Proton Transfer Pathways between Active Site and Proximal Cluster in

Article Cite This: J. Phys. Chem. B XXXX, XXX, XXX−XXX

pubs.acs.org/JPCB

Proton Transfer Pathways between Active Sites and Proximal Clusters in the Membrane-Bound [NiFe] Hydrogenase Daria Tombolelli and Maria Andrea Mroginski* Institut für Chemie, Technische Universität Berlin, Sekretariat PC 14, D-10623 Berlin, Germany

Downloaded via UNIV OF LOUISIANA AT LAFAYETTE on April 13, 2019 at 22:25:08 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

S Supporting Information *

ABSTRACT: [NiFe] hydrogenases are enzymes that catalyze the splitting of molecular hydrogen according to the reaction H2 → 2H+ + 2e−. Most of these enzymes are inhibited even by low traces of O2. However, a special group of O2-tolerant hydrogenases exists. A member of this group is the membrane-bound [NiFe] hydrogenase from Ralstonia eutropha (ReMBH). The ReMBH harbors an unusual iron sulfur cluster with composition 4Fe3S(6Cys) that is able to undergo structural changes triggering the flow of two electrons to the [NiFe] active site. These electrons promote oxygen reduction at the active site, preventing, in this way, aerobic inactivation of the enzyme. In the superoxidized state, the [4Fe3S] cluster binds to a hydroxyl group that originates from either molecular oxygen or water reaching the site. Both reactions, oxygen reduction to water at the [NiFe]- or [4Fe3S]-centers and oxygen evolution from water at the proximal cluster, require the delivery of protons regulated by a subtle communication mechanism between these metal centers. In this work, we sequentially apply multiscale modeling techniques as quantum mechanical/molecular mechanics methods and classical molecular dynamics simulations to investigate the role of two distinct proton transfer pathways connecting the [NiFe] active site and the [4Fe3S] proximal cluster of ReMBH in the protection mechanism against an oxygen attack. Although the “glutamate” pathway is preferred by protons migrating toward the active site to avoid inactivation by O2, the “histidine” pathway plays an essential role in delivering protons for O2 reduction at the proximal cluster. The results obtained in this work not only provide new pieces to the puzzling catalytic mechanisms governing O2tolerant hydrogenases but also highlight the relevance of dynamics in the proper description of biochemical reactions in general.

■

INTRODUCTION Hydrogenases are metalloenzymes that catalyze the cleavage of H2 in biological systems. The catalytic reaction takes place at a metal center whose composition determines the main classes in which hydrogenases are classified, namely, the [Fe]-, [FeFe]-, and [NiFe]-hydrogenases. Depending on their reactivity with molecular oxygen, the [NiFe] hydrogenases can be further divided into “oxygen tolerant” and “oxygen sensitive” (standard). In particular, the former group is very important for technological applications since these enzymes are able to perform catalysis at high rates also in the presence of air. The membrane-bound [NiFe] hydrogenase (MBH) from Ralstonia eutropha (Re), shown in Figure 1, is an oxygentolerant enzyme. ReMBH is made up of two subunits, a large one, where the nickel−iron active site (AS) is located, and a small one, where three iron−sulfur clusters acting as an electron relay are arranged. Ni−Fe Active Site. The active site (AS) of ReMBH consists of a bimetallic Ni−Fe center coordinated by four cysteine residues (Cys75, Cys78, Cys597, and Cys600) and one CO and two CN ligands coordinated with the Fe (Figure 1a). The bridging position between the two metals is alternatively unoccupied or occupied by different ligands during the catalytic cycle (Figure 1b). In standard hydrogenases, upon oxidation, two electron paramagnetic resonance (EPR)-active redox states called Ni-A and Ni-B are detected. Ni-B is characterized by a Ni(III)−Fe(II) center with a © XXXX American Chemical Society

bridging hydroxide ion and is known as the ready state because it reactivates rapidly under reducing conditions. Ni-A, instead, is also a Ni(III)−Fe(II) center, but it is considered as an unready state since it requires prolonged reactivation.1 The structure of this state is still under strong debate. The latest crystallography studies from Volbeda2 suggest a structure where a hydroxide ion occupies the bridging position between Ni and Fe, but, differently from Ni-B, a coordinating cysteine is oxidized to a bridging sulfanate. Previous crystallographic studies had assigned Ni-A to a hydroperoxide bridging ligand,3 whereas theoretical work from Breglia4 shows that the only ligand that can give a comprehensive explanation of both crystallographic structures3,5 is a peroxide O22−. However, the sulfanate species described by Volbeda2 is the most stable among all; therefore, the species coordinating peroxide and hydroperoxide ligands are probably intermediates along the mechanism of the oxidation in the presence of oxygen, leading to the Ni-A sulfanate state. Since Ni-A state has never been detected with EPR and Fourier-transform infrared spectroscopies6 in the oxygen-tolerant ReMBH7 and AaHase-1 hydrogenase from Aquifex aeolicus,8 this state is being considered as a trap preventing rapid reactivation of standard hydrogenases in the presence of oxygen. In the case of oxygentolerant hydrogenases, enzyme reactivation is suggested to Received: January 21, 2019 Revised: March 20, 2019 Published: April 1, 2019 A

DOI: 10.1021/acs.jpcb.9b00617 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

Figure 1. (a) Structure of membrane-bound [NiFe]-hydrogenase from R. eutropha. The protein scaffold is represented as ribbons (green for the large subunit and gray for the small subunit), whereas the four metal clusters are represented as spheres (S = yellow, Fe = pink, Ni = orange, C = cyan, and N = blue). The [NiFe] active site (AS) and [4Fe3S] proximal cluster (PC) are shown in detail on the right-hand side. (b) Sketch of the catalytic and deactivation cycle adapted from ref 9.

[NiFe] enzymes.11,12 The discovery of a unique structure of the PC cluster in various nonstandard hydrogenases,10,11,13,14 instead, opened the field to new interpretations. In fact, the special [4Fe3S] PC cluster, because of its structure and the structural changes it can encounter, is able to pass through two redox transitions within a narrow and physiologically accessible redox potential window.8,11 Hence, PC is able to establish a two-electron exchange with the active site. In this way, it can contrast the active site poisoning reaction, changing the equilibrium Ni-A ⇌ Ni-B toward the active Ni-B form. However, protons should also be provided to avoid the AS trapping in the inactive oxidized Ni-A state in the presence of oxygen. Redox Mechanism at the PC Cluster. The PC cluster can undergo two subsequent electron oxidation reactions, and from the available crystal structures, it is possible to assign the geometries of the reduced state and superoxidized state (SOX).10 A closed structure with Fe4 and S3 forming a bond is assigned to the reduced state of PC (PDB: 3RGW). The iron−sulfur center is described as a mixed valence state, with one iron atom with a formal charge of 3+ and three with a formal charge of 2+, for a total charge of the sole [4Fe3S] cluster of 3+. A so-called open structure instead is assigned to

occur through the fast and continuous supply of electrons and protons.9 Iron−Sulfur Clusters. The three iron−sulfur clusters of the enzyme are named according to their distance to the active site as distal cluster (DC), medial cluster (MC), and proximal cluster (PC) (see Figure 1a). DC is a standard cubane FeScluster with a three-cysteine and one-histidine coordination [4Fe4S](His)(Cys)3 first ligation sphere, whereas MC is a [3Fe4S](Cys)3 cluster. The PC cluster, shown in Figure 1b, is characterized by a novel uncommon structure consisting of a cubane cluster where one sulfur is substituted by one cysteine (Cys19) thiolate acting as a μ2-bridging ligand between Fe1 and Fe4, whereas a second supernumerary cysteine (Cys120) completes the tetrahedral coordination of the Fe3 site. In addition, a hydroxyl group attached to the Fe1 has been found in the oxidized structure of ReMBH.10 In this publication, an opening−closing mechanism has been postulated for the PC cluster, where the Fe4−S3 bond is broken and the deprotonated nitrogen of the backbone of Cys20 completes the coordination of Fe4. Oxygen Tolerance. In MBH, tolerance toward O2 is not due to modifications of the AS, where the coordination of the metal pair remains the same as in the standard O2-inactivated B


Article


Figure 2. Proton transfer pathways proposed for reduced (PDB: 3RGW, left) and superoxidized (PDB: 4IUC, right) states of ReMBH. Metal clusters are depicted as ball and stick, whereas Fe is green and Ni is blue. All other atoms are represented with standard colors (C = cyan, N = blue, O = red, and S = yellow). The orange arrows indicate the rotation of 180° around the C−C bond that is required for the proton transfer to take place. Two conformations of Glu16 were detected in both crystal structures.

reduction of oxygen to water. According to Dance,16 upon O2 binding, the AS signals the PC for delivering two electrons. Consequently, PC undergoes geometrical changes (Fe4−S3 bond breaking, NC20 deprotonation, and Fe4−NC20 bond formation) and becomes ready to transfer the two electrons. Dance proposed that the protonation of S3 at the PC site is causing the opening of the cluster and suggests that this proton comes from His229-Nε (or possibly from a water molecule in the vicinity of His229). Such a proton transfer involves the protonation of the Nδ atom of His229 and the presence of a “proton reservoir” in the vicinity composed of residues Glu72, Arg73, and water molecules. Finally, Dance identified the signaling mechanism by which the presence of O2 at the AS is communicated to the PC. Accordingly, there is a conserved link starting at Cys78, which is bridging between Ni and Fe at the AS, up to His229-Nδ, through Thr79, forming a hydrogen bond (H-bond). In this way, geometrical changes at the bridging region of the NiFe catalytic site caused by O2 binding can be communicated to His229, which, in turn, triggers the protonation of the proximal cluster at the S3 site. Thus, two mechanisms involving proton transfer at the PC are coupled to the RED ⇌ SOX interconversion: the NC20H deprotonation cycle and the S3 protonation cycle. Although these mechanisms are expected to be synchronous, details of the sequence of events are still to be explored. Besides the two protonation-coupled mechanisms during redox interconversion of the PC suggested by Dance, proton transfer events are also associated with the binding of a hydroxo ligand at the Fe1 upon double oxidation of the cluster. In fact, assuming that the hydroxyl group results from the dissociation of a water molecule, an additional proton would be released and further transferred to a proton-acceptor group. In the case the proton is derived from molecular oxygen O2, a total of three protons are required for the Fe + O2 → FeOH + H2O reaction to take place. Unfortunately, the source of the oxo-ligand in the superoxidized state of the PC remains elusive. Finally, protons have to be provided to the AS too, together with electrons, to protect it against a molecular oxygen attack. Proton Transfer Mechanisms. Two proton transfer mechanisms (see Figure 2) have been proposed to shuttle the protons back and forward between AS and PC. The first proton transfer mechanism, described first by Shomura et al.17

the superoxidized state (PDB: 4IUC, 4IUB), which is twoelectron oxidized with respect to the reduced state. Here, the Fe4−S3 bond is broken, whereas the tetrahedral coordination of the Fe4 is completed by the deprotonated nitrogen N of Cys20. Moreover, a hydroxyl ligand is bound to Fe1. In the superoxidized state (SOX), the total charge of the PC cluster is 4+, with three Fe(III) and one Fe(II). In addition, the structure of a partially reduced form of ReMBH has been published (PDB: 4IUD). Although in the superoxidized state structure the oxygen species at Fe1 has an occupancy ranging between 70 and 90%, this value is lower in the partially reduced structure, where the oxygen ligand is present in a fraction of only 30% of the proteins. Due to potential X-rayinduced damage, the fraction of clusters containing the oxoligand can be taken only as an estimation.10 Different computational studies have been published where the structural changes encountered by the PC upon two-electron oxidation15,16 have been analyzed. Pelmenschikov et al. concluded that the structural transformation of the PC is linked to the deprotonation of the Cys20 backbone amide by the Glu76 carboxylate and is responsible for providing two electrons from the PC to the AS. Dance,16 instead, proposed a mechanism where the protonation of S3 leads to the opening of the cluster. However, the mechanism of the redox-triggered structural changes at the PC as well as the structure of the partially reduced state remains under debate. Proton-Coupled Electron Transfer Mechanism. To quickly reactivate the Ni-A state at the proximal cluster, formed in the presence of air, electrons and protons have to be delivered in a continuous flow to the AS. As mentioned before, electrons are provided by the PC upon a change of its redox state. The close separation of reduction potentials allows the electron transfer to be fast. This process is accompanied by the formation of the Fe4−NC20 bond via deprotonation of Cys20 and the formation of the Fe1−OH bond, which stabilize PC in its superoxidized state. The redox capacity of the [4Fe3S] center permits the enzyme to move electrons in two different directions, depending on the type of the molecule approaching the AS. Under anaerobic conditions, the electron derived from the splitting of H2 is transferred from the AS via the iron− sulfur center chain. When O2 is approaching the AS, instead, the flow of electrons needs to be reversed to favor the C


Article


Figure 3. QM/MM setup. A water sphere of 40 Å centered on the Fe1 of the PC cluster (transparent gray surface) and protein atoms located in the space between 40 and 17 Å from Fe1 (transparent cyan ribbon diagram) are part of the MMfix layer. The second MM layer includes all atoms from the protein within a sphere of 17 Å from Fe1 [green (large subunit) and gray (small subunit) nontransparent ribbon diagram] and water molecules contained in the 17 Å sphere (not shown for clarity). The PC cluster, cysteinate residues, Glu76, and His229 form the QM region. A more detailed stick representation of the composition of the QM layer is also shown in the yellow circle (S = yellow, Fe = green, C = cyan, N = blue, O = red, and H = white).

for HmMBH and also proposed for ReMBH,10 is called Glupathway since it involves three glutamate residues besides one histidine and one threonine. This pathway connects the Fe4 center of the PC to the cysteine Cys597, which is terminally ligated to the Ni atom at the AS side. The second mechanism, suggested by Frielingsdorf et al., is known as the His-pathway since it involves two histidine residues (His229 and His82) and a water molecule (W83) connected to each other via a suboptimal H-bond network. Accordingly, proton transfer would require rotation of the imidazole rings of His229 and His82. This work addresses two fundamental issues that contribute to the elucidation of the complex molecular mechanism (MM) aiding the protection of the oxygen-tolerant ReMBH against O2: (a) the geometrical and chemical structure of the PC, specifically in its oxidized state and (b) the dynamic behavior of the ReMBH around the AS and PC sites and its influence on the proton transfer properties. For this purpose, hybrid quantum mechanical/molecular mechanic (QM/MM) methods and classical all-atom molecular dynamics (MD) simulation were combined in a sequential multiscale modeling approach.

according to specific setups described below (see Proton Transfer Pathways). Geometry optimization of the [4Fe3S] PC and its environment, consisting of the protein and explicit TIP3P water molecules within a distance of 40 Å around the geometrical center of the protein, was performed via hybrid QM/MM calculations. The system was divided into three layers, each of which was treated at a different level of theory and accuracy as depicted in Figure 3. Major details are given in Supporting Information (SI). PC Models. To investigate the structural transformation taking place at the PC cluster, upon redox change, three structural models of PC were built, starting from the reduced state structure (PDB: 3RGW). These structural models differ in the composition and oxidation state of the proximal cluster [4Fe3S]. Model Red (PC3+_NC20H_OE76) (see Figure S1a) represents the crystal structure of the reduced protein. PC is in the reduced (3+) electronic state, and Cys20 and Glu76 are modeled with standard protonation at pH 7. Model RedH instead (see Figure S1b) has also PC in the reduced state but with a neutral-charged Glu76 and deprotonated nitrogen of Cys20 (NC20). The structures of models Oxi and OxiH resemble those of models Red and RedH, respectively. However, the PC cluster is in the oxidized state with a total charge for the iron−sulfur moiety of 4+. Broken symmetry formulation has been used for the description of the electronic state of the PC cluster, and details are given in SI. Proton Transfer Pathways. The two different proton transfer pathways, namely, the Glu-pathway (Figure 4) and His-pathway (Figure 5), were characterized via computational methods under the assumption that proton translocation occurs via the Grotthuss mechanism. Accordingly, proton migration is explained as a relay process in which one proton binds a molecule causing the release of another proton from it that tries to find a new acceptor. Even though Grotthuss shuttling has been at first proposed to occur via water molecules, it can also take place through ionizable molecular groups such as titratable side chains of amino acids in proteins. Thus, this process is usually accompanied by slight structural rearrangements of the residues involved so that they become

■

METHODS Calculation Setup. The structural models of the MBH used for the calculations were built starting from the crystallographic structure of the reduced state (PDB: 3RGW)13 or oxidized state (PDB: 4IUC).10 Titratable side chains were protonated according to pH 7, except for His229, His82, and His553, which were protonated according to previous studies.10,15,18 In the reduced state of the enzyme, His229 is single protonated on the Nε and forms a hydrogen bond with the sulfur of the Cys17 ligand of the PC cluster.15 His82 is also single protonated on the Nε side and forms one hydrogen bond with the μ2-S of Cys600 at the AS and a second one with a proton residing on the Nε of His553, which is also single protonated.18 Moreover, for MD simulations, the protonation of residues involved in proton translocation steps has been adjusted D


Article


For the forward translocation, we assume that the active site, with a protonated Cys597 able to host a proton,22−24 is in the Ni-B state and the [4Fe3S] PC lies in an oxidation state (+5) as found in the crystal structure of the MBH in the superoxidized state (MD1, MD2). The backward translocation process, instead, is simulated considering the [NiFe] AS in the Ni-C state and the [4Fe3S] PC core in its reduced state (+3) as detected in the crystal structure of the reduced MBH (MD3, MD4). Furthermore, given the conformational flexibility of Glu16, the MD simulations for the forward (and the backward) proton translocation processes were performed considering the two possible rotamers of Glu16. The setups used for these four simulations are shown in Figure 4, and a summary is given in Table S1, together with a description of the technical details about the MD method used. Analysis of the stability of the H-bond network between AS and PC was done by counting the number of H-bonds that were formed between titratable groups of selected residues in 500 snapshots extracted from the 100 ns long MD trajectories. An H-bond is considered to be formed when the distance between the donor (D) and the acceptor (A), sharing a proton (H), is less than 3.0 Å and the angle D−H···A is wider than 120°. The probability of H-bond formation (PHF) between sites A and D was computed as

Figure 4. Protonation setups for (a) MD3 and MD4 and (b) MD1 and MD2. H-bonds indicated with numbers 1 to 5 and 1′ are evaluated by means of probability of H-bond formation (PHF) values. H-bond 1 is defined between Glu76 and His13, 2 between His13 and Glu16, 3 between Glu16 and Thr18, 4 is Thr18 interacting with Glu27, and 5 is the latter interacting with Cys597. Finally, 1′ indicates a possible cross H-bond interaction between Glu76 and Glu16.

PHF(AD) =

ready to host the next proton.19,20 Furthermore, proton translocation via Grotthuss mechanism is efficient if the hydrogen-bond network, connecting the initial proton donor and the final proton acceptor, is stable. Thus, a thorough statistical analysis of hydrogen-bond probability and stability provides indirect evidence of the proton transfer pathways. A similar approach has been applied by Ginovska-Pangovska to analyze the proton transfer pathway of Clostridium pasteurianum (CpI) [FeFe]-hydrogenase.21 To analyze the probability of formation of hydrogen bonds along the Glu-pathway, four independent MD simulations were performed. Although two MD simulations describe the forward proton translocation from the [NiFe] AS to the [4Fe3S] PC, the remaining two characterize the backward translocation of the proton from PC to the AS. The differences between the forward and the backward translocation processes are (a) the redox state and structure of the metal cofactors, (b) the protonation of the residues involved in the pathway, and (c) the starting crystal structure used for the MD simulations.

nH(AD) × 100 n

where nH is the number of snapshots in which an H-bond is formed between A and D and n is the total number of MD snapshots considered for the evaluation (n = 500). The higher the PHF value, the higher the probability for a proton to be transferred from D to A. Among the residues involved in the Glu-pathway, Glu16 deserves special attention. According to the crystal structure of the MBH in the reduced (3RGW) and superoxidized states (4IUC), this residue may adopt two different conformations named A and B. The A/B ratios are 54:46 and 50:50 in the reduced and the superoxidized enzyme, respectively. Due to its conformational flexibility, Glu16 is suggested to act as a gate in proton translocation processes. Thus, the conformational dynamics of this residue in the course of the MD simulations has been analyzed more deeply in terms of two dihedral angles: ψ (Cα−Cβ−Cγ−Cδ) and Φ (Cβ−Cγ−Cδ−Op), where pindex indicates the carboxylic oxygen proximal to the PC

Figure 5. Details of the His-pathway taken from molecular dynamics simulation MD3 (left) and the corresponding skeleton diagram (right). The cavity (Ω) between His229 and His82 is highlighted as a gray surface. The network is the same as that predicted using the crystal structure of the superoxidized state (4IUC); only the Nε of His229 is in H-bond distance with the hydroxo ligand bound to Fe1 (see Figure 2). The Nδ of His229 can form an H-bond with Thr79. E


Article


choice of the quantum mechanical (QM) approach and molecular mechanics force field but also the suitable assignment of the protonation state of titratable residues determining protein electrostatic. However, the largest displacements with respect to the crystal structure are predicted around Fe4, on Cys20 and Glu76, with a maximum of 0.50 Å deviation for the NC20H− OE76 distance. In this region, the proton on the backbone nitrogen of Cys20 adapts to the environment by establishing a hydrogen bond with the carboxylic oxygen of Glu76. The acceptor−donor (A−D) distance is reduced from 3.08 to 2.79 Å, whereas the acceptor−proton-donor (A−HD) angle adjusts to a value of 144°, upon QM/MM geometry optimization. The geometries of models Oxi, OxiH, and RedH were optimized (see Methods) and compared to that of the reference model Red structure. The QM/MM-optimized structures of all four models are depicted in Figure 6, whereas

moiety. To identify and characterize the Glu16 conformer occurring during the MD simulations, two-dimensional dihedral counting combination maps (CCMs) were built by monitoring the simultaneous evolution of ψ and Φ along the simulations and registering their occurrence. The CCM facilitates the visualization and localization of the most populated Glu16 conformers with their characteristic dihedral values. In the case of the His-pathway, it has been hypothesized that proton transfer between PC and AS can occur only upon 180° rotation of the imidazole rings of His82 and His229.10 However, such rotations are very unlikely to occur due to strong H-bonds between the Nε of His229 and the OH ligand on the Fe1 of the AS (4IUC) or the S of Cys17 (3RGW) on the one hand and between the Nε of His82 and Cys600.10 Our MD simulations (vide infra) support this argument. Therefore, if these two histidines remain in their crystallographic orientation, no stable H-bond network involving titratable side chains can be established between PC and AS. Consequently, the assumption of a Grotthuss mechanism for proton transfer cannot be applied as in the case of the Glupathway. Furthermore, according to the crystal structure, a relatively large space between His82 and His229 is occupied by a single water molecule, Wat83 (Figure 5). This conserved water, located in the vicinity of His82, is considered as an essential element of the His-pathway since it is a candidate to act as a shuttle for proton translocation between the two histidines. The space between His229 and the carbonyl functions of Glu72 and Cys78 although empty in the crystal structure is accessible to water molecules from the solvent. The occupation of this empty space by an additional water molecule would provide the missing link in the H-bond network between Wat83 and His229. Hence, verification of the plausibleness of the His-pathway has been done through a quantitative analysis of water dynamics and occupation of the cavity between His82 and His229 throughout the 100 ns long MD trajectories. Water occupancy was estimated by counting the number of times over 500 snapshots, extracted from MD simulations, in which water molecules were present within a defined region (Ω). Ω is an area resulting from the intersection of two spaces within a distance of 5.5 Å from each heavy atom of the side chains of His82 and His229. This space can be alternatively unoccupied or occupied by one or two water molecules throughout the simulations. Only when two water molecules are present in this space, proton transfer through the His-pathway is possible.

■

RESULTS Geometry Optimizations. As mentioned above, model Red (PC3+_NC20H_OE76) characterized by a deprotonated Glu76 side chain, a single protonated amide nitrogen of Cys20, and the PC with a total charge of 3+ represents the reduced state of the MBH protein. Starting from the crystal structure geometry (PDB: 3RGW), we used the QM/MM-optimized structure Red as a reference for evaluating the structural and electronic changes undergone by the PC upon one-electron oxidation and variation of the protonation pattern. Root-mean-square deviation (RMSD) values of the optimized model Red against the crystal structure are listed in Table S2. The relatively low RMSD estimated for the PC (less than 0.1 Å) and for all residues included in the QM region (0.35 Å) reflects only minor rearrangements of the PC and its environment. This demonstrates not only the appropriate

Figure 6. Optimized structures of the QM/MM models (a) Red, (b) RedH, (e) Oxi, and (f) OxiH represented with sticks and colored according to the standard codes (Fe = green, S = yellow, C = cyan, N = blue, O = red, and H = white). Skeleton diagrams of (c) close and (d) open conformations of the PC.

important structural parameters are given in Table S3. The geometry of PC in model Oxi (PC4+_NC20H_OE76) converges to a closed-form structure as observed in the crystal structure and in the reference Red model. However, a significant contraction of the Fe4−S3 bond length from 2.41 to 2.29 Å and a slight increase of the Fe4−NC20 distance of 0.1 Å are predicted with respect to our reference model. Although significant structural rearrangements are mainly predicted around Fe4, no proton translocation event took place upon F


Article


Figure 7. Distribution curves for dihedral angles ψ = Cα−Cβ−Cγ−Cδ and Φ = Cβ−Cγ−Cδ−Op throughout MD simulations of ReMBH in the (a) reduced form and in the (b) superoxidized form. Distribution patterns are obtained by counting the number of conformations with specific values for ψ and Φ (ranging from 0 to 360° in intervals of 5°) in 500 snapshots of the MD simulation.

The effect of one-electron oxidation and changes of the protonation pattern on the electronic properties of the PC was further investigated. Detailed information about charge distribution and electrostatic potential surfaces at PC of the four QM/MM-optimized models is provided in the Supporting Information (see Figure S2 and Table S4). MD Simulations. The dynamic properties of ReMBH in its reduced and superoxidized forms were investigated by means of classical molecular dynamics simulations. Special emphasis was given to the region connecting the AS with the PC. This region includes Glu16, Glu27, Glu72, Glu76, His13, His82, His229, Cys19, Cys20, Cys75, Cys597, and Thr18. Four independent simulations differing in the structure and redox state of PC, the protonation pattern of the protein, and the E16 rotamer were performed (see Table S1). Root-meansquare deviations (RMSD) of all heavy atoms in the region of interest have been calculated for the four MD simulations considering the corresponding starting crystal structure as a reference. RMSD for MD1 and MD2 (oxidized setup) shows average values of about 0.4 Å, which reflects the structural stability of the region between AS and PC. MD3, instead, shows a slightly increasing trend of the RMSD after 70 ns, with an average value of 0.5 Å and a maximum of 0.8 Å. MD4 shows an even more pronounced deviation in the last 10 ns, with an RMSD average value of 0.6 Å and a maximum of 1.3 Å. These relatively large RMSD values predicted for MD3 and MD4 can be explained in terms of the increased flexibility of Glu27 upon protonation of Cys597, which, in turn, weakens H-bonds between these two residues. Finally, flipping of His82 or His229 side chains around Cβ− Cγ is never observed throughout any of the four MD simulations. Although the orientation of His82 in the protein matrix is stabilized by strong H-bonds with Cys600 and His535, possible rotation of His229 is mainly hindered by Hbond interaction with Thr79 (Figure 5). Dynamic Behavior of Glu16. Particularly relevant for the description of the proton transfer pathway in the Glu-pathway is the analysis of the dynamic behavior of Glu16, which is

optimization. Furthermore, the H-bond established between Cys20 and Glu76 is only slightly weakened by the extraction of an electron from the cluster core as reflected by an A−D distance of 2.83 Å and an A-HD angle of 148°. The geometry of PC in model Oxi (PC4+_NC20H_OE76) converges to a closed-form structure as observed in the crystal structure and in the reference Red model. However, a significant contraction of the Fe4−S3 bond length from 2.41 to 2.29 Å and a slight increase of the Fe4−NC20 distance of 0.1 Å are predicted with respect to our reference model. Although significant structural rearrangements are mainly predicted around Fe4, no proton translocation event took place upon optimization. Furthermore, the H-bond established between Cys20 and Glu76 is only slightly weakened by the extraction of an electron from the cluster core as reflected by an A−D distance of 2.83 Å and an A-HD angle of 148°. In the case of model OxiH (PC4+_NC20_OE76H) where one proton has been additionally moved from the Cys20 amide group to the glutamate carboxylic oxygen proximal to the PC, the converged PC structure assumes an open form as previously reported for the superoxidized state of ReMBH.10 This open PC structure is characterized by a covalent bond Fe4−NC20 with a bond length of 2.03 Å and the cleavage of the Fe4−S3 bond as reflected by a large interatomic distance of 4.04 Å. Interestingly, the strong H-bond interaction between the acidic form of Glu76 and the deprotonated nitrogen of Cys20 is not maintained in the OxiH model since the hydrogen atom is unfavorably oriented with an A-HD angle of only 85°. Finally, the geometry of model RedH (PC3+_NC20_OE76H), with the same protonation pattern as in model OxiH, but PC in a reduced electronic state, also evolves during optimization to an open-form structure. Alike that of model OxiH, a bond is formed between Fe4 and NC20 (2.08 Å), whereas the Fe4−S3 bond breaks apart (4.11 Å). Again, H-bond interaction between NC20 and OE76 is lost, with an unfavorable A-HD angle of 82°. G


Article

The Journal of Physical Chemistry B found in two different conformations in the crystal structure. The conformational states of this residue can be efficiently described by two dihedral angles, ψ = Cα−Cβ−Cγ−Cδ and Φ = Cβ−Cγ−Cδ−Op. The occurrence of the rotameric states throughout the simulation can be estimated by computing the distribution curves for both dihedral angles and combining them together in a counting combination map (CCM) (Figure 7). In simulations performed using the reduced form of ReMBH, Glu16 can primarily be found in four different states, highlighted by empty blue circles in Figure 7. Three of these states are characterized by a ψ value of ∼175° and Φ values of 75, 175, and 250°, whereas the fourth state has ψ of 275° and Φ of 100°. In the crystal structure of the reduced form of ReMBH, two Glu16 rotamers can be identified: rotamer A (ψ = 178° and Φ = 41°) and rotamer B (ψ = 168° and Φ = 176°). Conformation 1 explored by Glu16 during the MD simulation resembles rotamer A, and conformation 2 can be assigned to rotamer B. Although the former conformation state is highly populated, the second one is only sporadically visited. Interestingly, the third conformational state (ψ = 175° and Φ = 250°) characterized by large dispersion of Φ values cannot be trapped in the crystal, probably because of high flexibility. A different picture is obtained when analyzing the dynamic properties of Glu16 in the MD simulation performed using the superoxidized form of ReMBH. The CCM in Figure 7b clearly shows that Glu16 lies in only two different conformational states, characterized by ψ = 275° and two different values of Φ (60 and 250°). Among them, only conformation 2 has a significant population and can be therefore assigned to rotamer B (ψ = 175°; Φ = 250°) detected in the crystal structure of the superoxidized state. (Note that despite the same nomenclature, rotamers are different in PDB = 4IUC and 3RGW.) The prediction of more conformational states of Glu16 in the reduced form of ReMBH compared to the superoxidized form suggests increased flexibility of the propionic side chain of this key residue in the former structural form. Hydrogen-Bond Network Analysis for the Glu-Pathway. The dynamics of the H-bond network throughout the Glu-pathway has been thoroughly analyzed in terms of PHF values quantifying the probability of H-bond formation between hydrogen acceptor (A) and donor (D) sites (vide supra). Table 1 lists the PHF values for selected AD pairs during MD1 and MD2 simulations with a protonation pattern favoring a forward proton translocation between PC and AS as well as during simulations MD3 and MD4 with a protonation pattern optimized for a backward proton translocation event.

To simplify the statistical analysis, the forward translocation pathway is subdivided into two sections. The first section extends from the PC to Glu16, whereas the second section starts at Glu16 and ends up at the AS. Independently from the simulation setup, the PHF values predicted for the individual steps of section 1 are significantly lower than those computed for section 2. Interestingly, the PHF values for H-bonds between Glu76 and His13 (step 1) are lower than for those between Glu76 and Glu16 (step 1′). Thus, the probability of direct proton transfer between the two glutamate residues is higher than that occurring via a two-step pathway through His13. However, if an excess proton reaches His13, it is very likely that it is further transferred to Glu16 (45%) when moving toward the AS. This is not the case in the opposite direction (AS → PC) where proton translocation from His13 back to Glu76 is most likely blocked since the conformation of the residues does not allow the formation of an H-bond. The second section, instead, presents significant higher PHF values than section 1, which reflect a stable H-bond network connecting Glu16, Thr18, and Glu27. Again, the PHF values for forward proton translocation are larger than those predicted for the backward reaction. This indicates that although proton translocation is possible in both directions, the transfer of a proton from Thr18 or Glu27 toward Cys597 coordinated to the AS is favored. H-bonds between Thr18··· Glu27 and Glu27···Cys597 are formed with PHF values of 100 and 95% in MD1 and MD2 against PHF values of 60 and 35% in MD3 and MD4, respectively. Hydrogen-Bond Network Analysis for the His-Pathway. The His-pathway, as described by Frielingsdorf et al.,10 cannot be ascribed within the Grotthuss-like mechanism of proton translocation. In fact, according to static models derived from crystal structures, the H-bond network is broken between His229 and crystal water (W83) that is close to His82. However, through visual inspection of the series of MD simulations, we noticed that the space around His229 can be transiently occupied by additional water molecules, thus closing the H-bond network between the two histidines. The percentage of water occupancy in the space Ω between His82 and His229 was evaluated throughout all four MD simulations. Interestingly, these values strongly depend on the starting structures used for the calculations. In MD1 and MD2, representing the enzyme in the superoxidized state, the probability of finding two water molecules in Ω is 40%, whereas in MD3 and MD4, representing the enzyme in the reduced state, this probability increases to 75%. The proton transfer mechanism through the His-pathway can be further analyzed in terms of the probability of H-bond formation (PHF) as done above for the Glu-pathway. This analysis was performed using the same four MD simulation runs described above. Alike in the Glu-pathway, the protonation states of the residues involved in the His-pathway remained the same in all four MD simulations, with His229 and His82 both single protonated at the Nε site. His229 is an essential element of the proton transfer chain in the His-pathway. Although its imidazole Nδ is capable of forming H-bonds with water molecules flowing into Ω (vide supra) or with the side chain of Thr79, the Nε is involved in H-bond interaction alternatively with the sulfur of Cys17, which is part of the PC unity, in the reduced enzyme and with the oxygen of the hydroxyl ligand at the Fe1 of PC in the superoxidized one. In particular, the NεH229−H···OPC hydrogen bond is remarkably short, with a distance between the

Table 1. Probability of H-Bond Formation (PHF) during MD Simulations for Residues Involved in the Glu-Pathway PC → AS

AS → PC

MD1

MD3

MD2

MD4

Section 1 step 1: E76···H13 step 2: H13···E16 step 1′: E76···E16 step 3: E16···T18 step 4: T18···E27 step 5: E27···C597

5 45 10 Section 2 85 100 95

0 10 10 35 60 35 H


Article

The Journal of Physical Chemistry B donor (NεH229) and the acceptor (OPC) of 2.41 Å. It can be therefore assigned to the group of transient low-barrier hydrogen bonds that are capable of efficient proton transfer.10,25 The PHF values predicted for the H-bonds formed by His229 are shown in Table 2. In the reduced state, His229

residuals of the superoxidized state conformation. It is clear that the crystal structure resolved for the oxidized state is a mixture of the two states: reduced and superoxidized. The QM/MM geometry optimizations of four models of the PC cluster differing in the redox state of the metals and the protonation state of key residues verify that one-electron oxidation of the reduced PC alone, represented by model Oxi, does not provide sufficient energy to the cluster to rearrange its structure toward superoxidized-like open conformation. Instead, the open conformation is reachable only upon deprotonation of the nitrogen at Cys20, as in models OxiH and RedH, which leaves an open site at NC20 ready to form a bond with Fe4. The formation of the NC20−Fe4 bond leads to the cleavage of the Fe4−S3 bond and consequently to the opening of the PC. Hence, deprotonation of the nitrogen is the necessary condition for the opening of the cluster, regardless of its oxidation state. With these calculations, we support the conclusion of Frielingsdorf et al.10 claiming that the structure of the oxidized state of the PC in ReMBH is similar to that of the reduced one. Proton Translocation at the PC. Based on quantum chemical calculations, Dance proposed a mechanism16 for the cluster structural morphing leading to the release of two electrons to the active site. Accordingly, three steps must occur at the PC site: the protonation of the S3 atom, the rupture of the Fe4−S3 bond, and the deprotonation of N C20 . Furthermore, Pelmenschikov et al.15 proposed that the proton that shuttles from NC20 to the S3 atom is transferred to Glu76. Our QM/MM calculations support in particular the last step since protonation of the carboxylate oxygen of Glu76 disrupts the H-bond between NC20H and OE76, which then leads to cluster morphing, as predicted in models OxiH and RedH. Although proton translocation to Glu76 is the first step of the Glu-pathway, the protonation of S3 may be part of the Hispathway mechanism. According to Dance,16 in fact, the protonation of S3 involves proton transfer from a nearby water molecule or from the NεH229, which is also the last step of the His-pathway. Role of the Proton Transfer Pathways. The prerequisite for a proton to be transferred from one point to another within the protein environment, following the Grotthuss mechanism, is the presence of a well-established hydrogen-bond network connecting the two points. By means of a series of MD simulations, we confirm the presence and solidity of an Hbond network connecting the PC and the AS through the Glupathway. This pathway, previously identified as part of one of the favorite routes for protons from the active site to the protein surface,26−28 is initiated by the transfer of a proton from the PC to Glu76 carboxylate side-chain oxygen upon deprotonation of the backbone nitrogen of Cys20. From the PHF values, obtained from the analysis of the four MD simulations, we can conclude that

Table 2. Probability of H-Bond Formation (PHF) during MD Simulations for Residues Involved in the His-Pathway

H229(Nε)···C17 H229(Nε)···OH(Fe1) H229(Nδ)···T79 H229(Nδ)···water H82(Nε)···C600 H82(Nδ)···H535 W83···C78

MD1

MD3

MD2

MD4

26 99 98 40 99 99 69

68 83 68 96 99 71

forms a bond with the sulfur of Cys17 with a PHF value of 68%, whereas this value decreases to 26% in the superoxidized state when NεH229 forms the hydrogen-bond interaction with the hydroxo ligand at Fe1 on the PC with a PHF value of 99%. The nitrogen Nδ of His229, instead, can form an H-bond with the polar hydroxylic side chain of a threonine residue, Thr79, with high PHF values of 83 and 98% for the reduced and the oxidized enzyme, respectively. Finally, His229 forms an Hbond with water molecules occupying the space Ω with PHF values of 40 and 68% for reduced and superoxidized states, respectively. Dance hypothesis16 of a possible H-bond between Glu72 and His229 is not confirmed by any of our MD calculations. RMSDs calculated for the His229 side chain are very low (average values of 0.440 and 0.483 Å for reduced and superoxidized states, respectively). RMSDs of the Glu72 side chain instead increase to 1.450 and 1.580 Å, but this is due to flipping movements around Cβ−Cγ axis, which, however, are not in the direction of His229 residue. His82 instead forms a very strong H-bond with the μ2-S of Cys600 on the Nε side and has another strong and stable H-bond with His535 on the δ side of the ring. PHFs for these two H-bonds, also shown in Table 2, are very high with values close to 100%, and His82 stays very close to its initial position in both setups. The flipping of His82 around Cβ−Cγ bond axes, which would favor proton transfer from W83 to Cys600 (Figure 2), is never observed throughout our calculations. However, an alternative link with AS could be the H-bond between W83 and the backbone oxygen of Cys78, which is predicted to be relatively stable (Table 2).

■

DISCUSSION Structural Analysis of the Oxidized State of the Proximal Cluster. Despite the availability of a threedimensional structure of the ReMBH in the oxidized state,10 the geometry of the PC remains elusive. According to the crystallographic data, the iron Fe4 in the PC shows two configurations, each with 50% of occupancy, and an oxygen atom coordinated to Fe1 is present with a reduced averaged occupancy of 30%. Moreover, the oxidized crystal was obtained by reducing the superoxidized enzyme making it impossible to distinguish between the structural and chemical features that are intrinsic to the oxidized state itself from those that are

(1) the H-bond network is stable along the Glu-Pathway, especially in section 2 extending between Glu16 and Cys597 where high PHF values are predicted; (2) His13 acts as an energy well for the proton transfer mechanism since a proton reaching this site while being translocated to PC has a higher probability to be retained there rather than to be further transferred; (3) the Glu16 residue behaves as a valve in the proton translocation event since it can assume different conformations (four in the reduced state and two in I


Article


Figure 8. Protection mechanism of ReMBH against a molecular oxygen attack. From 1 to 4: (1) the PC cluster in the closed (reduced) form, (2) the active site in the reduced Ni-C state with a bridging hydride between Ni and Fe, (3) the equilibrium between Ni-A and Ni-B oxidized states of the active site, and (4) the closed (superoxidized) form of the PC cluster. Yellow arrows indicate the Glu-pathway, whereas blue arrows indicate the His-pathway. Double-head arrows and dashed arrows indicate proton transfer pathways that are available, whereas solid arrows indicate most probable pathways, in a certain oxidation state of the protein, where the preferred direction of proton translocation is shown.

molecules trapped in it close the network by establishing Hbonds with the two histidines. Surprisingly, the double water occupancy is significantly low (40%) when the MD simulations are performed using the structure of the superoxidized form of ReMBH, compared to the values predicted for the reduced state (75%). This prediction can be understood by observing that in the superoxidized state His229 forms a very strong and stable H-bond with the hydroxo ligand at Fe1 of the PC, thus reducing the probability of H-bond formation with any other residues such as Cys17, Thr79, and incoming water molecules. The opposite is predicted when the enzyme is found in the reduced state. Here, the absence of a hydroxo ligand at Fe1 confers more mobility to His229, which, in turn, facilitates the capturing of water molecules via H-bond interactions through its NδH229. Thus, in the light of the results obtained with the MD simulations, we are able to suggest directionality also for the His-pathway since the H-bond network along this pathway, in particular between His229 and His82, is more stable when the enzyme lies in a reduced state. In this way, protons can be provided to PC in order for the structural morphing to take place. Mechanism of Protection of the Active Site against Oxygen. Based on the QM/MM calculations and MD simulations discussed above, we can postulate a mechanism responsible for the conservation of the catalytic activity of ReMBH in the presence of molecular oxygen. We present this mechanism in Figure 8. When the protein is in its reduced state (the upper part of Figure 8, states 1 and 2), the PC exhibits a closed form where Fe4 is bound to S3 and the

the oxidized state). This conclusion is confirmed by the higher rotation rate that has been observed for the corresponding residue (Glu13Aa) in AaHase-1;29 (4) a preferred directionality of the proton translocation might exist since the PHF values predicted for the PC → AS direction are significantly higher than those obtained for the backward direction. We can conclude that the proton translocation, which originates by the transfer of a proton from the PC to Glu76 and a successive relocation at the AS, takes place through the Glu-pathway. The classical MD simulations performed support also the existence of an H-bond network connecting PC and AS through the His-pathway. The existence of such a pathway has been hypothesized10 but not verified. A major drawback of the His-pathway from the static crystallographic point of view is the lack of a defined H-bond network connecting the key residues His229 and His82. Frielingsdorf et al.10 assumed that efficient proton transfer through the His-pathway requires a 180° rotation of the imidazole side chains of His82 and His229, which would orient the Nδ-nitrogens in favorable positions to form H-bonds. Reorientations of the histidine side chains were not observed in any of the MD simulations reported here. Although we cannot exclude this to happen at longer time scales, it seems very unlikely considering the high stability of the histidine residue in its pocket. Aided by the MD simulations, we were able to identify a large cavity located between His229 and His82, which can transiently host two water molecules. These two water J



■

nitrogen of Cys20 is protonated. Fe1 is coordinated only to SC17, SC19, S1, and S2 and bears no hydroxo ligand (see state 1). In this arrangement, His229 forms preferentially an H-bond with SC17. According to Dance,16 a proton at Cys17 can be further transferred through Fe4 to S3 of PC. The opening of the PC, triggered by the protonation of S3, is coupled with the proton transfer from NC20 to Glu76.16 This transformation allows the two-electron redox change of the PC to the superoxidized state (see steps 1−4 in Figure 8) in the presence of oxygen (see 1 in Figure 8, O2 highlighted in blue). In the protein structure of the superoxidized state, the PC is characterized by an open configuration where Fe4 is bound to NC20, hydroxyl ligand at the Fe1 atom center, and the absence of the S3−Fe4 bond (see state 4 in Figure 8). The proton sitting at the carboxylate side chain of Glu76 (4, H highlighted in red bound to E76) can be most likely further transferred, through the Glu-pathway, in the direction of the AS (see the yellow solid arrow in the lower part of Figure 8). In the presence of highly active molecular oxygen (see 3 in Figure 8, O2 highlighted in red), the AS can populate two different oxidized states, Ni-A and Ni-B.9 Hence, the protons transferred through the Glu-pathway (3, H+ highlighted in red), together with electrons released by the PC upon two-electron oxidation, can shift this equilibrium toward the Ni-B state, which is the catalytically active one. Based on the probability of water occupancy in the empty space around His229, proton transfer from the PC to the AS through the His-pathway is possible but less probable than in the opposite direction, as indicated by the transparent dashed double-headed arrow in the lower part of Figure 8. When the AS is in its reduced Ni-C state (2), protons can be transferred to the PC through the His-pathway (see the solid light blue arrow in the upper part of Figure 8). These protons have Cys17 as their last acceptor, which is bound to Fe1. They can then be used to protonate the S3 of PC as described above or can aid the formation of hydroxyl from molecular oxygen via a four-electron three-proton process. This OH− is then able to bind at the Fe1 of PC. However, the precise origin of this hydroxyl ligand is still under debate.30 When the AS is in its reduced Ni-C state, protons can be transferred to the PC through the His-pathway. These protons have Cys17 as their last acceptor, which is bound to Fe1. These protons can then be used to protonate the S3 of PC as described above or can aid the formation of hydroxyl from molecular oxygen via a four-electron three-proton process. This OH− is then able to bind at the Fe1 of PC. However, the precise origin of this hydroxyl ligand is still under debate.30

■

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.9b00617. QM/MM setup; schematic representation of starting models; setup for MD simulations; setup for classical MD simulations; root-mean-square deviations of QM/ MM models; structural parameters of QM/MMoptimized models; electron density distribution; partial Mulliken charges from QM/MM models; electrostatic potential surfaces of PC in optimized QM/MM models (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Maria Andrea Mroginski: 0000-0002-7497-5631 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is supported by the Deutsche Forschungsgemeinschaft [Clusters of Excellence “Unifying Concepts in Catalysis” (EXC 314) and “Unifying Systems in Catalysis” (EXC2008/ 1)]. The authors acknowledge the “Norddeutscher Verbund für Hoch- und Höchstleistungsrechnen” (HLRN) for providing HPC resources.

■

ABBREVIATIONS ReMBH, Ralstonia eutropha membrane-bound hydrogenase; AS, active site; PC, proximal cluster; QM/MM, quantum mechanical/molecular mechanics; MD, molecular dynamics; PHF, probability of hydrogen-bond formation; CCM, counting combination map

■

REFERENCES

(1) Fernandez, V. M.; Hatchikian, E. C.; Cammack, R. Properties and reactivation of two different deactivated forms of Desulfovibrio gigas hydrogenase. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1985, 832, 69−79. (2) Volbeda, A.; Martin, L.; Barbier, E.; Gutiérrez-Sanz, O.; de Lacey, A. L.; Liebgott, P. P.; Dementin, S.; Rousset, M.; FontecillaCamps, J. C. Crystallographic studies of [NiFe]-hydrogenase mutants: Towards consensus structures for the elusive unready oxidized states. J. Biol. Inorg. Chem. 2015, 20, 11−22. (3) Ogata, H.; Hirota, S.; Nakahara, A.; Komori, H.; Shibata, N.; Kato, T.; Kano, K.; Higuchi, Y. Activation process of [NiFe] hydrogenase elucidated by high-resolution X-ray analyses: Conversion of the ready to the unready state. Structure 2005, 13, 1635−1642. (4) Breglia, R.; Ruiz-Rodriguez, M. A.; Vitriolo, A.; GonzàlezLaredo, R. F.; de Gioia, L.; Greco, C.; Bruschi, M. Theoretical insights into [NiFe]-hydrogenases oxidation resulting in a slowly reactivating inactive state. J. Biol. Inorg. Chem. 2017, 22, 137−151. (5) Volbeda, A.; Martin, L.; Cavazza, C.; Matho, M.; Faber, B. W.; Roseboom, W.; Albracht, S. P. J.; Garcin, E.; Rousset, M.; FontecillaCamps, J. C. Structural differences between the ready and unready oxidized states of [NiFe] hydrogenases. J. Biol. Inorg. Chem. 2005, 10, 239−249. (6) Mouesca, J. M.; Fontecilla-Camps, J. C.; Amara, P. The structural plasticity of the proximal [4Fe3S] cluster is responsible for the O2 tolerance of membrane-bound [NiFe] hydrogenases. Angew. Chem., Int. Ed. 2013, 52, 2002−2006.

CONCLUSIONS

QM/MM and MD simulations support the existence of the Glu-pathway by which a proton is preferentially transferred from the PC to the AS. This process is initiated by the deprotonation of Cys20, as previously suggested,15,16 and involves the key residues Glu16 and His13. In addition, we verified the existence of a second H-transfer pathway connecting the AS with the PC by analyzing the dynamic behavior of water. In this pathway, His229 plays an important role in the final step shuttling protons to the PC. These protons can participate in the S3 protonation cycle16 and/or may be used for reducing molecular oxygen at the Fe1 site. K


Article

The Journal of Physical Chemistry B (7) Saggu, M.; Zebger, I.; Ludwig, M.; Lenz, O.; Friedrich, B.; Hildebrandt, P.; Lendzian, F. Spectroscopic insights into the oxygentolerant membrane-associated [NiFe] hydrogenase of Ralstonia eutropha H16. J. Biol. Chem. 2009, 284, 16264−16276. (8) Pandelia, M. E.; Fourmond, V.; Tron-Infossi, P.; Lojou, E.; Bertrand, P.; Léger, C.; Giudici-Orticoni, M. T.; Lubitz, W. Membrane-bound hydrogenase i from the hyperthermophilic bacterium Aquifex aeolicus: Enzyme activation, redox intermediates and oxygen tolerance. J. Am. Chem. Soc. 2010, 132, 6991−7004. (9) Lubitz, W.; Ogata, H.; Reijerse, E. Hydrogenases. Chem. Rev. 2014, 4081−4148. (10) Frielingsdorf, S.; Fritsch, J.; Schmidt, A.; Hammer, M.; Löwenstein, J.; Siebert, E.; Pelmenschikov, V.; Jaenicke, T.; Kalms, J.; Rippers, Y.; et al. Reversible [4Fe-3S] cluster morphing in an O2tolerant [NiFe] hydrogenase. Nat. Chem. Biol. 2014, 10, 378−385. (11) Goris, T.; Wait, A. F.; Saggu, M.; Fritsch, J.; Heidary, N.; Stein, M.; Zebger, I.; Lendzian, F.; Armstrong, F. A.; Friedrich, B.; et al. A unique iron-sulfur cluster is crucial for oxygen tolerance of a [NiFe]hydrogenase. Nat. Chem. Biol. 2011, 7, 310−318. (12) Fritsch, J.; Lenz, O.; Friedrich, B. Structure, function and biosynthesis of O2-tolerant hydrogenases. Nat. Rev. Microbiol. 2013, 11, 106−114. (13) Fritsch, J.; Scheerer, P.; Frielingsdorf, S.; Kroschinsky, S.; Friedrich, B.; Lenz, O.; Spahn, C. M. T. The crystal structure of an oxygen-tolerant hydrogenase uncovers a novel iron-sulphur centre. Nature 2011, 479, 249−252. (14) Lukey, M. J.; Roessler, M. M.; Parkin, A.; Evans, R. M.; Davies, R. A.; Lenz, O.; Friedrich, B.; Sargent, F.; Armstrong, F. A. Oxygentolerant NiFe-hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster. J. Am. Chem. Soc. 2011, 133, 16881−16892. (15) Pelmenschikov, V.; Kaupp, M. Redox-Dependent Structural Transformations of the [4Fe-3S] Proximal Cluster in O 2 - Tolerant Membrane-Bound [NiFe]- Hydrogenase: A DFT Study. J. Am. Chem. Soc. 2013, 11809−11823. (16) Dance, I. What is the trigger mechanism for the reversal of electron flow in oxygen-tolerant [NiFe] hydrogenases? Chem. Sci. 2015, 6, 1433−1443. (17) Shomura, Y.; Yoon, K.-s.; Nishihara, H.; Higuchi, Y. Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase. Nature 2011, 479, 253−256. (18) Buhrke, T.; Brecht, M.; Lubitz, W.; Friedrich, B. The H2 sensor of Ralstonia eutropha: Biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site. J. Biol. Inorg. Chem. 2002, 7, 897−908. (19) Agmon, N. The Grotthuss mechanism. Chem. Phys. Lett. 1995, 244, 456−462. (20) Cukierman, S. Et tu, Grotthuss! and other unfinished stories. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 876−885. (21) Ginovska-Pangovska, B.; Ho, M.-h.; Linehan, J. C.; Cheng, Y.; Dupuis, M.; Raugei, S.; Shaw, W. J. Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase. Biochim. Biophys. Acta, Bioenerg. 2014, 1837, 131−138. (22) Siebert, E.; Horch, M.; Rippers, Y.; Fritsch, J.; Frielingsdorf, S.; Lenz, O.; Escobar, F. V.; Siebert, F.; Paasche, L.; Kuhlmann, U. Resonance Raman Spectroscopy as a tool to monitor the active site of hydrogenases. Angew. Chem., Int. Ed. 2013, 5162−5165. (23) Kampa, M.; Pandelia, M. E.; Lubitz, W.; van Gastel, M.; Neese, F. A metal-metal bond in the light-induced state of [nife] hydrogenases with relevance to hydrogen evolution. J. Am. Chem. Soc. 2013, 135, 3915−3925. (24) Dong, G.; Ryde, U. Protonation states of intermediates in the reaction mechanism of [NiFe] hydrogenase studied by computational methods. J. Biol. Inorg. Chem. 2016, 21, 383−394. (25) Ishikita, H.; Saito, K. Proton transfer reactions and hydrogenbond networks in protein environments. J. R. Soc., Interface 2014, 11, No. 20130518.

(26) Galván, I. F.; Volbeda, A.; Fontecilla-Camps, J. C.; Field, M. J. A QM/MM study of proton transport pathways in a NiFe hydrogenase. Proteins 2008, 73, 195−203. (27) Teixeira, V. H.; Soares, C. M.; Baptista, A. M. Proton pathways in a NiFe-hydrogenase: A theoretical study. Proteins 2008, 70, 1010− 1022. (28) Sumner, I.; Voth, G. A. Proton transport pathways in NiFehydrogenase. J. Phys. Chem. B 2012, 116, 2917−2926. (29) Oteri, F.; Baaden, M.; Lojou, E.; Sacquin-Mora, S. Multiscale simulations give insight into the hydrogen in and out pathways of NiFe-hydrogenases from Aquifex aeolicus and Desulfovibrio fructosovorans. J. Phys. Chem. B 2014, 118, 13800−13811. (30) Kalms, J.; Schmidt, A.; Frielingsdorf, S.; Utesch, T.; Gotthard, G.; et al. Tracking the route of molecular oxygen in O 2 -tolerant membrane-bound [ NiFe ] hydrogenase. Proc. Natl. Acad. Sci. U.S.A. 2018, E2229−E2237.

L


Proton Transfer Pathways between Active Site and Proximal Cluster in

Recommend Documents