| 1. |
- Cao, Lili
(författare)
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Computational Studies of Nitrogenase
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- AbstractNitrogenase is the only enzyme that can convert the inert nitrogen molecule to ammonia, so that it can be used for biomass production and in biosynthetic pathways. It contains a complicated acitve site, composed of eight metal ions, nine sulfur ions and one carbide ion (the FeMo cluster). Although it has been thoroughly studied with crystallographic, biochemical, kinetic, spectroscopic and computational methods, the reaction mechanism is still not known and many conflicting hypotheses have been presented. To solve some of these problems, we have performed a thorough and systematic study of nitrogenase with various computational approaches, based on a combination of quantum mechanics (QM), molecular mechanics (MM) and sometimes also crystallographic refinment. We have: • Decided the protonation states of eight key amino acid residues around the active site and showed that the homocitrate ligand is singly prontated on the hydroxide group. • Studied how the the broken-symmetry (BS) state for the FeMo cluster depends on the QM method, the basis sets, the surrounding protein and the protonation and oxidation state of the cluster. Thereby, we could propse a practical prodecure to deal with these states in computational studies.• Predicted the most stable protonation state for the E0–E4 states of nitrogenase with two density functional theory (DFT) methods: The TPSS functional perfers the protonation of Fe, whereas B3LYP prefers protonation of the carbide ion.• Showed that different DFT methods give relative eneriges that can differ by ~1100 kJ/mol for nitrogenase. This is the main reason for the diverging computational results. Pure functionals and TPSSh predict the best geometries of the E0 state, whereas B3LYP and PBE0 give more reliable H2 dissociation energies.• Showed that the most stable E4 structure obtained with pure functionals has two hydride ions bridging between two pairs of iron ions and two protons on the sulfide ions, in agreement with experiments. We also found a new low-energy BS state in which only two Fe ions have minority spin.• Predicted that the most stable binding mode of N2H2 to nitrogenase involves trans-HNNH binding to Fe2. However, other binding modes, e.g. involving cis-HNNH or NNH2 and binding to Fe6, are rather low in energy. • Suggested an alternating reaction mechanism for nitrogenase with a dissociated S2B ligand.• Decided the most stable BS states for the P-cluster in four oxidation states and showed that the one-electron oxidised state involves a protonated Cys-88, but a deprotonated Ser-188.• Developed a novel quantum-refinement approach allowing for disorder in the QM system and applied it to the P-cluster in two crystal structures of nitrogenase.• Shown by quantum refinement that a recent crystal structure of V-nitrogenase does not involve a N-derived ligand, but rather a hydride-inhibited state with a significant amount of the undissociated S2B ligand.Together, these studies have taken us a significant step closer to an atomic understanding of nitrogenase, showing that experiments and calculations start to converge, illustrating the strength of quantum refinement, but also pointing out important problems that need to be solved in computational studies of nitrogenase.
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| 2. |
- Cao, Lili, et al.
(författare)
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Does the crystal structure of vanadium nitrogenase contain a reaction intermediate? Evidence from quantum refinement
- 2020
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:6, s. 847-861
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Tidskriftsartikel (refereegranskat)abstract
- Abstract: Recently, a crystal structure of V-nitrogenase was presented, showing that one of the µ2 sulphide ions in the active site (S2B) is replaced by a lighter atom, suggested to be NH or NH2, i.e. representing a reaction intermediate. Moreover, a sulphur atom is found 7 Å from the S2B site, suggested to represent a storage site for this ion when it is displaced. We have re-evaluated this structure with quantum refinement, i.e. standard crystallographic refinement in which the empirical restraints (employed to ensure that the final structure makes chemical sense) are replaced by more accurate quantum–mechanical calculations. This allows us to test various interpretations of the structure, employing quantum–mechanical calculations to predict the ideal structure and to use crystallographic measures like the real-space Z-score and electron-density difference maps to decide which structure fits the crystallographic raw data best. We show that the structure contains an OH−-bound state, rather than an N2-derived reaction intermediate. Moreover, the structure shows dual conformations in the active site with ~ 14% undissociated S2B ligand, but the storage site seems to be fully occupied, weakening the suggestion that it represents a storage site for the dissociated ligand. Graphic abstract: [Figure not available: see fulltext.]
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| 3. |
- Cao, Lili, et al.
(författare)
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N2H2 binding to the nitrogenase FeMo cluster studied by QM/MM methods
- 2020
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Ingår i: Journal of Biological Inorganic Chemistry. - : Springer Science and Business Media LLC. - 0949-8257 .- 1432-1327. ; 25:3, s. 521-540
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Tidskriftsartikel (refereegranskat)abstract
- We have made a systematic combined quantum mechanical and molecular mechanical (QM/MM) investigation of possible structures of the N2 bound state of nitrogenase. We assume that N2 is immediately protonated to a N2H2 state, thereby avoiding the problem of determining the position of the protons in the cluster. We have systematically studied both end-on and side-on structures, as well as both HNNH and NNH2 states. Our results indicate that the binding of N2H2 is determined more by interactions and steric clashes with the surrounding protein than by the intrinsic preferences of the ligand and the cluster. The best binding mode with both the TPSS and B3LYP density-functional theory methods has trans-HNNH terminally bound to Fe2. It is stabilised by stacking of the substrate with His-195 and Ser-278. However, several other structures come rather close in energy (within 3–35 kJ/mol) at least in some calculations: The corresponding cis-HNNH structure terminally bound to Fe2 is second best with B3LYP. A structure with HNNH2 terminally bound to Fe6 is second most stable with TPSS (where the third proton is transferred to the substrate from the homocitrate ligand). Structures with trans-HNNH, bound to Fe4 or Fe6, or cis-HNNH bound to Fe6 are also rather stable. Finally, with the TPSS functional, a structure with cis-HNNH side-on binding to the Fe3–Fe4–Fe5–Fe7 face of the cluster is also rather low in energy, but all side-on structures are strongly disfavoured by the B3LYP method.
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| 4. |
- Cao, Lili, et al.
(författare)
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Putative reaction mechanism of nitrogenase after dissociation of a sulfide ligand
- 2020
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Ingår i: Journal of Catalysis. - : Elsevier BV. - 1090-2694 .- 0021-9517. ; 391, s. 247-259
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated the implications of the recent crystallographic findings that the m2-bridging S2B sulfide ligand may reversibly dissociate from the active-site FeMo cluster of nitrogenase. We show with combined quantum mechanical and molecular mechanical (QM/MM) calculations that once S2B has dis- sociated, N2 may bind in that position and can be protonated to two NH3 groups by thermodynamically favourable steps. The substrate forms hydrogen bonds with two protein ligands, Gln-191 and His-195. For all steps, we have studied three possible protonation states of His-195 (protonated on either ND1, NE2 or both). We find that the thermodynamically favoured path involves an end-on NNH2 structure, a mixed side-on/end-on H2NNH structure, a side-on H2NNH2 structure, a bridging NH2 structure and a bridging NH3 structure. In all cases, His-195 seems to be protonated on the NE2 atom. Dissociation of the NH3 pro- duct is often unfavourable and requires either further reduction or protonation of the cluster or rebinding of S2B. In conclusion, our calculations show that dissociation of S2B gives rise to a natural binding and reaction site for nitrogenase, between the Fe2 and Fe6 atoms, which can support an alternating reaction mechanism with favourable energetics.
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| 5. |
- Cao, Lili, et al.
(författare)
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Quantum refinement with multiple conformations : Application to the P-cluster in nitrogenase
- 2020
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 76, s. 1145-1156
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Tidskriftsartikel (refereegranskat)abstract
- X-ray crystallography is the main source of atomistic information on the structure of proteins. Normal crystal structures are obtained as a compromise between the X-ray scattering data and a set of empirical restraints that ensure chemically reasonable bond lengths and angles. However, such restraints are not always available or accurate for nonstandard parts of the structure, for example substrates, inhibitors and metal sites. The method of quantum refinement, in which these empirical restraints are replaced by quantum-mechanical (QM) calculations, has previously been suggested for small but interesting parts of the protein. Here, this approach is extended to allow for multiple conformations in the QM region by performing separate QM calculations for each conformation. This approach is shown to work properly and leads to improved structures in terms of electron-density maps and real-space difference density Z-scores. It is also shown that the quality of the structures can be gauged using QM strain energies. The approach, called ComQumX-2QM, is applied to the P-cluster in two different crystal structures of the enzyme nitrogenase, i.e. an Fe 8 S 7 Cys 6 cluster, used for electron transfer. One structure is at a very high resolution (1.0 Å) and shows a mixture of two different oxidation states, the fully reduced P N state (Fe 8 2+, 20%) and the doubly oxidized P 2+ state (80%). In the original crystal structure the coordinates differed for only two iron ions, but here it is shown that the two states also show differences in other atoms of up to 0.7 Å. The second structure is at a more modest resolution, 2.1 Å, and was originally suggested to show only the one-electron oxidized state, P 1+. Here, it is shown that it is rather a 50/50% mixture of the P 1+ and P 2+ states and that many of the Fe - Fe and Fe - S distances in the original structure were quite inaccurate (by up to 0.8 Å). This shows that the new ComQumX-2QM approach can be used to sort out what is actually seen in crystal structures with dual conformations and to give locally improved coordinates.
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| 6. |
- Cao, Lili, et al.
(författare)
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What Is the Structure of the E4 Intermediate in Nitrogenase?
- 2020
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Ingår i: Journal of Chemical Theory and Computation. - : American Chemical Society (ACS). - 1549-9618 .- 1549-9626. ; 16, s. 1936-1952
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Tidskriftsartikel (refereegranskat)abstract
- Nitrogenase is the only enzyme that can cleave the strong triple bond inN2. The active site contains a complicated MoFe7S9C cluster. It is believed that itneeds to accept four protons and electrons, forming the E4 state, before it can bind N2.However, there is no consensus on the atomic structure of the E4 state. Experimentalstudies indicate that it should contain two hydride ions bridging two pairs of Fe ions,and it has been suggested that both hydride ions as well as the two protons bind on thesame face of the cluster. On the other hand, density functional theory (DFT) studieshave indicated that it is energetically more favorable with either three hydride ions or with a triply protonated carbide ion, depending on the DFT functional. We have performed a systematic combined quantum mechanical and molecular mechanical (QM/MM) study of possible E4 states with two bridging hydride ions. Our calculations suggest that the most favorable structure has hydride ions bridging the Fe2/6 and Fe3/7 ion pairs. In fact, such structures are 14 kJ/mol more stable than structures with three hydride ions, showing that pure DFT functionals give energetically most favorable structures in agreement with experiments. An important reason for this finding is that we have identified a new type of broken-symmetry state that involves only two Fe ions with minority spin, in contrast to the previously studied states with three Fe ions with minority spin. The energetically best structures have the two hydride ions on different faces of the FeMo cluster, whereas better agreement with ENDOR data is obtained if they are on the same face; such structures are only 6−22 kJ/mol less stable.
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| 7. |
- Ding, Yunmei, et al.
(författare)
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Factors influencing kinesiophobia during the “blanking period” after radiofrequency catheter ablation in patients with atrial fibrillation by the fear-avoidance model
- 2022
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Ingår i: International Journal of Cardiology. - : Elsevier BV. - 0167-5273. ; 363, s. 49-55
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Tidskriftsartikel (refereegranskat)abstract
- Background: The influencing factors of kinesiophobia (fear of movement) in patients with atrial fibrillation(AF)during the post-operative “Blanking Period” are not known. The aims were to investigate the status of kinesiophobia in patients with AF during the post-operative “Blanking Period”, then further describe the occurrence and analyze the influencing factors of patients' kinesiophobia by the Fear-Avoidance Model. Materials and methods: In total,400 patients diagnosed with atrial fibrillation, during the post-operative “Blanking Period” from the selected hospital were included in this study. The Tampa Scale for Kinesiophobia Heart (TSK-SV Heart), the Self-efficacy for Exercise (SEE) scale, and the Geriatric Locomotive Function Scale (GLFS) were used to assess kinesiophobia, exercise self-efficacy, and physical function. The study adopted a cross-sectional design. Results: The score of kinesiophobia during the “Blanking Period” after operation in patients with atrial fibrillation was (44.06 ± 10.77), and the rate of high kinesiophobia was 71.61%.Logistic regression results showed that age, education, household monthly income, resting heart rate, EHRA symptom classification, exercise self-efficacy, and physical function influenced the kinesiophobia of patients during the post-operative “Blanking Period”(p<0.05， p<0.01). Conclusions: Kinesiophobia is common in patients with atrial fibrillation during the postoperative “Blanking Period”, and the fear of movement is related to age, education, household monthly income, resting heart rate, EHRA symptom classification, exercise self-efficacy, and physical function. Clinical and nursing staff should pay close attention to the psychological problems in the post-operation “Blanking Period” of exercise rehabilitation in patients with atrial fibrillation, make timely interventions to reduce patients' fear of movement, and improve patients' compliance with exercise rehabilitation.
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| 8. |
- Jiang, Hao, et al.
(författare)
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Proton Transfer Pathways in Nitrogenase with and without Dissociated S2B
- 2022
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Ingår i: Angewandte Chemie (International edition). - : Wiley. - 1521-3773. ; 61:39
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Tidskriftsartikel (refereegranskat)abstract
- Nitrogenase is the only enzyme that can convert N2 to NH3. Crystallographic structures have indicated that one of the sulfide ligands of the active-site FeMo cluster, S2B, can be replaced by an inhibitor, like CO and OH−, and it has been suggested that it may be displaced also during the normal reaction. We have investigated possible proton transfer pathways within the FeMo cluster during the conversion of N2H2 to two molecules of NH3, assuming that the protons enter the cluster at the S3B, S4B or S5A sulfide ions and are then transferred to the substrate. We use combined quantum mechanical and molecular mechanical (QM/MM) calculations with the TPSS and B3LYP functionals. The calculations indicate that the barriers for these reactions are reasonable if the S2B ligand remains bound to the cluster, but they become prohibitively high if S2B has dissociated. This suggests that it is unlikely that S2B reversibly dissociates during the normal reaction cycle.
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| 9. |
- Vysotskiy, Victor P., et al.
(författare)
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Assessment of DFT functionals for a minimal nitrogenase [Fe(SH)4H]− model employing state-of-the-art ab initio methods
- 2023
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Ingår i: The Journal of chemical physics. - 0021-9606. ; 159:4
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Tidskriftsartikel (refereegranskat)abstract
- We have designed a [Fe(SH)4H]− model with the fifth proton binding either to Fe or S. We show that the energy difference between these two isomers (∆E) is hard to estimate with quantum-mechanical (QM) methods. For example, different density functional theory (DFT) methods give ∆E estimates that vary by almost 140 kJ/mol, mainly depending on the amount of exact Hartree–Fock included (0%–54%). The model is so small that it can be treated by many high-level QM methods, including coupled-cluster (CC) and multiconfigurational perturbation theory approaches. With extrapolated CC series (up to fully connected coupled-cluster calculations with singles, doubles, and triples) and semistochastic heat-bath configuration interaction methods, we obtain results that seem to be converged to full configuration interaction results within 5 kJ/mol. Our best result for ∆E is 101 kJ/mol. With this reference, we show that M06 and B3LYP-D3 give the best results among 35 DFT methods tested for this system. Brueckner doubles coupled cluster with perturbaitve triples seems to be the most accurate coupled-cluster approach with approximate triples. CCSD(T) with Kohn–Sham orbitals gives results within 4–11 kJ/mol of the extrapolated CC results, depending on the DFT method. Single-reference CC calculations seem to be reasonably accurate (giving an error of ∼5 kJ/mol compared to multireference methods), even if the D1 diagnostic is quite high (0.25) for one of the two isomers.
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| 10. |
- Zhai, Huanchen, et al.
(författare)
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Multireference Protonation Energetics of a Dimeric Model of Nitrogenase Iron-Sulfur Clusters
- 2023
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Ingår i: Journal of Physical Chemistry A. - 1089-5639. ; 127:47, s. 9974-9984
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Tidskriftsartikel (refereegranskat)abstract
- Characterizing the electronic structure of the iron-sulfur clusters in nitrogenase is necessary to understand their role in the nitrogen fixation process. One challenging task is to determine the protonation state of the intermediates in the nitrogen fixing cycle. Here, we use a dimeric iron-sulfur model to study relative energies of protonation at C, S, or Fe. Using a composite method based on coupled cluster and density matrix renormalization group energetics, we converge the relative energies of four protonated configurations with respect to basis set and correlation level. We find that accurate relative energies require large basis sets as well as a proper treatment of multireference and relativistic effects. We have also tested ten density functional approximations for these systems. Most of them give large errors in their relative energies. The best performing functional in this system is B3LYP, which gives mean absolute and maximum deviations of only 10 and 13 kJ/mol with respect to our correlated wave function estimates, respectively, comparable to the uncertainty in our correlated estimates. Our work provides benchmark results for the calibration of new approximate electronic structure methods and density functionals for these problems.
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