intermediate InC has an energy above the substrate level (3−29 kcal/mol). Taken together, feasible reaction barriers for the first proton transfer were found for ligand X (SH−/SH2/OH−/OH2) as a proton donor, or for H260 as the proton donor with ligand X− (SH−, OH− and CN−). But in the case where CN− would coordinate the tungsten ion, it is thermodynamically favorable to protonate CN− to CNH, which would be a dead end for the reaction. This protonation occurs moreover with a low barrier and is thus kinetically fast. This finding makes CN− an unlikely candidate for ligand X. First Reaction StepScenario II: Starting from W(V). In the following considerations, we assume that the reduction of the substrate occurs starting from the W(V) state of the enzyme. On the basis of our electrostatic considerations discussed above, we consider negatively charged ligands X (SH−, OH−, and CN−) in this scenario. After geometry optimization at the QM/MM model, we find that the unpaired spin density is located mainly at the tungsten center (Table S5, SI). As can be seen from Table 2, SH− as the ligand X can donate the proton to the substrate (Figure 4A) with the lowest barrier (11.3 kcal/mol) among all variants starting from W(V). The energy of the intermediate is reasonably low (5.1 kcal/ mol). Proton transfer from OH− shows a high barrier (18.5 kcal/mol), albeit the formed intermediate has a reasonably low energy (3.3 kcal/mol). The reaction barriers are unfavorable for H260 (16−20 kcal/mol) or E251 (28−32 kcal/mol) as proton 14494


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reprotonated before the reaction can occur. A one-step proton transfer from E251 to H260 virtually converts InB to InC, which is associated with a significant rise in energy (9 to 10 kcal/mol). The conversion from InC back to InB is associated with a negligible barrier, making InC unstable. Nevertheless, we probed the possible protonation of C3 by H260 starting from the InC state and found a high barrier (13−16 kcal/mol) and a high energetic product state with both E251 and H260 deprotonated (13−14 kcal/mol). Another option to proceed from InB is adding one more proton to the system and thus restoring a protonated H260. We addressed the energy associated with the reprotonation using continuum-electrostatic calculations on QM/MM optimized structures.30 The free energy for reprotonating H260 was found to be 3.8, 1.6, −1.6, and 4.6 kcal/mol for SH−, OH−, CN−, and CO, respectively. Once H260 carries a proton, the proton transfer from H260 to the radical intermediate in the presence of X− (SH−, OH−, or CN−) proceeds with a reasonable barrier (3−7 kcal/mol). The reaction is, however, much more difficult with CO as ligand X (barrier 10.2 kcal/ mol, reaction energy 7.5 kcal/mol), which together with the reaction energy of the first step and protonation energy results in an overall reaction barrier of 29.9 kcal/mol (Figure 5D). According to the Mulliken population analysis of the unpaired spin density (Tables S2−S4, SI), a pure W(VI) state is only obtained in the case of full sulfur and oxygen deprotonation (S2− and O2−). In other cases, where ligand X has charge −1, the pyranopterin cofactors form a radical species and the W(V) state is retained (see also ProA in Figure 6). Taken together, the second step of the benzoyl-CoA reduction, which involves a proton transfer from H260 to C3, is energetically feasible provided that E251 remains protonated. Reprotonation of the Ligand X. As the ligand X serves as a proton donor in several mechanistic scenarios considered in this study, its initial protonation needs to be restored after the reaction finishes. Our computational analysis of the binding pocket of the Zn-bound structure and of the ligand-bound structure in which we deleted the bound monoenoyl-CoA indicates that, in both cases, the binding pocket is large enough to hold water molecules. Thus, ligand X can be reprotonated either in the substrate-free state via water molecules that fill the cavity or in the substrate-bound state if E251 protonates C322 and the proton is further transferred to X. We first address this issue in a QM/MM model of a modeled substrate-free active site, supposing that the product has left and the cavity was filled with water. We suggest that ligand X gets reprotonated through a chain of water molecules that we found in our calculations. This chain connects ligand X to E461, which is protonated in the substrate-bound state and hydrogen-bonded to the carbonyl group of benzoyl-CoA (see Figure S4, SI). The barrier for reprotonating W(V)−[X2−] from E461 is 4.6 kcal/mol for S2− and 2.3 kcal/mol for O2−, and the reaction energy is 0.6 kcal/ mol for sulfur and −10.0 kcal/mol for oxygen. However, the reprotonation is not possible with W(VI), where the state W(VI)−[XH−] is not stable and the proton is always transferred back to E461. In a similar fashion, reprotonation of SH− or OH− to SH2 or OH2 is possible only in the presence of W(IV). Interestingly, the barrier and reaction energy for the protonation of OH− to OH2 are, respectively being 3.0 and −0.5 kcal/mol, favorable, while the SH− can be hardly protonated to SH2 using the same mechanism (barrier of 18.2 kcal/mol and reaction energy of 13.4 kcal/mol). For completeness, we also consider protonation of CN− by the

Figure 6. Initial (Sub), intermediate (InA), and product (ProA) structures from mechanism A, where ligand X donates the first proton and H260 the second proton. The preferred variant for X = OH2 and W(IV) starting oxidation state of the tungsten is shown. Unpaired spin density is represented by an isosurface: α spin in orange, β spin in violet (isovalue 0.005 e/Å3). In the ProA state, the radical is distributed over both pyranopterin cofactors.

kcal/mol) as well the reaction energy (0.5 to −0.6 kcal/mol) are relatively low. In contrast, the anionic intermediate (InA′) gets protonated with almost no barrier (0.3−0.5 kcal/mol). The energy drops by 13−15 kcal/mol, making this step virtually irreversible. If the reaction proceeds from InB, which received its first proton already from H260, this histidine residue needs to get 14495


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electron transfer? In order to answer these questions, we first relate to the results of our continuum-electrostatic calculations on BamB together with a search for water channels using McVol. First, realistic scenarios for the charge of the active site had to be chosen. The tungsten ion is coordinated by five thiolate ligands (four from the two pyranopterin cofactors and one from C322), giving rise to a charge of −5. Given the fact that the reaction has to start from a reduced state, i.e., either W(IV) or W(V), it is highly unlikely that the ligand X will be a sulfido (S2−) or oxo (O2−) ligand, since an overall charge of −3 or −2 buried in the cavity would cause destabilization of the structure. Instead, a sulfido or oxo ligand is expected to stabilize the fully oxidized W(VI) state. We thus considered SH−/SH2, OH−/OH2, CN−/CNH, and CO as the starting states of ligand X. Furthermore, continuum electrostatics shows that E461, a residue hydrogen bonded to the carbonyl oxygen of benzoylCoA, is protonated when substrate is bound. In addition, a proton transfer chain (K325, E257, E251, and H260) connects two solvent channels to the active site. As described above, lower oxidation states of the tungsten attract the protons to the proximity of substrate. We performed extensive mechanistic searches using QM/ MM calculations in order to find which combination of ligand X (SH−/SH2, OH−/OH2, CN−/CNH, and CO), proton donors (H260, E251, or ligand X), and starting tungsten oxidation state [W(V) or W(IV)] leads to the most plausible mechanism. Many combinations were shown to be energetically unfeasible (Tables 1 and 2). CO can directly be ruled out as a candidate for ligand X, since no reaction profile with feasible barriers and reaction energies could be found (Table 1 and Figure 5). Apparently, carbon monoxide stabilizes the tungsten strongly in the reduced state [W(IV)], since no W(V) state is formed alongside the benzoyl radical and the electron is donated by the pyranopterin cofactors instead (see Mulliken spin populations in Table S3, SI). For other candidates (sulfur, oxygen, and cyanide), we found at least one realistic energy profile. In the search for potential proton donors, E251 is clearly surpassed by both H260 and protonated ligand X in all the cases and thus E251 can be ruled out as a direct proton donor (Tables 1 and 2). E251 certainly retains an important role in the proton transfer chain, since it can reprotonate Nε2 of H260. Moreover, E251 is coordinating the zinc ion in the substratefree resting state and may thus play an important role in regulating the enzyme activity. When comparing W(V) and W(IV) as starting oxidation states, it is evident that the energy barriers and reaction energies starting from W(IV) (Table 1) are much more favorable than alternatives involving W(V) (Table 2). Thus, a reaction mechanism in which both reduction steps start from W(IV), which would also be a possibility, does not seem to be required. The second reduction step is easily possible directly after the first reduction without the need of an intermediate reduction of the tungsten center with a radical intermediate in the cavity. After these initial observations, seven variants remain starting from W(IV). H260 can donate both protons with negatively charged ligand X (SH−, OH−, or CN−), or ligand X can be the proton donor with SH−, OH−, SH2, or OH2. Isotope-labeling experiments showed the full reversibility of benzoyl-CoA reduction by BamBC.8 This reversibility needs to be reflected in a realistic energy profile of the catalytic mechanism. In the cases where ligand X− (SH− or OH−) donates the first proton, an enormous drop in energy (>20

same mechanism. Like SH2 and OH2, CNH is not stable with W(V) present. With W(IV), the W(IV)−[CN−] state is still slightly more stable than CNH (by 2.7 kcal/mol), and CNH can transfer its proton to E461 with a negligible barrier (0.1 kcal/mol). We next tested an option of the reprotonation of ligand X with the substrate bound in the cavity assuming that substrate binding triggers tungsten reduction to W(IV). We found a mechanism where the proton of the protonated E251 is transferred via the tungsten ligand C322 to the ligand X. The presence of W(IV) is necessary for the protonation of C322 with X = SH−, OH−, or CN−. E251 first transfers its proton to C322 with a barrier of 8.6, 13.7, and 13.6 kcal/mol for SH−, OH−, and CN−, respectively. The energy change is uphill by 6.7, 11.9, and 5.2 kcal/mol, respectively, for SH−, OH−, and CN−. Subsequently, the proton from C322 is transferred to the ligand X with a high barrier (16 kcal/mol and more dependent on the nature of ligand X), which makes the process prohibitive given the previous thermodynamic penalty for C322 protonation. A protonated C322 can be possibly stabilized by adding one more proton to the system and reprotonating E251. Starting from such a state, SH2 and CNH could be obtained with a reasonable barrier (11.8 and 12.7 kcal/mol for SH2 and CNH, respectively). The energy drops to −5.9 and −16.6 kcal/ mol for SH2 and CNH, respectively. We were not able to find any reasonable mechanism for protonating OH− to OH2 in the presence of bound substrate, presumably due to much shorter oxygen−tungsten bonds. In fact, the protonated C322 always had to dissociate from the tungsten in order to protonate the OH−. Note that already a big thermodynamic penalty (11.9 kcal/mol) hinders C322 protonation in the case of OH−. Another potential problem of the reprotonation pathway through C322 is a competitive futile formation of H2. Starting from protonated C322 and W(IV)−[SH−], the barrier for H2 formation is only 6.8 kcal/mol (compared to 11.8 kcal/mol for the protonation of SH−; see Figure S5, SI), and the reaction is highly downhill (−15.9 kcal/mol). In an equivalent situation with protonated C322 and W(IV)−[OH−], the H2 formation is hindered by a huge energy barrier (>30 kcal/mol), apart from the unlikeliness of C322 protonation discussed above. Thus, formation of H2 is a potential side reaction in the case ligand X that would involve a sulfur atom. This H2 formation is much less likely if ligand X would involve an oxygen atom. From the calculations discussed above, we can conclude that the reduction of the tungsten center to W(IV) always has to precede the reprotonation of ligand X. After the reduction of tungsten, it is energetically feasible to protonate the tungsten coordinating S2− or O2− to SH− or OH−, as well as OH− to OH2 from a water chain in the substrate channel. However, protonation to SH2 from solvent is unlikely and CNH is also unstable when the substrate is not bound in the cavity. Moreover, we found no feasible ligand X reprotonation mechanism when the substrate is bound in the cavity. Thus, reprotonation of ligand X seems easily possible in the case of an oxygen ligand and much harder in the case of all other ligands.

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DISCUSSION Reflections on the Results of the Calculations. Before the catalytic mechanism of the tungsten-based BCR BamB can be discussed, three major questions have to be answered: (i) What is the nature of ligand X? (ii) Which oxidation state of the active site is necessary to get a feasible reaction energy profile? (iii) Which proton donors are most suitable to complement the 14496


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Figure 7. Proposed catalytic cycle of radical benzoyl-CoA reduction by BamB derived from our calculations. (1) The cycle starts from a water-filled binding pocket with a fully reduced W(IV) center with a water ligand. (2) The binding of benzoyl-CoA replaces the water and causes the protonation of E461. (3) H260 protonates and the reaction can start to proceed. (4) In the first reaction step, a hydrogen atom is formally transferred to benzoyl-CoA and a radical intermediate is formed. (5) Benzoyl-CoA gets reduced to dienoyl-CoA. The electrons for the reduction are at least partially provided by the pyranopterin rings and eventually from the [4Fe−4S] cluster in the vicinity of the tungsten center. (6) The cationic pyranopterin radical gets reduced and the product leaves the binding pocket. To restore the starting state, the tungsten center gets reduced and the OH− ligand gets reprotonated. Steps 3−5 represent the steps for which the energy profile (solid green line in Figure 5C) was calculated and which is shown in the movie provided in the Supporting Information.

substrate is bound to the protein, but this path has relatively high energy barriers and can thus be ruled out. Moreover, it would be more favorable to form H2 from the W(IV)−[SH−] state than from protonating SH− via C322 (see Figure S5, SI). The findings could suggest that in the fully reduced, substratebound state, a mechanism where W(IV)−[SH−] protonates the substrate directly could be followed instead, but this mechanism involving W(IV)−[SH−] has already been ruled out because of its irreversibility, which contradicts the experimental findings. These results make SH2 an unlikely candidate for ligand X. On the basis of the above observations, we rule out sulfur to be a likely candidate for ligand X. This conclusion is in line with the results from EXAFS,11 which preclude the presence of six sulfur ligands. Two scenarios with an oxygen ligand remain after the above considerations, namely, H260 donates both protons with W(IV)−[OH−], or W(IV)−[OH2] donates the first proton and H260 the second one. From an electrostatic point of view, it is more likely that the reduction/oxidation of the tungsten will be coupled to the protonation/deprotonation of the ligand X in order to change the charge at the active site as little as possible. Furthermore, the spin population observed on the aromatic ring of benzoyl-CoA (Table S3, SI) before any proton transfer happens points to the instability of the W(IV)−[OH−] state. It might thus be difficult to reduce the tungsten with OH− as ligand X. Moreover, the tungsten reduction coupled with the protonation of OH− to OH2 from solvent should be much easier.

kcal/mol) is observed starting from W(IV). This energy drop is in contradiction to the expected reversibility, since it would imply an energy barrier of about 20 kcal/mol for the backward reaction. These results thus indicate that the W(IV)−[X−] starting states are unrealistic. Furthermore, it is unlikely that H260 would donate a proton if SH− coordinates the tungsten, since the SH− would protonate the substrate with a lower barrier first. H260 could donate both protons starting from CN− or OH−. In the case of CN− as ligand X and H260 as the first proton donor, the radical intermediate formed after the first proton-coupled electron transfer can be easily converted back to benzoyl-CoA by transferring the proton from the radical intermediate to CN− and the electron to the tungsten ion (barrier of 2.3 kcal/mol). The resulting W(IV)−[CNH] state is very stable, since our calculations show that its energy is lower by 22 kcal/mol compared to that of the radical intermediate and by 23 kcal/mol compared to that of the initial W(IV)−[CN−] state. Note that W(IV)−CNH is in general a bad starting state (see Table 1). Since the formation of CNH, a thermodynamic trap, cannot be avoided, we consider that ligand X is unlikely to be CN−. SH2 and OH2 could serve as good proton donors with reversible energy profiles. In order to close the reaction cycle, ligand X needs to get reprotonated from the solvent. Our calculations show that W(IV)−[OH−] can be easily protonated through a water channel after the product leaves the binding pocket. The corresponding reprotonation of SH− is, however, thermodynamically hindered by 13.4 kcal/mol. An alternative could be to protonate SH− via protonated C322 when the 14497


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moiety50 similar to what we suggest here for BamB. This finding might lead to new and exciting insights into the function of this unusually complex cofactor. Moreover, the pyranopterin cofactor is hydrogen bonded to three conserved, negatively charged residues (namely, E178, D352, and D529), which can potentially stabilize a positively charged pyranopterin radical, which would form after oxidation of the pterin by the tungsten center (see Figure 8; for conservation, see the

Nature of the Unknown Tungsten Ligand. After exploring many different possibilities, a reaction starting from W(IV) with OH2 as ligand X appears most plausible. Seemingly, the electron density of the crystal structure speaks against an oxygen as the ligand X.11 However, due to the large number of electrons at the tungsten center, Fourier transform artifacts render the estimation of distances from the metal center unreliable at resolutions lower than about 1 Å.12 The best crystal structure of Bam(BC)2 has a resolution of 1.85 Å, which may not be high enough to reliably determine the distance between the tungsten and ligand X. Thus, it is difficult to decide on the chemical nature of ligand X solely from the electron density. The EXAFS spectrum of the BamB tungsten site11 contains a small peak at 2.0 Å, which could also be explained by a hydroxo (OH−) ligand coordinating to a W(V) iona stable state in the possible mechanism that will be discussed in the following paragraph. In our calculations, we find a distance of 1.97 Å for a OH−-coordinated W(V) center, which is inline with the EXAFS data. If this interpretation is correct, the third Fourier transform peak seen in the EXAFS spectrum at about 3.2 Å may be due to an artifact and not due to a diatomic ligand. This interpretation is inline with some experimental data, as discussed in the Introduction. Moreover, the presence of an exchangeable proton in the vicinity of tungsten is evident from some preliminary EPR studies.13 Combined with the EXAFS data, which show that only five sulfur atoms coordinate the tungsten,11 these data support an oxygen-based ligand X. According to our calculations, the reaction would start from a W(IV) state with a coordinated water molecule, which is formed after proton-coupled reduction of the W(V) center. Acetylene hydratase from Pelobacter acetylenicus has the same tungsten coordination sphere: two pyranopterins, one cysteine residue, and an aqua ligand.46 Interestingly, acetylene hydratase has to be fully reduced in order to be active,47 which makes it likely that the fully reduced state is also accessible in BamB. Potential Mechanism of Benzoyl-CoA Reduction with an Aqua Ligand. On the basis of our calculations, we propose a reversible mechanism with an aqua ligand starting from W(IV) (Figure 7). Figure 6 and a movie provided in the Supporting Information depict the chemical steps of the reaction. The first electron transfer from W(IV) to the substrate is coupled to a proton transfer from the aqua ligand. Thus, formally a hydrogen atom is transferred from the tungsten center to the substrate, resulting in a neutral radical intermediate, which represents a minimum on the energy landscape. The second electron transfer from the tungstopterin center triggers a proton transfer from H260 to the substrate. The energy profile of the reaction is depicted by the solid green line in Figure 5C. In our calculations, we observe delocalized spin density formation on the pyranopterin parts of the cofactor after this step and a partial retainment of the W(V) character (see Mulliken population analysis in Table S2, SI). The unpaired spin on the pyranopterins can be a result of the limited size of our QM/MM model, since the electron can be in reality promptly provided by the FeS chain of the Bam(BC)2 complex. Including the FeS clusters to the QM region was beyond our computational feasibility. Note, however, that a pyranopterin radical was observed in EPR experiments with the molybdoprotein aldehyde dehydrogenase.48 The redox activity of pyranopterin cofactors and their role in catalysis are currently discussed in the literature.22,49 For the enzyme YedY, there is strong evidence for a redox-active pterin

Figure 8. Hydrogen bonding of the tungsto-pyranopterin cofactor. The numbers indicate the hydrogen bond distances in angstroms. Interestingly, there are three conserved, negatively charged residues (namely, E178, D352, and D529) that can potentially stabilize a positively charged pyranopterin-centered radical.

Supporting Information). The mechanism of benzoyl-CoA reduction by BamB that we propose here on the basis of our calculations is to the best of our knowledge not in conflict with experimental data and provides a good basis for further experimental mechanistic investigations.

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CONCLUSIONS In this work, we have presented an intensive search for the mechanism of the benzoyl-CoA reduction by BamB, a tungsten enzyme that is part of the larger enzyme complex BamB-I, which drives the benzoyl-CoA reduction. The major problem for understanding the mechanism of BamB is that the nature of one crucial ligand of the catalytic tungsten center is not known. We investigated a large variety of possibilities and tried to derive mechanism of benzoyl-CoA reduction on the basis of our calculations. From all the possibilities that we have investigated, a water molecule seems to be the most plausible candidate for the unknown ligand. The mechanism of BamB that we have obtained from our calculations starts from a fully reduced and protonated W(IV) complex, and formally a hydride, i.e., one proton and two electrons, is transferred from the tungsten center to benzoyl-CoA. The remaining proton to neutralize the negative charge is donated by a histidine of the active site of the protein, leading to dienoyl-CoA. The reaction proceeds, however, not in one step. Instead, according to our calculations, a hydrogen atom is transferred from the tungsten center to the benzoyl ring in a first step, leading to a relatively stable benzoyl-CoA radical intermediate. A second electron transfer from the tungsten center to the radical intermediate 14498


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(9) Buckel, W.; Thauer, R. K. Biochim. Biophys. Acta, Bioenerg. 2013, 1827, 94−113. (10) Kung, J. W.; Löffler, C.; Dörner, K.; Heintz, D.; Gallien, S.; Van Dorsselaer, A.; Friedrich, T.; Boll, M. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 17687−17692. (11) Weinert, T.; Huwiler, S. G.; Kung, J. W.; Weidenweber, S.; Hellwig, P.; Stärk, H.-J.; Biskup, T.; Weber, S.; Cotelesage, J. J. H.; George, G. N.; Ermler, U.; Boll, M. Nat. Chem. Biol. 2015, 11, 586− 591. (12) Burger, E.-M.; Andrade, S. L.; Einsle, O. Curr. Opin. Struct. Biol. 2015, 35, 32−40. (13) Huwiler, S. G. Structure and function of the tungsten-containing active site of class II benzoyl-CoA reductases. Albert-LudwigsUniversität Freiburg, Ph.D. thesis, https://freidok.uni-freiburg.de/ data/11074, 2015. (14) Arnoux, P.; Ruppelt, C.; Oudouhou, F.; Lavergne, J.; Siponen, M. I.; Toci, R.; Mendel, R. R.; Bittner, F.; Pignol, D.; Magalon, A.; Walburger, A. Nat. Commun. 2015, 6, 6148. (15) Boll, M.; Einsle, O.; Ermler, U.; Kroneck, P. M.; Ullmann, G. M. J. Mol. Microbiol. Biotechnol. 2016, 26, 119−137. (16) Pushie, M. J.; Cotelesage, J. J.; George, G. N. Metallomics 2014, 6, 15−24. (17) Hille, R.; Hall, J.; Basu, P. Chem. Rev. 2014, 114, 3963−4038. (18) Neese, F. Wiley Interdiscip. Rev. Comput. Mol. Sci. 2012, 2, 73− 78. (19) Becke, A. J. Chem. Phys. 1993, 98, 5648−5652. (20) Weigend, F.; Ahlrichs, R. Phys. Chem. Chem. Phys. 2005, 7, 3297−305. (21) Andrae, D.; Häußermann, U.; Dolg, M.; Stoll, H.; Preuß, H. Theor. Chim. Acta 1990, 77, 123−141. (22) Rothery, R. A.; Stein, B.; Solomonson, M.; Kirk, M. L.; Weiner, J. H. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 14773−14778. (23) Breneman, C.; Wiberg, K. J. Comput. Chem. 1990, 11, 361−373. (24) Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M. J. Comput. Chem. 1983, 4, 187−217. (25) MacKerell, A. D.; Bashford, D.; Dunbrack, R. L.; Evanseck, J. D.; Field, M. J.; Fischer, S.; Gao, J.; Guo, H.; Ha, S.; Joseph-McCarthy, D.; Kuchnir, L.; Kuczera, K.; Lau, F. T. K.; Mattos, C.; Michnick, S.; Ngo, T.; Nguyen, D. T.; Prodhom, B.; Reiher, W. E.; Roux, B.; Schlenkrich, M.; Smith, J. C.; Stote, R.; Straub, J.; Watanabe, M.; WiórkiewiczKuczera, J.; Yin, D.; Karplus, M.; Bellott, M. J. Phys. Chem. B 1998, 102, 3586−3616. (26) Torres, R. A.; Lovell, T.; Noodleman, L.; Case, D. A. J. Am. Chem. Soc. 2003, 125, 1923−1936. (27) Till, M. S.; Ullmann, G. M. J. Mol. Model. 2010, 16, 419−429. (28) Ullmann, G. M.; Knapp, E. W. Eur. Biophys. J. 1999, 28, 533− 551. (29) Bashford, D.; Gerwert, K. J. Mol. Biol. 1992, 224, 473−486. (30) Ullmann, R. T.; Ullmann, G. M. J. Comput. Chem. 2012, 33, 887−900. (31) Beroza, P.; Fredkin, D. R.; Okamura, M. Y.; Feher, G. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 5804−5808. (32) Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10037−10041. (33) Taly, A.; Sebban, P.; Smith, J. C.; Ullmann, G. M. Biophys. J. 2003, 84, 2090−2098. (34) Field, M. J. J. Chem. Theory Comput. 2008, 4, 1151−1161. (35) Becke, A. D. Phys. Rev. A: At., Mol., Opt. Phys. 1988, 38, 3098− 3100. (36) Perdew, J. P. Phys. Rev. B: Condens. Matter Mater. Phys. 1986, 33, 8822−8824. (37) Schäfer, A.; Horn, H.; Ahlrichs, R. J. Chem. Phys. 1992, 97, 2571. (38) Gisdon, F. J.; Culka, M.; Ullmann, G. M. J. Mol. Model. 2016, 22, 242. (39) Fischer, S.; Karplus, M. Chem. Phys. Lett. 1992, 194, 252−261. (40) Peters, B.; Heyden, A.; Bell, A. T.; Chakraborty, A. J. Chem. Phys. 2004, 120, 7877−7886. (41) Elschenbroich, C. Organometallics; Wiley-VCH: Weinheim, Germany, 2006.

follows. This electron transfer is accompanied by a protonation from the protein matrix, namely, from H260. Such a two-step reduction is similar to a Birch reduction known from organic chemistry.4,6 The noninnocent nature of the pyranopterin cofactor, the electrostatic environment in the active site, and the positioning of the proton donor H260 may help to facilitate the stepwise Birch-like reaction. Namely, the protein stabilizes the W(V) state. The first reduction of benzoyl-CoA occurs from a transient W(IV) state, and the second electron is then taken from the pyranopterin cofactor. The positively charged, oxidized pyranopterin radical is stabilized by conserved residues that form hydrogen bonds to the cofactor. The strict coupling of the first electron transfer to a proton transfer originating from the sixth ligand of the tungsten center lowers the barrier for breaking the aromaticity and allows the enzyme to reduce the aromatic ring under mild conditions.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07012. Five supporting figures, 32 supporting tables, and a description of the sequence alignment together with an interpretation (PDF) Movie of the proposed mechanism (AVI) A FASTA file containing the alignment of 133 sequences similar to BamB (TXT)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Martin Culka: 0000-0002-3944-152X Simona G. Huwiler: 0000-0002-4876-9308 Matthias Boll: 0000-0001-8062-8049 G. Matthias Ullmann: 0000-0002-6350-798X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Ulrich Ermler for providing experimental data ahead of publication and for fruitful discussions. This work was supported by the DFG Priority Program 1927 through the grants UL 174/9 and BO 1565/15-1 to G.M.U. and M.B., respectively.

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REFERENCES

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