Human Ferrochelatase: Insights for the Mechanism of Ferrous Iron

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Human Ferrochelatase: Insights for the Mechanism of Ferrous Iron Approaching into Protoporphyrin IX by QM/MM and QTCP Free Energy Studies Jingheng Wu, Sixiang Wen, Yiwei Zhou, Hui Chao, and Yong Shen J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00216 • Publication Date (Web): 01 Nov 2016 Downloaded from http://pubs.acs.org on November 10, 2016

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Journal of Chemical Information and Modeling

Human Ferrochelatase: Insights for the Mechanism of Ferrous Iron Approaching into Protoporphyrin IX by QM/MM and QTCP Free Energy Studies Jingheng Wu, Sixiang Wen, Yiwei Zhou, Hui Chao, and Yong Shen

*

School of Chemistry, Sun Yat-sen University, 510275, Guangzhou, P R China

Abstract: Ferrochelatase catalyzes the insertion of ferrous iron into protoporphyrin IX, the terminal step in heme biosynthesis. Some disputes in its mechanism remain unsolved, especially for human ferrochelatase. In this paper, high-level quantum mechanical/molecular mechanics (QM/MM) and free-energy studies were performed to address these controversial issues including the iron-binding site, the optimal reaction path, the substrate porphyrin distortion and the presence of sitting-atop (SAT) complex. Our results reveal that the ferrous iron is probably at the binding site coordinating with Met76 and His263 plays the role of proton acceptor. The rate-determining step is either the first proton removed by His263 or the proton transition within the porphyrin with an energy barrier of 14.99 or 14.87 kcal/mol by the quantum mechanical thermodynamic cycle perturbation (QTCP) calculations respectively. The fast deprotonation step with the conservative residues rather than porphyrin deformation found in solution provides the driving force for biochelation. The SAT complex is not a necessity for the catalysis though it induces a modest distortion on the porphyrin ring. Keywords: ferrochelatase, QM/MM, free energy, mechanism, biosynthesis 1.

Introduction

Tetrapyrroles like heme is vital to almost all living organisms serving as an essential cofactor in cytochromes, catalases, peroxidases, hemoglobin and myoglobin.1 The biosynthesis of heme in animals, fungi, and α-proteobacteria belonging prokaryotes is a conserved pathway consisting eight steps, of which the final step is the insertion of the ferrous iron (Fe2+) into protoporphyrin IX by ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) to form protoheme IX (Fig. 1).2 Ferrochelatases widely exist in bacteria and eukaryotes of plants and animals for its critical role.3 Gene mutation in ferrochelatase may cause pathological changes like erythropoietic protoporphyria (EPP), an autosomal dominant disease which can develop into cholelithiasis and varying degrees of liver diseases.4 Many efforts have been made to better understand the pathogenetic mechanism of ferrochelatase for the development of treatments. Crystallographic and mutagenetic studies suggested a mechanism including the binding of the substrate protoporphyrin IX and Fe2+, the desolvation of Fe2+, the deprotonation of two pyrrole nitrogens in protoporphyrin IX, the insertion of Fe2+, and, the formation of protoheme IX.5,6 However, some key problems including the binding site, the reacting sequence and the intermediate states remain unsolved.

*

Corresponding author: [email protected] (Y. Shen).

ACS Paragon Plus Environment


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Fig. 1 The catalyzed reaction by ferrochelatase with the position of rings A, B, C, and D shown. Human ferrochelatase, a membrane-associated homodimer, exhibits a high structural diversity with only 7% similarity in protein sequences with Bacillus subtilis ferrochelatase, a water-soluble monomeric. 6,7,8 A 36º tilt angle was found in one of the pyrrole rings of substrate N-methyl mesoporphyrin for B. subtilis ferrochelatase,9 which implies that when ferrochelatase works, the planar porphyrin ring undergoes a distorted intermediate of sitting-atop (SAT) complex, which has been characterized in the non-enzymatic porphyrin metallation.10 In human ferrochelatase, however, only a modestly (10º–12º) macrocyclic distortion is observed for the substrate protoporphyrin and its position is approximately 100º rotated and 4.5 Å deeper in the active site.8 In addition, the largely hydrophilic surface inside the active-site pocket in human ferrochelatase, which may be associated with the efficient proton transfer, is not found in B. subtilis ferrochelatase. In fact, it has been proposed that these two ferrochelatases have distinguishing mechanisms in metal binding and inserting.11 It is strongly believed that the metal binding site in B. subtilis ferrochelatase involves highly conserved histidine and glutamic acid residues,12 but the corresponding histidine residue His263 in human ferrochelatase is characterized to be a pyrrole protons acceptor instead of a metal ligand.13,14 Interestingly, the mutagenesis of an indirect ligand of the ferrous iron made B. subtilis ferrochelatase select the same metal substrate as human ferrochelatase.15 These finding imply that the catalytic mechanism of human ferrochelatase is quite different from that of B. subtilis ferrochelatase and need to investigate comprehensively. Since the entry pathway and the binding site of the ferrous iron remain in dispute, the catalytic mechanism is far from well determined.16 Because of some known experimental difficulties, many assays were indeed distinct from the physiological reaction and might bring quite doubtful data.6 On the other hand, some theoretical studies have been carried out to shed light on the mechanistic particulars. In these studies, the quantum mechanics/molecular mechanics (QM/MM) methodology, which takes the advantage of the accuracy of QM method and the efficiency of MM method, provides some useful information in describing biological reactions. Sigfridsson and Ryde established a QM/MM model in solution, in which only one pyrrole ring tilts 13–15º while the other three are 1–10º out of the porphyrin plane.17 Their calculation is consistent with the crystal structure of substrate protoporphyrin IX in human ferrochelatase. It also raises a query whether a larger distortion of the original porphyrin substrate exists in ferrochelatase. Besides, our earlier QM/MM study proposed a reasonable binding site and the significant role that the first


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Fe-N bond formation plays in B. subtilis ferrochelatase,18,19 which is in good agreement with crystallographic analysis.12 However, few theoretical approach concerns on the catalytic pathway of human ferrochelatase. In this paper, we focused on understanding where and how Fe2+ approaches into the native substrate protoporphyrin IX. By QM/MM method, we expanded the view of the driving force in the human ferrochelatase catalysis and discussed some prominent questions about the metallic binding site, the porphyrin distortion and the SAT complex. In addition, the accurate reacting energy barriers were estimated by the quantum mechanical thermodynamic cycle perturbation (QTCP) method. 2. 2.1.

Computational details QM/MM approach

The QM/MM approach was performed with the ComQum program20,21 which utilizes Turbomole 5.922 for the QM calculation and Amber 723 with the Amber 1999 force field24 for the MM calculation. The QM/MM system comprised three parts: (1) System 1, containing the protoporphyrin ring, the ferrous iron and its coordinating water molecules, His263, Glu343 and other relevant residues, is treated at the QM level. (2) System 2, which consists of all residues within 6 Å of any atom in system 1, is relaxed by MM minimization in every step of QM/MM optimization (protein-free calculations) or kept fixed (protein-fixed calculations). (3) System 3, the remaining part of all the atoms, is frozen still in all QM/MM calculations. The QM region is presented by a wave function and polarized by the surrounding point charges of all the atoms in system 2 and 3 derived from Amber libraries. The hydrogen link-atom approach was introduced to handle the covalent bonds between system 1 and system 2. In this approach, the positions of the capped hydrogen atoms in QM system are linearly related to corresponding carbon atom in the MM system.20 The point charges of these conjunctive carbon atoms are cleared and the remaining charges on the truncated amino acid are adjusted to keep the fragment neutral.25 The QM/MM energy for the whole system is given as: EQM/MM=EQM+EMM123-EMM1 (1) In this formula, EQM is the QM energy of the quantum system with the hydrogen atoms truncated for the substitution of the link atoms between system 1 and system 2, excluding the self-energy from the surrounding point charges. EMM1 is the MM energy from the quantum system with the hydrogen atoms for truncation, excluding the electrostatic interactions. EMM123 is the classical energy of all the atoms with normal conjunctive carbon atoms. In the calculation, any charge of the quantum system in MM part was zeroed to avoid double counting the electrostatic interactions. This approach is similar to the ONIOM method26 and the error from truncation in the quantum system should cancel out. The geometry optimizations were first carried out by optimizing quantum system only. Afterwards, system 2 were allowed to relax (this process would not be carried out in protein-fixed calculations), and then frozen proceeding with the optimization of system 1. A looser convergence criterion (10-4 a.u. for the energy change and 10-2 a.u. for the maximum norm of the Cartesian gradient) was performed in the optimization of system 2, in which the charges of atoms in the QM region were updated every iteration.21 All the energies referred in the following text are the QM/MM energies mentioned in (1). 2.2.

The protein

The initial structure was constructed from the crystal structure of human ferrochelatase, in which



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PDB 2HRE with variant E343K and bound substrate protoporphyrin IX was used since protoporphyrin IX did not present in PDB 2HRC.8 One of the monomer was extracted from the four symmetrical polymerized subunits. The mutated Lys343 was recovered back to Glu343 and a ferrous iron was added. The hydrogen atoms were added by the module leap in AMBER software, assuming all the residues were in a normal protonation at neutral pH that all residues Asp and Glu were negatively charged and residues Lys and Arg were positively charged. The protonation states of histidine residues were assigned by considering the hydrogen bond pattern and the surroundings: protonation of Nδ1 atom for His230 and His341; protonation of Nε2 atom for His167 and His388; and protonation on both Nδ1 and Nε1 atoms for the other histidine residues. The model of [2Fe-2S] cluster was built as our previous study, where Fe2+ was bonded with two sulfur atoms in the middle and the two sulfur atoms in the cystine residues.27 The crystal structure was then charged neutrally by 11 Cl- ions and then be solvated in a sphere box with a radius of 37 Å. The entire system contains 16851 atoms including 365 residues and 3653 waters molecule. The position of all the hydrogen atoms and solvent water were optimized by energy minimization and 300 ps equilibration using a simulated annealing molecular dynamics calculation. 2.3.

Quantum chemical calculations

All the quantum chemical calculations were performed with density function theory (DFT). In the analysis of reaction pathway, the protein-free calculations were carried out by using Becke-Perdew86 (BP86) method28,29 and expedited by the resolution-of-identity (RI) approximation.30,31 The DZpdf basis set, an enhanced DZP basis set32 with d, and f functions with coefficients 0.1244 and 1.339, was utilized for Fe and the basis set 6-31G(d) was employed for all other atoms.17 It has been known that this computational level underestimates the energy barriers. More accurate single-point energies were estimated by Becke’s three-parameter hybrid functional method (B3LYP)33,34 and the 6-311+G(2d,2p) basis set except Fe, for which the DZpdf basis set was enhanced with s and f functions. As mentioned in our earlier study, all the iron complexes were assumed high-spin quintet state and treated by unrestricted open-shell theory.35 2.4.

QM region and reaction coordinates

The starting position of Fe2+ greatly affects the pathway of its insertion into protoporphyrin. Similar with our previous study on B. subtilis ferrochelatase,18,19 there are two possible sites sitting at the two sides of the protoporphyrin IX respectively for the binding of Fe2+. Besides, the coordinating states of Fe2+ should be considered as well. In our models, the QM region consists of the substrate protoporphyrin ring without side chains, the Fe2+, the coordinating water molecules and some truncated residues such as Met76 (truncated as dimethylsulfide), His263 (truncated as protonated imidazole) and Glu343 (truncated as CH3COO-). The additional parts forming hydrogen bonds to any atom in the QM region were included in the QM region as well. The alternative sites for Fe2+ were modeled as the site A and site B in Fig. 2. For the presence of Fe2+ in the binding site A, the Fe2+ coordinated with Nδ2 of His263, Oδ1 of Glu343, and two coordinating waters, forming a planar quadridentate metal center (Fig. 3(a)). This model supports the suggested mechanism for B. subtilis ferrochelatase, in which the conserved histidine and glutamic acid helped the Fe2+ insertion. Although His263 is above the porphyrin central and close to Glu343, it may not play the coordinating role with Fe2+ but serve as a proton acceptor. We thus established another model where the metal ligand His263 was substituted by a water molecule (Fig. 3(b)). In this model, the Fe2+ linked with Oδ2 of Glu343, and His263 formed a hydrogen bond


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with a coordinating water molecule. When the Fe2+ is captured by the sulfur atom in Met76 in the binding site B (Fig. 4), the coordinating water molecules may form hydrogen bonds with Tyr165 or one of the water molecules nearby. These water molecules as well as the nearby residues such as Leu92 and Leu98 were thus included in an enlarged QM region in the model. Accounting for the presence of a hydrogen bond network among His263, His341, and Glu343,36 His263 should participate in proton abstraction if it is not a ligand of Fe2+. As a result, His263 was not protonated in our initial models. Glu343 was thus determined neutralized due to the short distance from Oδ2 of Glu343 to Nδ2 of His263. In this study, the reacting coordinate was set as the distance between the moving proton and the atom being protonated in the proton acceptor, or the distance between the Fe2+ and its approaching nitrogen atom on the porphyrin rings for the formation of Fe-N bond.

Fig. 2 The two binding sites in human ferrochelatase. The subscripts of the nitrogen atoms indicate which pyrrole ring they are from.



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Fig. 3 The optimized geometries when Fe2+ bound in the binding site A, in which His263 servers as a ligand of Fe2+ (a) or not (b). The hydrogen bonds are shown by dash lines in yellow.

Fig. 4 The optimized geometry when Fe2+ bound in the binding site B. The hydrogen bonds are shown by dash lines in yellow.

2.5.

QTCP

The QTCP approach is a high-level free energy calculating method at the QM/MM level.37,38 This method has been estimated by the theoretical study of the proton transfer at metal sites in macromolecular.39 It based on a thermodynamics cycle described in Fig. 5. The QM/MM free energy from reactant (R) and the product (P), ∆AQM/MM(R→P), was calculated by performing three


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free energy perturbations: ∆AQM/MM(R→P) = ∆AMM(R→P) + ∆AMM→QM/MM(P) - ∆AMM→QM/MM(R) (2) They are the corresponding energy change at the MM level (the vertical bottom line in Fig. 5), ∆AMM(R→P), and the change from the MM level to the QM/MM level of R and P states (the horizontal line in Fig. 5), ∆AQM/MM(R)/∆AQM/MM(P). In the standard QTCP calculation, the QM system should be fixed because the MM→QM/MM perturbation is a single-step perturbation. In the simulation, sampling in the phase space is done at the MM level, thereby avoiding the problems of time-consuming and convergence at the QM/MM level.39 A smaller refined QM system is introduced to reduce the partly ignored effect from the entropy in the QM part. This contribution has been tested at the semiempirical level inducing insignificant change to the final free energy.40 Besides, before the QTCP approaches, the models were evaluated by the QM/MM optimizations with the snapshots from the MD simulations (described in the following) to make sure that the QM part did not have a major change induced to the QM/MM energy by the environment.

Fig. 5 The QTCP thermodynamic cycle and energies between reactant (R) and product (P). For the QTCP calculation, the reacting states were redescribed (from the states with a free system 2 in the MM level) by the protein-fixed calculation at the B3LYP/6-31g(d) (DZpdf for Fe) level. The systems were then further solvated in a 9 Å extending octahedral box of TIP3P water molecules.41 For the state with the highest energy in the path, the protein was relaxed by a 1000 step minimization for all the hydrogen atoms and water molecules with a restrained force constant of 100 kJ·mol-1·Å-2. Then, it went through with two 20 ps MD simulations at the condition of constant volume and constant pressure respectively with heavy atoms restrained. A 60 ps equilibration with constant pressure was subsequently carried out. With the MM coordinates from the final structure of the above MD simulations, all the states run a 600 ps equilibration with constant volume. Totally 200 snapshots at 2 ps intervals in the last 400 ps of the trajectories were collected for each state. All the MD simulations were performed at 300 K and 1 atm. The long-range electrostatic interactions were calculated by employing the particle mesh Ewald (PME) method with a 8 Å cut-off. Finally, the free energy perturbations were done by the software calcqtcp. These procedures were actually the same as the standard QTCP approach.37 The final free energies were corrected by adding the zero-point energies calculated in frequency calculation at the same level. 2.6.

QM/MM with MD snapshots

To achieve reliable pathways for the determination of the rate-determining step, we carried out the MD simulations to acquire various snapshots for the MM part. For the most possible reaction states, a set of coordinates of the MM part were sampled from the total 1080 ps MD simulations



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(as mentioned in the QTCP part). The average QM/MM energies were collected with 80 set of MD trajectories in the last 400 ps equilibrations at 5 ps intervals with the same computational level in the QM/MM calculations. Moreover, we also carried out QM/MM optimizations by B3LYP with 10 snapshots from the MD equilibrations (at 40 ps intervals). By this way, the possible structural reorganizations in the QM part and the effect from the MM surrounding were considered in our calculations. 3.

Results

The steps for Fe2+ approaching to protoporphyrin include (1) the formation of the Fe-N bond, (2) the transferring of the protons within the porphyrin ring and (3) the deprotonation of protoporphyrin. The protonation state was carefully checked for the possibility that the two protons transfer to any nitrogen atoms within the protoporphyrin ring. All the intermediates were named by the sites of the protons bonding with any pyrrole nitrogen atom, and the nitrogen atoms bonding with Fe2+ were marked with subscripts. The starting structures were named HNAHNC or HNBHND, and the final states were FeNANBNCND. The barrier energies were calculated at the BP86/6-31G(d) level if no interpretation is given. Some findings should be noted at first: (1) the Fe2+ could hardly bond with a protonated pyrrole ring; (2) the removal of the closer proton to the acceptor had lower barrier energy than the farther one (the higher one was not presented); (3) the formation of ferroporphyrin completed soon after the secondary deprotonation of the porphyrin ring. As the metallation of protoporphyrin IX was fast,42 the reaction paths with calculated barrier energies higher than 30 kcal/mol would not be appropriate. 3.1.

The acceptors of protoporphyrin protons

As the mouth of the active site is closed during the insertion of ferrous iron in human ferrochelatase, a proton transfer channel within the hydrophilic active site should thus be involved for porphyrin deprotonation. His263 has been conjectured to play the role of proton acceptor by Dailey et al.13 In our study, the Nδ2 atom of His263 can accept a porphyrin proton in both the binding sites. Glu343 was able to accept protons with Oδ2 for the insertion in the binding site A, but not in the binding site B (see the following results in detail). For Met76, our study showed it needed quite a large barrier energy (>70 kcal/mol) for its sulfur atom to acts as a proton acceptor. The water molecule was also proposed to take part in the catalytic process. As no water molecule was found around the center of protoporphyrin IX in the crystal structure,7,8 the Fe2+ coordinating water molecules may serve as the proton acceptor. Relatively, the hydrophilic pocket within the binding site A was considered far more superior for deprotonation to the hydrophobic Met76/Leu92/Leu98 residues within the site B. To find out the possible proton transfer paths, additional models were made by adding one water molecule into the hydrophilic site and the results were summed up in Table 1. The water molecule being a proton acceptor could be an axial ligand, an equatorial ligand or a free water molecule in site A or site B. In most of the cases, the pyrrole proton could not be removed successfully by the water molecule except for the two paths with determined ∆EPD in Table 1. The small space in the binding site A consists of His263, Glu343, the Fe2+ and the porphyrin ring can only accommodate one water molecule as an axial metal ligand, which formed two hydrogen bonds with the pyrrole nitrogen atoms after geometry optimization (Fig. S1 RC). This conformation prevents the transfer of the porphyrin protons or the approach of Fe2+ towards the porphyrin ring (Fig. S1 PD). In case that the added water did not bind with Fe2+, it could not take any porphyrin protons away. When it serves as an intermediate in


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the hydrogen network, (Fig. S2) a high barrier energy of 36.8 kcal/mol was observed in the second deprotonation step. As to the case that Fe2+ binds in site B, the added water molecule stayed between His263 and Glu343. Forming a hydrogen bond with Nδ2 in His263, that water molecule accepted one of the protons from protoporphyrin with a 13.5 kcal/mol barrier energy (Fig. S3), which was still higher than the one of the directly removal by His263. However, in case of removing HNB, although the barrier energy is low, the site of HNB was soon filled by a proton from the coordinating water (Fig. S4). Table 1 Reactions for proton abstraction by the water molecule and their relative energies (in kcal/mol) by BP86/(Fe:DZpdf; other atoms:6-31G(d)). Binding site of Fe2+

Reactant/ Reaction

Position of proton acceptor axial ligand

HNBHND/HND→H2O

HNAHNC/HNA→H2O

HNAHNCFeND/HNA→H2O site B 1

∆EPD1

5.3

-

13.4

-

37.6

-

site A near protoporphyrin

11.3

-

site A near protoporphyrin

36.8

22.9

site B near protoporphyrin

8.2

-

site B near protoporphyrin equatorial ligand

site A

∆ETS

(without added water)

HNBHND/HND→H2O

site A near His263

13.5

-0.8

HNBHND/HNB→H2O

site A near His263

6.6

-

∆EPD is not given if the protoporphyrin was not successfully deprotonated.

3.2.

Approach of Fe2+ from the binding site A

For the Fe2+ bonded with His263 (Fig. 3(a)), it was 3.32 Å and 3.43Å from its closest nitrogen atom on protoporphyrin for the two initial states HNAHNC and HNBHND, respectively. Despite these similar distances, the step from HNAHNC to HNAHNCFeND was exothermic, whereas the bonding of Fe2+ with any porphyrin ring of HNBHND led to an unstable conformation. Therefore, the protonation state of HNAHNC was more reasonable. Starting with this state, the possible steps before the second deprotonation were described in Fig. 6. After HNAHNCFeND formed, neither the secondary Fe-N bonding nor the removal of any porphyrin was probable for the strong planar coordination with the Fe2+. The transfer of HNA to NB needed only 13.0 kcal/mol energy forming HNBHNCFeND. It made Glu343 succeed in removing HNC with 15.4 kcal/mol barrier energy and the Fe2+ spontaneously bond to NA producing a stable intermediate of HNBFeNAND (red line in Fig. 6). The relative energies of subsequent intermediates and transition states from HNBFeNAND are arranged in Fig. 7. After HNB transfered to NC by overcoming 7.8 kcal/mol barrier energy forming HNCFeNAND, HNC could be removed with a barrier energy of 10.1 kcal/mol by the deprotonated Glu343. Afterwards, the Fe2+ bonded with NC and NB immediately, forming the product protoheme IX with a large decline of the system energy.



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Fig. 6 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site A until the first deportation step of protoporphyrin. Possible pathways are connected by soild line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The path with the lowest energy barrier is colored in red.

Fig. 7 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site A for HNBFeNAND. Possible pathways are connected by soild line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The path with the lowest energy barrier is colored in red. There is another possible reacting route starting with the state HNAHNCFeND. After HNC transferred to NB overcoming 18.6 kcal/mol barrier energy, a stable state of HNAHNBFeND formed as shown in Fig. 6. With the removal of HNB by His263, the intermediate HNAFeNCND automatically formed in this state. The subsequent pathways are arranged in Fig. S5. Owing to the steric effect of the second porphyrin proton, the formation of Fe-NB bond with a 6.6 kcal/mol barrier energy caused


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the cleavage of Fe-ND bond forming HNAFeNBNC. With the transition of the captured proton on His263 to the nearest water molecule by overcoming 9.7 kcal/mol energy for HNAFeNCND (3.9 kcal/mol for HNAFeNBNC), the removal of HNA by His263 required 20.9 kcal/mol energy (14.9 kcal/mol for HNAFeNBNC) to form the final structure FeNANBNCND. Alternatively, HNA may transfer to NB overcoming a barrier energy of 11.4 kcal/mol to achieve the state HNBFeNCND, but the barrier energy for the following path was larger than the one in the first proton transfer process. What would happen if His263 did not coordinate with Fe2+ (as shown in Fig. 3(b))? The Fe2+ was 4.04 Å away from the pyrrole nitrogen atoms for HNAHNC, and no local minimum was found for the following steps. As for HNBHND, the related pathways were presented in Fig. S6. The Fe2+ connected with NC with a barrier energy of 6.5 kcal/mol. The leaving of HND by a water molecule was endothermic forming an intermediate with a barrier energy 18.5 kcal/mol. Meanwhile, His263 was soon protonated by the water molecule and the Fe2+ connected with ND giving intermediate HNBFeNCND. The possible following steps have been described above in Fig. S5. 3.3.

Approach of Fe2+ from the binding site B

When the Fe2+ was in site B, the initial state HNAHNC did not favor the insertion of the ferrous iron. HNBHND was preferred for the Fe2+ to approach NC after the desolvation of Fe2+. It required only 1.6 kcal/mol energy for the leaving of equatorial water molecule and 1.3 kcal/mol for the axial one. The Fe-NC bond formed spontaneously and the following route was presented in Fig. 8. Starting with the intermediate HNBHNDFeNC, the direct formation of the second Fe-N bond is quite hard due to the steric effect from two porphyrin protons. Compared with a proton transferring to NA, His263 could easily accept the HNB/HND with lower barrier energies of 9.6 and 5.8 kcal/mol. Due to the lower barrier energies in the processes of direct formation of the Fe-NC bond and His263 removing HND (5.8 kcal/mol), they should be optimal paths to reach the state HNBFeNCND. In the following steps, HNB could be removed directly by His263 by 15.1 kcal/mol barrier energy, or first move to NA atom via 9.2 kcal/mol barrier energy and then be captured by His263 by 5.1 kcal/mol barrier energy after Fe-NB bond formed. It is obviously that the latter one is the optimal path. The rate-determining step of this path (red line in Fig. 8) can be concluded as the HNB-NA transition for HNBFeNCND with a barrier energy 9.2 kcal/mol. Relatively, another pathway that HNB was first removed by His263 (blue line in Fig. 8) with a little higher energy barrier (9.6 kcal/mol) provided another choice.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 8 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site B when Fe2+→ND is the first step. Possible pathways are connected by solid line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The two paths with the lowest energy barriers are colored in red and blue. On the other hand, if the desolvation of Fe2+ is not the first step, there are two types of routes including proton transferring within the porphyrin ring and His263 removing the pyrrole proton of the porphyrin (Fig. S7). The barrier energies of HNB transition to NA, NC and His263 were calculated as 11.0, 11.8 and 15.6 kcal/mol respectively, and 21.0, 14.7, 7.0 kcal/mol for HND. Although the barrier energy of HND being removed by H263 is lower than the one of the optimal path in Fig. 8, the product HNBFeNCND located before the rate-determining step in Fig. 8, so did the product HNAHNBFeNCND from HND to NA and HNAHNDFeNC from HNB to NA. 3.4.

Energy refinement

The most probable reacting paths for both binding sites were summarized in Fig. 9 and Fig. 10. Their detail parameters including the coordination bond lengths, the distances between Fe2+ and the pyrrole nitrogen atoms, and the distortion angle of porphyrin ring were summarized in Table 2-5. The highest energy barrier for the binding site A is 15.4 kcal/mol and 9.2 or 9.6 for the binding site B (in Fig. 9 and Fig. 10). To obtain more reasonable barrier energies, the reactants and intermediates were recalculated by the B3LYP method. The basis set were enlarged by combining an enhanced DZpdf for the ferrous iron and 6-311+G(2d,2p) for the other atoms. The refined barrier energy for the bind site A was 17.9 kcal/mol and 11.6 or 12.4 kcal/mol for the bind site B (Table 6). Obviously, the insertion from the binding site B is more favorable, especially considering that the metallation of protoporphyrin IX was fast.42


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Fig.9 The most probably pathway of the Fe2+ approaching from the binding site A. The relative energies of reactant (RC), intermediates (INT), product (PD) and the energy barriers are achieved by BP86/6-31G(d)/DZpdf. The refined energies by B3LYP/6-311+G(2d,2p) are given in parenthesis.



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Fig. 10 The most probably pathway of the Fe2+ approaching from the binding site B. The relative energies of reactant (RC), intermediates (INT), product (PD) and the energy barriers are achieved by BP86/6-31G(d)/DZpdf. The refined energies by B3LYP/6-311+G(2d,2p) are given in parenthesis. The two possible pathways are marked as blue and red respectively.


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Table 2 The coordination bond lengths and the distances between Fe2+ and the pyrrole nitrogen atoms (in Å) for the reactant (RC), intermediates (INT) and product (PD) in the most probable pathway for Fe2+ insertion from the binding site A. Structures

Fe-NA

Fe-NB

Fe-NC

Fe-ND Fe-NHis Fe-OGlu Fe-OWat1 Fe-OWat2

RC(HNAHNC)

4.174

4.462

3.973

3.318

2.099

1.946

2.027

2.071

INT-1(HNAHNCFeND)

3.551

4.270

3.749

2.297

2.195

2.005

2.111

2.085

INT-2(HNBHNCFeND)

3.728

4.346

3.470

2.238

2.201

2.013

2.118

2.093

INT-3(HNBFeNAND)

2.389

3.472

3.146

2.021

2.084

3.328

2.136

2.146

INT-4(HNCFeNAND)

2.190

3.373

3.450

2.196

2.120

3.457

1.977

2.247

PD(FeNANBNCND)

2.088

2.099

2.120

2.079

2.262

3.784

3.760

4.330

Table 3 The coordination bond lengths and the distances between Fe2+ and the pyrrole nitrogen atoms (in Å) for the reactant (RC), intermediates (INT) and products (PD) in the most probable pathways I and II for Fe2+ insertion from the binding site B. Fe-NA

Fe-NB

Fe-NC

Fe-ND Fe-SMet Fe-OWat1 Fe-OWat2 Fe-OWat3

RC(HNBHND)

Structures

4.265

4.787

4.268

4.108

2.409

2.092

2.209

2.160

INT-1(HNBHND)

4.061

4.410

3.526

3.530

2.416

2.062

2.193

2.062

INT-2(HNBHND)

3.952

3.609

2.070

3.125

2.371

2.132

2.267

2.194

INT-3-I(HNDFeNBNC)

3.356

2.200

2.046

3.233

2.380

2.058

3.503

2.219

INT-4-I(HNDFeNANBNC)

2.331

2.080

2.119

2.455

3.108

2.301

3.601

2.238

PD-I(FeNANBNCND)

2.316

2.122

2.110

2.135

3.556

2.324

3.527

2.283

INT-3-II(HNBFeNCND)

3.455

3.262

2.031

2.242

2.429

2.089

3.030

2.177

INT-4-II(HNAFeNCND)

3.212

2.591

2.005

2.327

2.448

2.138

3.400

2.226

INT-5-II(HNAFeNBNCND)

3.175

2.437

2.010

2.415

2.497

2.139

3.437

2.233

PD-II(FeNANBNCND)

2.296

2.127

2.111

2.135

3.683

2.355

3.541

2.256



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Table 4 The distortion angles of porphyrin ring (in °) for the reactant (RC), transition states (TS), intermediates (INT) and product (PD) in the most probable pathway for Fe2+ insertion from the binding site A. ringA

ringB

ringC

ringD

RC(HNAHNC)

Structures

6.63

4.21

8.82

5.05

INT-1(HNAHNCFeND)

7.89

6.12

11.65

5.55

TS-1

7.65

6.41

12.00

6.71

INT-2(HNBHNCFeND)

6.90

6.60

13.01

6.69

TS-2

12.10

3.75

19.43

8.05

INT-3(HNBFeNAND)

12.99

3.25

16.47

3.50

TS-3

16.73

1.58

7.37

6.35

INT-4(HNCFeNAND)

17.00

2.42

10.18

5.82

TS-4

12.92

5.18

24.15

4.11

7.68

3.44

16.29

4.21

PD(FeNANBNCND)


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Table 5 The distortion angles of porphyrin ring (in °) for the reactant (RC), transition states (TS), intermediates (INT) and products (PD) for Fe2+ approaching from the binding site B in pathways I and II. Structures

ringA

ringB

ringC

ringD

6.51

6.75

11.70

9.77

TS-1

6.00

6.64

11.80

9.49

INT-1(HNBHND)

5.85

6.72

10.51

8.84

TS-2

6.08

6.91

10.41

8.87

INT-2(HNBHND)

5.26

8.03

6.07

8.15

TS-3-I

3.79

5.13

6.40

4.75

INT-3-I(HNDFeNBNC)

7.15

10.61

7.40

3.98

TS-4-I

3.62

16.44

11.41

8.42

INT-4-I(HNDFeNANBNC)

5.74

14.39

12.39

9.06

TS-5-I

6.00

14.20

13.22

10.29

PD-I(FeNANBNCND)

9.44

9.13

11.84

2.43

TS-3-II

4.69

9.99

6.39

10.82

INT-3-II(HNBFeNCND)

4.71

5.26

6.52

6.22

TS-4-II

6.54

5.06

5.91

7.58

INT-4-II(HNAFeNCND)

8.91

7.68

6.13

4.69

INT-5-II(HNAFeNBNCND)

9.53

8.18

6.38

3.88

TS-6-II

12.93

10.56

5.16

4.33

PD-II(FeNANBNCND)

11.25

8.69

12.31

1.67

RC(HNBHND)

3.5.

QM/MM energies from the MD snapshots

From the QM/MM study, the differences of barrier energies of possible paths for the binding site B are quite small making it hard to tell the most probable reaction path. To make the actual reaction path clear, the MD simulations of the MM part were performed for the key reaction paths and their QM/MM barrier energies averaged on 80 MD snapshots. Shown in Table 6, the barrier energy of path I (blue line in Fig. 8) is almost as the same as the one of path II (red line in Fig. 8). It suggests that the movements of the surrounding residues do not have significant influence on the reaction inside the porphyrin ring. The leaving of HNA (HNA→His) revealing the largest energy difference did not change the rate-determining step as well. To further verify the QM/MM results with a free QM region, QM/MM geometry optimization with the protein fixed under BP86/6-31G(d) level were carried out from 10 MD snapshots for the steps with higher barrier energies (e.g. path I: HNB→His, HND→His and path II: HNB→NA). As can be seen in Table 6, the calculated QM/MM energy barriers are almost the same as those obtained from 80 MD snapshots without geometry optimization with a very small deviation. This could be easily explained as the proton transferring process is localized and the effect from the MM part on the QM system is very limited. Results support that the assumption of a fixed QM region in the QTCP sampling is reasonable, for the conformations from the QM part stayed similar.



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Page 18 of 36

Table 6 The QM/MM energies deviations between the intermediates and the transition states for Fe2+ approaching from the binding site B in pathways I and II (in kcal/mol). The standard deviations are listed in parentheses. Calculation approach

QM/MM Opt. Aver. on 80 MD snapshots Aver. on 10 MD snapshots

3.6.

QM level

∆EQM/MM (pathway I)

∆EQM/MM (pathway II)

HNB→His

HND→His

HND→His

HNB→NA

HNA→His

BP86/6-31G(d)

9.6

7.6

5.8

9.2

5.1

B3LYP/6-311+G(2d,2p)

12.4

9.6

7.4

11.6

5.6

BP86/6-31G(d)

11.6(1.2)

6.2(1.8)

3.3(1.3)

11.5(1.1)

1.2(0.8)

BP86/6-31G(d)

10.0(1.2)

6.3(1.1)

-

10.8(0.7)

-

B3LYP/6-31G(d)

14.0(1.6)

8.3(3.3)

-

14.5(0.8)

-

QTCP calculations

To obtain the free energy profiles for path I (HNB→His) and path II (HNB→NA) with high accuracy, the QTCP approaches were carried out. The QM structures were refined, which contained Fe2+, water ligands and protoporphyrin without side chains for path I (HNB→His). For path II (HNB→NA) a nearby imidazole was added. The refined barrier energies, 16.5 kcal/mol for path I (HNB→His) and 18.6 kcal/mol for path II (HNB→NA), were a bit higher than the ones with 10 MD snapshots listed in Table 6 since more atoms were fixed. The calculated QTCP free energies for path I (HNB→His) were shown in Table 7 and path II (HNB→NA) in Table 8 respectively. In the calculations, the proton transfers within the porphyrin ring reveal higher ∆AMM→QM/MM than one towards His263. The larger standard deviations from ∆AMM→QM/MM and negative accumulated ∆AMM may due to the movement of truncated His263 in the MD simulations. Actually, the contributions from the MM part in both paths were small (within 1 kcal/mol to the energy barrier). After the correction by zero-point energy (-2.72 kcal/mol for path I (HNB→His) and -3.23 kcal/mol for path II (HNB→NA)), the energy barrier for path I (HNB→His) was evaluated to be 14.99 kcal/mol, and, for the step of path II (HNB→NA) it was 14.87 kcal/mol. Both energy barriers are not large. In view of the tunnel effect (usually decline the energy barrier by 1-2 kcal/mol), the actual reaction barrier energies would be smaller than the calculated ones. Since proton transferring within the porphyrin ring is a fast step, the approach of Fe2+ into porphyrin should be a fast step as well. It is in good agreement with the suggestion that in the conversion of free porphyrin to free metalloporphyrin occurs after chelation the rate-determining step is most probably product release.43


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Table 7 The QTCP free energies and its individual contributions to the free energy barriers based on the ensemble averages for HNB→His for Fe2+ approaching from the binding site B in the pathway I. The standard deviations are given in parentheses. d(HNB-His)/Å

∆AMM→QM/MM

∆AMM

Path

Forward

Reverse

Avg.

Acc.

∆AQM/MM

2.25

0.00(0.10)

-

-

-

-

-

0.00

1.95

-0.47(0.26)

2.25→1.95

0.02(0.03)

-0.31(0.03)

-0.15

-0.15

-0.62

1.80

4.60(0.19)

1.95→1.80

-0.02(0.02)

0.06(0.02)

0.02

-0.12

4.47

1.65

4.29(0.21)

1.80→1.65

0.25(0.02)

-0.02(0.02)

0.12

0.00

4.28

1.50

10.09(0.23)

1.65→1.50

0.01(0.02)

0.22(0.02)

0.12

0.11

10.21

1.40

15.11(0.23)

1.50→1.40

-0.03(0.02)

0.10(0.02)

0.03

0.14

15.25

1.35

15.14(0.22)

1.40→1.35

0.05(0.01)

0.04(0.01)

0.05

0.19

15.33

1.25

16.22(0.19)

1.35→1.25

0.59(0.04)

0.77(0.05)

0.68

0.87

17.09

1.20

-1.76(0.15)

1.25→1.20

0.07(3.00)

2.61(0.91)

1.34

2.21

0.45

1.05

-7.32(0.15)

1.20→1.05

-3.43(1.01)

-2.08(0.09)

-2.76

-0.54

-7.86

Table 8 The QTCP free energies and its individual contributions to the free energy barriers based on the ensemble averages for HNB→NA for Fe2+ approaching from the binding site B in the pathway II. The standard deviations are given in parentheses. d(HNB-NA)/Å

4. 4.1.

∆AMM→QM/MM

∆AMM

Path

Forward

Reverse

Avg.

Acc.

∆AQM/MM

2.00

0.00(0.73)

-

-

-

-

-

0.00

1.85

6.67(0.50)

2.00→1.85

0.00(0.04)

-0.35(0.04)

-0.17

-0.17

6.50

1.65

9.44(0.62)

1.85→1.65

-0.22(0.02)

-0.28(0.02)

-0.25

-0.42

9.02

1.55

15.05(0.43)

1.65→1.55

-0.11(0.01)

-0.09(0.01)

-0.10

-0.52

14.53

1.45

17.59(0.58)

1.55→1.45

-0.09(0.01)

-0.02(0.01)

-0.06

-0.58

17.01

1.35

18.89(0.56)

1.45→1.35

-0.26(0.02)

-0.16(0.02)

-0.21

-0.79

18.10

1.25

14.34(0.72)

1.35→1.25

-0.22(0.09)

0.26(0.02)

0.02

-0.77

13.57

1.10

1.56(0.47)

1.25→1.10

0.08(0.06)

-0.78(0.06)

-0.35

-1.12

0.44

1.00

-2.21(0.73)

1.10→1.00

-0.06(0.01)

-0.09(0.01)

-0.07

-1.19

-3.40

Discussion The role of porphyrin proton acceptor

Although water molecules are good lewis bases in many catalysis processes, our study suggests that they are not required for porphyrin deprotonation in the catalysis by human ferrochelatase. As described in Table 1, the removal of pyrrole proton by a water molecule is an endothermic process. In most cases, it accompanied with the return of another proton of water to the porphyrin ring or failing to achieve a stable intermediate (Fig. S1-S4). Only when His263 was neither a metallic ligand nor protonated it can accept a proton from the intermediate water molecule. In a way, it makes sense when His263 is far from the porphyrin center, but there is not enough space to hold many water molecules in the binding site to build a hydrogen network to transfer protons.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Furthermore, the surrounding hydrophobic residues in the binding site B such as methionine and leucine residues prevented the water molecules from exchanging protons with other residues. Both Glu343 (for Fe2+ in site A) and His263 (for Fe2+ in site B) were so close to the porphyrin ring that it was hard for a water molecule to approach the protons. It suggests that Glu343 and His263 are prior to the water molecule in removing pyrrole protons of porphyrin. His263 was supposed to be the direct proton acceptor since at least one hydrogen bond is involved between the pyrrole proton and His263,36 which could be found when the Fe2+ was in the binding site B in our models. Results reveal a lower energy barrier for this configuration. In another model that His263 does not act as a ligand for Fe2+ (Fig. 3(b)), His263 formed hydrogen bond with one of the coordinating waters rather than the porphyrin ring. As shown in Fig. S6, at least 35.1 kcal/mol energy is need for His263 to accept a proton in this case. As we can see without the help of hydrogen bond the deprotonation step by His263 needed more energy than that by Glu343 coordinated in a monodentate mode when the Fe2+ was in the binding site A (15.4 kcal/mol at most). 4.2.

The formation of the Fe-N bonds

The protoporphyrin metallation was prevented by the steric effect from the porphyrin protons and largely depended on the state of porphyrin. The first metallic bond with the pyrrole nitrogen atoms was rapid, but the Fe2+ would not fall into the protoporphyrin immediately. The deprotonation step was in demand for the followings binding steps. Once the two protons on the porphyrin ring were removed, the ferrous iron approached into the porphyrin ring and the product protoheme IX formed immediately. These results were in good agreement with the fact that a rapid step of binding and a slower following step were involved in the Fe2+ chelation.43 In fact, the Fe-N bond bonding resulted in a more stable state. Our results propose that the Fe-N bond formation is exothermic, and the rate of protoporphyrin metalation depends on the transition step of protons. Similar conclusion can be found from kinetic measurements in solution that the fast deprotonation step for porphyrin rather than the exchange of the metallic ligands is the major driving force in the catalysis.44 On the other hand, the bonding of the Fe2+ with the pyrrole nitrogen affected the proton transition steps by pressing the proton(s) on the porphyrin ring. For instance, the barrier energy of the deprotonation of HNBFeNANCND by Glu343 in the binding site A (56.1 kcal/mol) is much higher than the one for HNBFeNCND (25.4 kcal/mol). From the structure analysis (shown in Fig. S8), the HNB was pushed by the Fe2+ and exposed towards the binding site B after the Fe-NA bond formed. As a result, Glu343 could not remove it without breaking of the Fe-NC bond. Another example can be found in the binding site B for HNBHNCFeNAND that its 6.4 kcal/mol barrier energy of the HNB deprotonation step was lower than 9.0 kcal/mol for HNBHNCFeND. Therefore, a more favorable pathway can be concluded when the metal insertion and the proton deprotonation takes place from the other site of porphyrin (Fe2+ in the site B). 4.3.

Which binding site is the selected one?

The mechanism of porphyrin metallation has long been discussed for the suspensive binding site of the divalent metal ion. Our study proposes that the barrier energies primarily depended on the deprotonation steps. As figured out in the paths for Fe2+ approaching from the site B, the deprotonation by His263 proceeded without any difficulty. However, if the Fe2+ approached from the opposite site, it needed at least 28 kcal/mol energy, which almost doubled the one by Glu343.


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Is it the breakage of the metallic bond with equatorial ligand His263 (Fig. 3(b)) which makes His263 hard to remove protons in the site A? The high barriers of energy from the models without bonding between Fe2+ and His263 negate this viewpoint (Fig S6). So what should be the main reason causing the difference? Another point of view is introduced to answer this issue. The study on the reaction path in B. subtilis ferrochelatase suggests that the bending of H-N bond and the long H-NHis183 distance (4Å) lead to a high barrier energy of deprotonation.19 Similarly, in human ferrochelatase when Fe2+ was in the binding site A, the porphyrin deprotonation by His263 required more than 40 kcal/mol energy. The starting distances between His263 and the leaving proton were at least 3.24 Å when His263 was the one of the ligands of Fe2+, and 3.40 Å when His263 was not. Relatively, because a hydrogen bond made the protoporphyrin distort and expose its protons towards His263, shorter His263-proton distances (2.57 and 2.60 Å) were found for Fe2+ binding in the site B. Consequently, the distance that the porphyrin proton travels should be a more important factor that affects the porphyrin deprotonation. Our calculations proposed that His263 plays the alternative role of coordinating with Fe2+ or accepting protons. If it is a ligand of the ferrous iron in the binding site A, Glu343 rather than His263 will undertake the task of removing porphyrin protons. Otherwise, His263 can help the protons leave with a water molecule. It is obvious that Glu343 binding with the divalent metal ion by both of its carboxyl oxygen atoms failed to accept the porphyrin protons in this case. This finding is consistent with the observation that the mutation of His263 inactivates the catalysis of human ferrochelatase. Since the aspartic acid has a similar structure with the glutamic acid, the aspartic acid can play the role of metallic coordinator or proton acceptor. It supports the study that there is no significant change in Kmproto occurred in E343D mutation.13 What’s more, the porphyrin metallation without enzyme revealed a 12.2 kcal/mol barrier energy by Fe2+.35 The energy barrier 17.9 kcal/mol for its insertion from the binding site A in our calculations is somehow too high in this catalysis process. On the other hand, the barrier energy (~12 kcal/mol) from the metallic binding site B was in accordance with the 10.90 kcal/mol activation energy for the metallic chelation by zinc, which could be inhibited competitively by Fe2+ in ferrochelatase.45 Furthermore, experiment studies confirmed that there was a hydrogen bond formed by His263 with one of the porphyrin protons by the mutation of Arg164/Tyr165,13,36 which is consistent with our model for Fe2+ approach from the site B. As a result, the reaction path, in which the ferrous iron approached into the porphyrin ring from the site B is a more probable route. 4.4.

The SAT complex and the distortion of porphyrin ring

The SAT complex is a reaction intermediate in porphyrin metalation found in vitro that the pyrrolenine ring is weakly chelated with the metal ion by its two nitrogen atoms.46 An out-of-plane distortion for the porphyrin ring was believed to enhance the reaction rate by facilitating the metal insertion.47 The ~36º distorted N-MeMP substrate in crystal structure of the B. subtilis ferrochelatase was thought to be a direct support.9 However, this conformation was not found in the crystal structure of ferrochelatase with natural substrate, and only slight distortion was observed in the theoretical studies so far. In this study, as Table 4 and Table 5 shown, the protoporphyrin IX distorted within 11.70º (the angles between the least-squares plane of the porphyrin ring and four pyrrole rings, the same as follows) under the induction by human ferrochelatase (HNBHND and HNAHNC). The largest bending angles were all detected during the second deprotonation steps. They were found moderate (slightly more than 15º) for the insertion



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from the binding site B, and 24.15º from the binding site A (due to the far distance 3.21 Å between the leaving proton with Glu343). As proposed before, the protoporphyrin tilts about 1-16º under the protein interaction while a larger tilt angle (~30º) requires about 5 kcal/mol extra energy.17 Predictably, the system should overcome such a barrier if a larger distortion of porphyrin is induced. In our study, the tilted angles did not alter significantly in the reaction. It suggests a large distortion did not exist otherwise a higher binding energy barrier was involved. Can the SAT complex be an immediate in the chelation without a large distortion of protoporphyrin? The ferrous iron could not bind with two pyrrole nitrogen atoms at the same time in our study. It supposes that in vivo the SAT formation is not determined by the rate of ligand exchange just as what is in solution.35,47 It requires the protons on protoporphyrin being exposed towards the outer side, and it does not provide a faster reaction path as well. Altogether, our study suggests that the distortion is indeed involved in the catalysis of human ferrochelatase, however, totally differed from our general belief, it does not provide the driving force for the metal ion insertion. The moderate protoporphyrin distortion did not assist Fe-N bonding, but was just the result of the secondard deprotonation step. We suppose that in the protein the fixing of the ferrous iron by the conserved residues helps the chelating process. In solution, the unoriented movement makes it hard for the hydrated divalent metal to get close to the porphyrin center. In this case, an obvious distortion of the porphyrin ring, which has been proven to bring a high barrier of energy, is necessary for its first metallic bonding. 5.

Conclusion

In this study, QM/MM and QTCP free energy calculations were performed to investigate the porphyrin metallation in human ferrochelatase. It suggests a most reasonable pathway including the steps the ferrous iron approaching from the site with Met76 coordinated and His263 playing the role of accepting proton. The overall reaction path can be described as following steps: (1) Fe2+ loses two of its coordinated water molecules and approaches to the pyrrole nitrogen atom on ring C; (2) the proton on ring B leaves the porphyrin ring and the ferrous iron forms bond with ring B; (3) the deprotonation of ring D proceeds; (4) the ferrous iron inserts into the porphyrin ring completely to form ferroporphyrin. The rate-determining step is the first porphyrin deprotonation with 14.99 kcal/mol barrier energy. The ring B proton transition step within the porphyrin ring with 14.87 kcal/mol barrier energy could be a possible rate-determining step in another path if the deprotonation of ring D proceeds soon after step (1). Some interesting findings are distinctive for human ferrochelatase, in which the exchanges of the metallic ligands with pyrrole nitrogen atom on porphyrin ring were fast, and the typical SAT structure hardly formed due to the small deformation of the porphyrin ring. Our results reveal a mechanism completely different from what have been suggested in solution that the distortion of the porphyrin ring is the driving force for catalysis, that is to say, the distortion induced by the deprotonation is not a trigger in the metal chelation. Acknowledgment This project is supported by the National Science Foundation of China (No. 21577179) and High Performance Computing Center (HPCC) at Sun Yat-sen University. We are very grateful to Prof. Ulf Ryde (Lund University, Sweden) for his COMQUM and QTCP software. Supporting Information This information is available free of charge via the Internet at http://pubs.acs.org.


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For Table of Contents Use Only Human Ferrochelatase: Insights for the Mechanism of Ferrous Iron Approaching into Protoporphyrin IX by QM/MM and QTCP Free Energy Studies Jingheng Wu, Sixiang Wen, Yiwei Zhou, Hui Chao, and Yong Shen


†

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Fig. 1 The catalyzed reaction by ferrochelatase with the position of rings A, B, C, and D shown. Fig. 1 181x76mm (300 x 300 DPI)



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Fig. 2 The two binding sites in human ferrochelatase. The subscripts of the nitrogen atoms indicate which pyrrole ring they are from. Fig. 2 98x84mm (300 x 300 DPI)


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Fig. 3 The optimized geometries when Fe2+ bound in the binding site A, in which His263 servers as a ligand of Fe2+ (a) or not (b). The hydrogen bonds are shown by dash lines in yellow. Fig.3 148x88mm (300 x 300 DPI)



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Fig. 4 The optimized geometry when Fe2+ bound in the binding site B. The hydrogen bonds are shown by dash lines in yellow. Fig. 4 74x84mm (300 x 300 DPI)


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Fig. 5 The QTCP thermodynamic cycle and energies between reactant (R) and product (P). Fig. 5 111x37mm (300 x 300 DPI)



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Fig. 6 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site A until the first deportation step of protoporphyrin. Possible pathways are connected by soild line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The path with the lowest energy barrier is colored in red. Fig. 6 304x157mm (300 x 300 DPI)


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Fig. 7 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site A for HNBFeNAND. Possible pathways are connected by soild line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The path with the lowest energy barrier is colored in red. Fig. 7 304x152mm (300 x 300 DPI)



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Fig. 8 Relative energies of intermediates and transition states of the Fe2+ approaching from the binding site B when Fe2+→ND is the first step. Possible pathways are connected by solid line, while the importable ones (>30 kcal/mol) are marked with dash lines in grey. The two paths with the lowest energy barriers are colored in red and blue. Fig. 8 457x209mm (300 x 300 DPI)


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Fig. 9 The most probably pathway of the Fe2+ approaching from the binding site A. The relative energies of reactant (RC), intermediates (INT), product (PD) and the energy barriers are achieved by BP86/631G(d)/DZpdf. The refined energies by B3LYP/6-311+G(2d,2p) are given in parenthesis. Fig. 9 216x170mm (300 x 300 DPI)



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Fig. 10 The most probably pathway of the Fe2+ approaching from the binding site B. The relative energies of reactant (RC), intermediates (INT), product (PD) and the energy barriers are achieved by BP86/631G(d)/DZpdf. The refined energies by B3LYP/6-311+G(2d,2p) are given in parenthesis. The two possible pathways are marked as blue and red respectively. Fig. 10 198x384mm (300 x 300 DPI)


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Human Ferrochelatase: Insights for the Mechanism of Ferrous Iron

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