computational modelling of metalloenzymes
TRANSCRIPT
Division Of Chemistry And Biological Chemistry
School of Physical & Mathematical Sciences
Computational Modelling of Metalloenzymes
To Understand the Mechanism of L-Tryptophan Nitration in P450 TxtE
RASHMI HIRANYA SENEVIRATNE
N1403520J
ASST. PROF. HAJIME HIRAO
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Academic Year 2015 Semester 2
Acknowledgements
Many thanks to Assistant Professor Hirao for giving me the opportunity to take a project under his supervision in a topic that I am interested in and for his help and guidance throughout the semester. His enthusiasm for his work and clear explanations during lectures in Computational Chemistry (CM4043) inspired me to take a project in biological computational chemistry.
Thank you to Kai Xu, a PhD student in Asst. Prof. Hirao’s group who answered all my questions, helped me understand the computational techniques I was using and patiently checked through my calculations when I encountered errors. Despite his own work demands, he was dedicated to helping me with my project.
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AbstractCytochrome P450 TxtE is a metalloenzyme containing an iron metal complex that can catalyse the nitration of L-Tryptophan. The mechanism of the nitration of L-tryptophan using TxtE is discussed in this report.
Molecular modelling and quantum mechanical calculations were carried out on the whole P450 enzyme (Protein Data Bank ID: 4TPO) using AMBER parameters for L-tryptophan and the haem group to define the quantum mechanical region.
Further quantum mechanical calculations were carried out to observe how nitration occurs in P450 TxtE enzyme using geometry optimisation and potential energy surface scanning in Gaussian09w between ferric (iii) superoxide with nitric oxide and L-tryptophan with nitrogen dioxide (positive and neutral forms).
All calculations were carried out from Nanyang Technological University, BoonLay Linux Terminal.
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ContentsAcknowledgements.....................................................................................................................................2
Abstract.......................................................................................................................................................3
Background..................................................................................................................................................6
Gaussian09..............................................................................................................................................6
Geometry Optimisation.......................................................................................................................6
Potential Energy Surface Scanning......................................................................................................6
Basis Sets.............................................................................................................................................6
Modelling of Large Biological Molecules.............................................................................................7
AMBER.....................................................................................................................................................8
Linux Terminal.........................................................................................................................................8
Introduction.................................................................................................................................................9
Method......................................................................................................................................................10
1. QM Calculations on Ferric (III) Superoxide and Nitric Oxide..............................................................10
Optimisation......................................................................................................................................10
Potential Energy Surface Scanning....................................................................................................10
2. QM Calculations on L-Tryptophan and Nitrogen Dioxide..................................................................14
Optimisation......................................................................................................................................14
Potential Energy Surface Scanning....................................................................................................14
3.QM/MM Calculations on P450 TxtE Enzyme [13]...............................................................................16
Preparation of Topology Files for each Molecule..............................................................................16
Determining Charges for MM and QM Regions.................................................................................16
Preparation for ONIOM Calculation [13]............................................................................................18
Work in Progress [13]............................................................................................................................20
Full ONIOM Calculation [13]..............................................................................................................20
Results.......................................................................................................................................................21
1.QM Calculations on Ferric (III) Superoxide and Nitric Oxide...............................................................21
2.QM Calculations on L-Tryptophan and Nitrogen Dioxide...................................................................24
L-Tryptophan and Neutral Nitrogen Dioxide......................................................................................24
L-Tryptophan and Positive Nitrogen Dioxide.....................................................................................27
Conclusion.................................................................................................................................................28
Appendix...................................................................................................................................................29
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A1: Some Linux Commands [16] [17].....................................................................................................30
A2: Some Errors Encountered Setting Up ONIOM.................................................................................31
A3: Table to Show Energy/kcalmol-1 Against Distance/Å between Nitric Oxide and Ferric Superoxide.32
A4: Table To Show How Spin Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide.............................................................................................................................................32
A5: Table To Show How Charge Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide...................................................................................................................................32
A6: Table To Show How Energy/kcalmol-1 Varies with Distance/Å between L- Tryptophan and Neutral Nitrogen Dioxide....................................................................................................................................33
A7: Tables To Show How Spin Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide....................................................................................................................................34
A8: Tables To Show How Charge Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide....................................................................................................................................35
A9: Table to Show Energy/kcalmol-1 Against Distance/Å between Positive Nitrogen Dioxide and L-Tryptophan............................................................................................................................................36
Works Cited...............................................................................................................................................37
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BackgroundTo determine the nitration mechanism of L-tryptophan via P450 TxtE enzyme several concepts from quantum mechanics and physical chemistry are used. The calculations are run on Gaussian09w with the input files prepared using GaussView and Vim editor on Linux Terminal. Other manipulation and visualisation software like AMBER and Visual Molecular Dynamics (VMD) are also used for generation of data and analysis of results.
Gaussian09GaussView and Gaussian09w are part of a series of programs used for electronic structure modelling. Large molecules such as enzymes can be opened from protein data bank (*.pdb, * denoting any name) files available from the online Protein Data Bank using GaussView. GaussView is the visualisation program in Gaussian09 package used for building input files and setting up the route section of the input file to run calculations using Gaussian09w. Input files are in *.com format and results are gathered in an output file, usually in *.log and *.chk (checkpoint) formats. Generally, log files are used to visualise the results of the calculation while checkpoint files contain the results of the calculation that can be used in further calculations [1].
Geometry OptimisationGeometry optimisation is used to find the lowest energy geometry of a molecule. The molecule is built in GaussView and used to create an input file. Gaussian09w program then varies the initial co-ordinates of the molecule to find the lowest energy geometry of the molecule using the potential energy surface (PES) of the molecule [1]. The PES contains information about the molecule such as positions of atoms of the molecule and energies of molecular conformations. Geometry Optimisation is also known as energy minimisation as the lowest energy conformation of the molecule is found at the closest, lowest energy minimum point to the initial geometry on the potential energy surface. Note that this point is only the local minimum on the potential energy surface and not the global minimum (the lowest energy conformation of the molecule for the complete reaction) [1].
Potential Energy Surface Scanning Potential energy surface scanning is another type of Gaussian09 calculation that uses the optimised geometry as a starting point to scan the potential energy surface of the reaction at different distances between reactant molecules. The scans will indicate the geometry of the reactant molecules at selected intervals or distances in the reaction [1]. There are two types of PES Scanning: rigid, where the structures produced at each interval in the scan have not been optimised; and relaxed, where the geometry of the reacting molecules at each interval has been optimised. Rigid scanning is easier and takes less computational time to perform, although these scans can contain less information on reaction dynamics than relaxed scans [2].
Basis Sets For each type of calculation, a basis set needs to be specified to define the molecular orbitals of the reactant molecules. A basis set is a group of mathematical functions that approximate the molecular orbitals of a molecule. The more mathematical functions used, the more accurate the basis set, so a more exact model of the molecular orbital is produced. However, greater computational time is
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required to calculate the approximation if a large basis set is used. A compromise between computer resources and type of basis set is needed to finish calculations with speed and accuracy [1].
Molecular Orbitals (MOs) are a linear combination of atomic orbitals (LCAOs). Using a large number of atomic orbitals can give a more accurate representation of MOs. These general atomic orbitals can be approximated by Slater functions, which are suitable because they give the predicted shape of the atomic orbitals [2]. However Slater functions are very complex so require considerable computational time. Gaussian functions are used instead, as they are much simpler and so require less computational time. Several Gaussian functions, fixed in a linear combination called contracted Gaussian, can be used to approximate a Slater function. These approximations are called Slater Type Orbital-nGaussian (STO-nG) basis sets, where n is the number of Gaussian functions used in the approximation. The approximations can be modified to account for polarisation and diffusion of molecular orbitals in a molecule that are due to atomic properties. Basis sets formed from STO-nG use the Density Functional Theory (DFT); a method of solving the Schrödinger equation by using the electron density of a many electron system [2].
A common basis set used, for example, is 6-31g(d), a split-valence basis set used to expand the 1s core of second period elements and to describe valence shells. The (d) letter means that a polarization function "d" is added to the heavy atoms’ p orbitals [3].
GenECP can be used specify basis sets for individual elements in a molecule. For example, ferric superoxide contains iron, carbon, hydrogen, oxygen, nitrogen and sulphur. LanL2DZ basis set is used for iron while 6-31g(d) is used for the other elements. It is designed for use in ONIOM calculations in which you want to use a general basis set within one ONIOM layer [3].
Modelling of Large Biological MoleculesUsing high accuracy models such as DFT/6-31g(d) for optimisation on large biological molecules is not practical as applying this method on a large system can drain computer resources. Gaussian09 software package contains a method called ONIOM that can overcome the limitations produced from optimising large molecules. ONIOM stands for Our own N-layered Integrated molecular Orbital and molecular Mechanics and uses layers of Molecular Mechanics (MM) and DFT Quantum Mechanics (QM) calculations to compute optimisations for a large molecule, say an enzyme, with reasonable accuracy without extensive computational time [4].
In ONIOM, the system can be defined as two layers: MM method is used for whole system while a QM method is used for a site of interest such as a receptor site of an enzyme. The QM region is also known as the HIGH layer and is the smallest layer, and as such is treated with the most accurate method. The LOW layer is the entire molecule in a two layer model [5]. A computationally inexpensive method such as MM is used with a smaller, less accurate basis however the calculation in the MM region corresponds to the environmental effects of the molecular environment in the QM region [4].
ONIOM works by calculating the whole molecule with MM calculations. Then the high-level zone is saturated with hydrogens and calculated with both QM and MM calculations [5].
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≈ + –
DFT Amber DFT Amber
AMBERAssisted Model Building with Energy Refinement (AMBER) is a software package containing molecular simulation programs for biological molecules [6]. The programs included in the package can be used to generate force fields that are used for the simulation of the biomolecules. These AMBER force fields are generated from topology files, which contain data on equilibrium bond length, bond angles, torsions and improper torsions as well as atom Van Der Waals figures.
Some of the programs used from AMBER software package include: LEaP, which is used for preparation of input files for simulation; Antechamber, which automatically creates parameters for small organic molecules using the GAFF database included with AMBER; and SANDER (Simulated Annealing with NMR-Derived Energy Restraints) which is the main simulation program that can perform energy minimization and molecular dynamics [6].
Linux TerminalThe Linux Terminal is the command line program used by the Linux Operating System, however the terminal can be accessed from any computer using a different operating system like Windows via PuTTY, a terminal emulator [7], and Xming, a display server for programs accessed on the Linux Terminal [8]. This means the terminal can be accessed from any computer with PuTTY and Xming installed.
A Linux Terminal network can be used to carry out commands on programs such as GaussView and AMBER via PuTTY and Xming. An advantage of using Linux Terminal is that the terminal allows the user to carry out calculations via a network of computers available on the server. Using the Terminal allows a larger amount of disk space and memory to be used, allowing calculations to be completed more quickly or within a greater timeframe without being impeded by the limitations of an individual computer.
A list of some Linux Terminal commands can be found in the Appendix (A1).
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FIGURE 1 SHOWS GRAPHICALLY HOW THE COMPUTATIONS FOR A TWO-LAYER ONIOM CALCULATION PROCEEDS [4]
Introduction Metalloenzymes are proteins that have a metal complex embedded within the protein structure [9]. They have a variety of functions from transportation of small molecules to signal transduction via electron transfer. Cytochromes are a type of membrane bound metalloenzyme generally used for ATP generation via electron transport within eukaryotic cells [10]. The electron transfers are carried out by oxidising and reducing the metal ion centre, the most common metals in cytochromes being iron and copper. Cytochrome P450 TxtE uses an iron complex (haem group) to catalyse the nitration of the aromatic group in L-tryptophan, an amino acid [11].
The iron complex comprises of a central iron ion bound to porphyrin and tethered to the enzyme by a cysteine ligand. This ligated iron ion is the active site of the P450 enzyme and binds to molecular oxygen to catalyse a two electron reduction process [11].
The iron complex in the enzyme is initially in ground state form. In the presence of the substrate L-tryptophan the iron complex has high spin and with the addition of molecular oxygen, ferrous dioxygen is formed. Ferrous dioxygen resonates with ferric superoxide, a high spin radical complex. Nitrogen dioxide is added to ferric superoxide to form ferric peroxynitrile. Nitrogen dioxide is cleaved and used to eventually nitrate L-tryptophan. The low spin ground state ferric complex is reformed. Another possible mechanism for nitration involves proton-triggered cleavage of the ferric-peroxynitrite complex to yield NO2
+, followed by classical electrophilic aromatic substitution of L-tryptophan [12].
Regardless of the nitration mechanism, protonation of Compound III is required to restore the low spin ferric resting state of the enzyme [12]
In this study, both the positive and neutral forms of nitrogen dioxide are analysed to determine which form of nitrogen dioxide is used in the mechanism of L-tryptophan using P450 TxtE enzyme.
Generally in organic synthesis, the mechanism of nitration of an aromatic ring uses a positively charged nitronium ion formed from nitric acid and sulphuric acid. These conditions are very harsh as the reagents used are not
tolerant to many functional groups. By studying the nitration mechanism of P450 TxtE, the potential of TxtE as a nitration biocatalyst can be explored [9].
MethodThe mechanism of L-tryptophan nitration in P450 TxtE was determined using:
1. Quantum Mechanical calculations on ferric(iii)-superoxide and nitric oxide 2. Quantum Mechanical calculations on nitration of L-tryptophan and nitrogen dioxide 3. Quantum Mechanical/Molecular Mechanical calculations on P450 TxtE enzyme
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FIGURE 2 SHOWS THE CONVERSION OF L-TRYPTOPHAN TO THE NITRATED FORM VIA TXTE ENZYME, NITROGEN
OXIDE, THE REDUCING AGENT, NADPH AND AN OXYGEN DIRADICAL [18]
FIGURE 3 SHOWS THE MECHANISM OF L-TRYPTOPHAN NITRATION WITH HAEM GROUP OF
P450 TXTE ENZYME [12]
Results are collected from Steps 1 and 2.
1. QM Calculations on Ferric (III) Superoxide and Nitric OxideOptimisationThe spin multiplicity and charge of ferric (iii) superoxide was determined as doublet spin and -1 charge and the structure of ferric (iii) superoxide was provided by 4TPO.pdb. The ferric superoxide was built in GaussView and the input file for calculation was saved as Ferric.Superoxide.com.
The input file was opened in vim editor on the Linux terminal to check details in the route section for the calculation. A geometry optimisation calculation was carried out using DFT B3LYP/GENECP basis set: LanL2DZ for iron and 6-31G for carbon, hydrogen, oxygen, nitrogen, and sulphur atoms to give the optimised geometry in an output file.
Potential Energy Surface ScanningAfter geometry optimization calculations were performed, the results give the lowest energy geometry for the molecule. The results were taken from the output Ferric.Superoxide.log file and used to create a new input file, Ferric.Superoxide.scan.com. Potential energy surface scans were performed on the molecule to observe how the ferric (iii) peroxynitrile was formed and then how nitrogen dioxide was cleaved.
There were two types of scans that were performed: multiple steps of the PES in one calculation or multiple calculations of one step in the PES. The individual scans are differentiated by distance between reactant molecules. The distance between nitric oxide and ferric (iii) superoxide was reduced at intervals of 0.2Å for a Potential Energy Surface Scanning calculation.
Ferric.Superoxide.com file used for geometry optimisation is shown below. The route section was at the beginning of the input and indicates which calculation was used on the data, how much processing power was required as well as other details specifying parameters used on the data.
TABLE 1 TABLE CONTAINING DETAILS ON ROUTE SECTION OF FERRIC.SUPEROXIDE.COM INPUT FILE
%mem=24GB 24GigaBytes (GB) of memory was used to compute calculation
%chk=Ferric.chk A checkpoint file containing results was created with the name Ferric.chk
%nprocshared=2 2 processors were used to compute calculation
#p an extended printout is generated
opt=loose sets optimisation convergence limit
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FIGURE 4 SHOWS INPUT FILE FOR FERRIC
SUPEROXIDE AND NITRIC OXIDE
GEOMETRY OPTIMISATION
CALCULATION USING GAUSSIAN
ub3lyp/genecp defines basis setnosymm prevented molecule
reorientation and caused all computations to be performed in the input orientation
gfoldprint prints basis set in Gaussian format
iop(5/6=5) sets convergence of optimisation
iop(1/8=7) sets maximum step size for optimisation
iop(5/7=128) sets maximum number of iterations for optimisation
Ferric Superoxide Geometry Optimisation
Title
-1 2 Charge and multiplicity, respectively
S 8.027… 24.212… -0.568…
Atom x y zco-ordinates
Fe 0LanL2DZ****C H O N S 06-31G(d)****
Fe0LanL2DZ
Indicated basis set for atoms: Iron has basis set LanL2DZ and atoms carbon, hydrogen, nitrogen, oxygen and sulphur use 6-31G(d) basis set
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The commands used in Linux Terminal to create the potential energy surface scan are outlined below. The route section specifies a scan calculation and the atom co-ordinates were replaced with the co-ordinates from results of the geometry optimization calculation. A single scan or multi-step scan was specified at the end of the input file.
TABLE 2 : TABLE TO SHOW COMMANDS USED TO GENERATE POTENTIAL SURFACE SCANNING INPUT FILE
$cat *.com Read only input file*.com file$xyz *.log View results of geometry
optimisation calculation. Optimised distance between NO2 and L-tryptophan had changed from 5.054Å to 4.135Å
$xyzlast.exe *.log Transfers data on the last geometry in output *.log file to h.xyz
$cp *.com *.scan.com Copies information in *.com to new input file *scan.com
$vi *.scan.com:i (to insert characters)
Opens Vim editor in Linux to edit route section in input file *scan.com
:r h.xyz Reads and writes data from h.xyz containing final optimised geometry to *.scan.com
opt(modredunant,loose) Placed in route section, redundant internal co-ordinates used to perform relaxed potential energy surface scan
11 28 4.1 f Placed at end of input file,atom number 1atom number 2distance between atoms 1 and 2 (f) one step scan
11 28 S 14 -0.2 Placed at end of input file,atom number 1atom number 2(S) several step scannumber of scansdistance intervals
:wq writes and saves changes, then exits vim editor
$g09boon !$ Runs calculation for *.scan.com input file in Gaussian09w on BoonLay Network Server.
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The energy, spin and charge of the system at each distance were recorded and can be used to indicate whether a positive or neutral form of nitrogen dioxide is formed and which form is used for nitration of L-tryptophan.
To find the spin and charges of each step in the potential energy surface scan, the reactant molecules had to be placed into groups based on assigned number labels assigned by Molden:
TABLE 3 TABLE TO SHOW WHICH ATOMS WERE ASSIGNED TO EACH GROUP BASED ON NUMBER LABEL FOR FORMATION OF NITROGEN DIOXIDE USING FERRIC SUPEROXIDE
Group Number Atoms Number LabelGroup 1 Sulphur-Hydrogen 1, 2Group 2 Iron 3Group 3 Oxygen 4Group 4 Porphyrin ligand 5-40Group 5 Oxygen 41Group 6 Nitric oxide 42,43
For each group the spin and charge density was recorded using the spin2012.exe *.log file command. Results on the energy of each step were found in output *.log files which were viewed on GaussView.
2. QM Calculations on L-Tryptophan and Nitrogen Dioxide The same method used for QM calculations on ferric superoxide and nitric oxide was also used for QM calculation on L-tryptophan with nitrogen dioxide (positive and neutral forms).
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FIGURE 5 SHOWS ATOM AND NUMBER LABEL FOR EACH ATOM IN FERRIC SUPEROXIDE AND NITRIC OXIDE
OptimisationThe spin multiplicity and charge of neutral nitrogen dioxide was determined as doublet spin and neutral (0) charge, while the spin and charge of positive nitrogen dioxide was determined as singlet spin and +1 charge. The geometry of L-tryptophan was provided from 4TPO.pdb and nitrogen dioxide was added using GaussView. The structures were optimized to find a local minimum point on the energy surface to give a structure with the lowest energy using DFT B3LYP/6-31G(d) basis set. This geometry optimization was carried out for both positive and neutral forms of nitrogen dioxide with L- tryptophan under anisotropic solvated conditions.
FIGURE 6 SHOWS THE
INPUT FILE FOR L-TRYPTOPHAN AND NITROGEN
DIOXIDE GEOMETRY
OPTIMISATION CALCULATION
USING GAUSSIAN
Potential Energy Surface Scanning After geometry optimization calculations were performed, the results were the lowest energy geometry for the molecule. The results were taken from the output (positive.)nitrogen.solvent.log file and used to create a new input file (positive.)nitrogen.solvent.scan.com. The new input file was used to perform potential energy surface scans on the nitrogen dioxide and L-tryptophan molecules to observe how the nitration of L-tryptophan mechanism proceeds.
Initially individual one step scans at intervals of 0.2Å were carried out to find stable intermediate structure for L-tryptophan nitration. Once a stable transition state was found, the distance between nitrogen dioxide (positive and neutral forms) and L-tryptophan was reduced from intervals of 0.2Å to 0.1Å in a Potential Energy Surface Scanning using multiple steps in one calculation.
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The energy, spin and charge of the system at each distance were recorded and can be used to indicate whether a positive or neutral form of nitrogen dioxide is used for nitration of L-tryptophan.
To find the spin and charges of each step in the potential energy surface scan, the reactant molecules had to be placed into groups based on assigned number labels:
TABLE 4 TABLE TO SHOW WHICH ATOMS WERE ASSIGNED TO EACH GROUP BASED ON NUMBER LABEL FOR NITRATION OF L-TRYPTOPHAN WITH NITROGEN DIOXIDE
Group Number Atoms Number Label1 Nitrogen 12 Carbon 23 Carbon-Oxygen 3,44 Carbon 55 (aromatic) Carbons 6-146 Oxygen 157 Hydrogen 16,178 (aromatic) Hydrogens 18-239 Hydrogen 24
10 Hydrogens 25-2711 Nitrogen Dioxide 28-30
For each group the spin and charge density was recorded using the spin2012.exe *.log file command. Results on energy of each step were found in output *.log files which were viewed on GaussView.
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FIGURE 7 SHOWS ATOM AND NUMBER LABEL FOR EACH ATOM IN L-TRYPTOPHAN AND NITROGEN
3.QM/MM Calculations on P450 TxtE Enzyme [13]Preparation of Topology Files for each MoleculeThe optimised geometry of L-tryptophan was used to calculate a topology file from AMBER parameters using tleap. The topology file for the heme group was copied from parameters given in a previous study [14].
The geometry of L-tryptophan was optimised using g09a2boon. The optimised structure in the output *.log file was then used to create an *.ac file using antechamber from the AMBER software package [6]. Bold indicates the program execution command, words in green indicate the initial file, and words in blue indicate the converted file.
/usr/local/amber10/bin/antechamber –i *.log –fi gout –o *.ac –fo –c resp
*.ac file is then used to form *.prepi using prepgen in AMBER.
/usr/local/amber10/bin/prepgen –i *.ac –o *.prepi –f prepi
Finally *.prepi is converted to an *.frcmod file.
/usr/local/amber10/bin/parmchk –i *.prepi –f prepi –o *.frcmod
These *.prepi and *.frcmod files contain details on bonds, bond angles, torsion and improper torsions for L-tryptophan and were used to identify the parameters of the QM region in the ONIOM calculation.
The parameters for ferric superoxide were already provided in the supporting information of a previous study in the form of a *.mol2 file. This file was converted to *.ac to then generate *.prepi and *frcmod files for ferric superoxide.
/usr/local/amber10/bin/antechamber –fi mol2 –fo –i *.mol2 –o *.ac –c rc –cf qout2
All of these files were used to identify the parameters in the QM region of an ONIOM calculation [13].
Determining Charges for MM and QM RegionsThe neutral or protonated forms of each amino acid residue in P450 TxtE were determined using PROPKA to find the charge of the whole enzyme. 4TPO was the identification tag assigned by the Protein Data Bank for the high-resolution structure (1.23 Å resolution) of P450 TxtE with bound L-tryptophan substrate [15]. 4TPO consists of protein chain A, glycogen molecule (GOL), sulphate molecule (SO4) and an iron complex group (HEM). Only chain A and the HEME group were required, GOL and SO4 were removed.
The missing internal residues were added and saved in 4TPO.2.pdb. PROPKA3.0 was then used to determine the charges of amino acid residues in 4TPO.2.pdb and the total charge of 4TPO. Particular care was take with the amino acid histidine, which has four forms of protonation: HID, where histidine has hydrogen on the delta nitrogen; HIE, where histidine has hydrogen on the epsilon nitrogen; HIP, where histidine has hydrogens on both nitrogens and is positively charged [6].
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TABLE 5 TO SHOW CHARGES ASSIGNED TO AMINO ACID RESIDUES AND OVERALL CHARGE OF ENZYME P450 TXTE [13]
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Preparation for ONIOM Calculation [13] 4TPO.02.pdb contains the co-ordinates and connectivity of each atom in the P450 TxtE enzyme. The hydrogen atoms were manually removed from 4TPO.02.pdb using GaussView and saved as 4TPO_no_H.03.com. The hydrogens were added again using tleap, part of the AMBER software package.
The L-tryptophan and ferric superoxide AMBER parameter files, and 4TPO_no_H.03.com input file were used to create topology files for the whole enzyme. Words in blue indicate the file used for the process. These files can be found in NTU Boon Lay Terminal under /home/guest/4tpo_RHS/QMMM/.
=== “leaprc” ===source leaprc.ff03.r1source leaprc.gaff
> loadamberprep Tryptophan.amber.prepi> loadamberparams Tryptophan.amber.frcmod> loadamberprep CpdI.prepi> loadamberparams Ferric.Superoxide.frcmod> mol = loadpdb 4TPO_no_H.03.pdb> charge mol (-4.000001)> saveAmberParm mol no-Solv1.top no-Solv1.crd> quit
The no-Solv.top and no-Solv.crd contain parameter information for the whole enzyme. These files were converted to no-Solv.pdb. % ambpdb –p no-Solv.top < no-Solv.crd > no-Solv.pdbA problem with this PDB was that the residue numbers were different from those in the original PDB so the residue numbers were modified and put into resnum.pdb. resnum.pdb contained hydrogen atoms and modified residue numbers.% resnumorg.exe 4TPO_no_H.03.pdb no-Solv.pdb
resnum.pdb is generated
ONIOM set up was then prepared in GaussView using resnum.pdb% gview resnum.pdbUsing the multi-layer ONIOM command in the calculation set up in GaussView, basis sets for QM And MM region were defined. The porphyrin ligand in ferric superoxide group, aromatic group of L-tryptophan and sulphur of cysteine residue of enzyme were indicated as part of the high layer (QM region) for QM calculations and the other residues were labelled as part of the low layer for MM calculations. High layer had a ball-and-stick bond appearance while Low layer was shown in stick bond form.
The calculation type was changed to amber=softfirst using VIM editor on the Linux terminal and the charges for each atom were inserted using information from the parameter files created in tleap.
% q_amber2oniom.exe no-Solv.top resnum.com oniom1.com is generated
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FIGURE 8 SHOWS HIGH AND LOW LAYERS IN GAUSSVIEW FOR
ONIOM CALCULATION
As a result of replacement of charges oniom1.com file was no longer well formatted, therefore oniom1.com had to be reformatted. % oniom_format.exe oniom1.com
oniom2.com is generatedThe charges were checked using the following command. % oniom_qcheckG09.exe oniom2.com The total charge of the molecule came to -3.999999
A nitric oxide molecule was added to the oniom2.com file. The changes were saved in oniom2.NO.com.The atom types of HEM were changed to match OH.CpdI.ASH-THR.RC.com. The atom types of MOL were also changed so they were unique in oniom2.NO.com file. Parameters for VDW, bond, angle, torsions and improper torsions for HEM were added from OH.CpdI.ASH-THR.RC.com. The same parameters for MOL were taken from gaff.dat, an information file in AMBER software package. The changes were saved as H-opt.com% oniom_fixHEAVY.exe oniom2.NO.com
h1.com generated% cp h1.com H-opt.04.com
Hydrogen atoms were minimised first, excluding the hydrogen atoms of water molecules. At this stage, residues 186 and 188 were also optimised because they had very strained geometries. New input files containing information on initial and final geometries of P450 enzyme were generated in oniom0.pdb and oniom1.pdb. % g09boon H-opt.04.com % gv H-opt.04.log% oniom_pdb2014.exe no-Solv.pdb H-opt.04.log
oniom0.pdb (initial geometry same as H-opt.04.com) oniom1.pdb (final geometry same as H-opt.04.log)
% vmd oniom0.pdb L-tryptophan substrate, nitrogen dioxide and one superoxide oxygen were missing from oniom0.pdb and oniom1.pdb. total 7631 (7661 including missing atoms).
A new input file was created using the results of H-opt.04.log. % oniom_com2014.exe H-opt.04.com H-opt.04.log
h.com generated
Using h.com, all other atoms were fixed and the water molecules were optimised only. % mv h.com water_opt.comTo do this, for all amino acid residues, 0 became -1 and for water molecules, -1 became 0. % vi water_opt.com % g09boon water_opt.com
A new input file was created using the results of water_opt.com. % oniom_com2014.exe water_opt.com water_opt.log
h.com generatedThen the backbone atoms of each amino acid residue and the oxygen atom of water molecules were fixed in position and the rest of the structure was optimised. % oniom_fixBB2014.exe h.com
h1.com generated
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% mv h1.com BBfix.com% g09boon BBfix.com
At this point the MM microiterations failed to converge. These microiterations are required to ensure that the system remains in the same local valley during the course of optimization [5].
Work in Progress [13]The set up for the ONIOM calculation was not completed due to errors in the calculation, but the following outlines the method to continue the ONIOM Calculation set up. Some errors encountered can be found in the Appendix (A2).
The optimised structure from fixing the backbone atoms were used to generate a new input file where atoms in the QM region are fixed. The structure was optimised again. % oniom_fixHIGH2014.exe h.com
h1.com generated% mv h1.com QMfix.com% g09boon QMfix.com
Finally all the atoms were optimised together as a whole in MMopt2.com. % g09boon MMopt2.com
Once MMopt2.log file had been generated, a pdb file was created. Residues surrounding the iron atom (atom number 5495) at a distance of 15Å from the HEM group were fixed in their positions. From the results of MMopt2 calculation, a new input file was generated. % oniom_pdb2014.exe resnum.pdb MMopt2.log% distRES2014.exe oniom1.pdb 5495 15% oniom_com2014.exe MMopt2.com MMopt2.log% oniom_fixdistRES2014.exe h.com
h1.com generated
This new input file, h1.com, was used for the full ONIOM calculation. Full ONIOM Calculation [13]h1.com was then used to generate an input file for the full ONIOM calculation. This route section of h1.com was changed to indicate separate calculations for High layer and Low layer. High layer uses DFT/Genecp with LanL2DZ specified for iron and 6-31g for carbon, hydrogen, oxygen, nitrogen and sulphur atoms while the Low layer used Amber for all atoms.
20 | P a g e
Results1.QM Calculations on Ferric (III) Superoxide and Nitric OxideThe optimized structure of ferric superoxide with nitric oxide shows the nitric oxide was initially at a distance of 2.4Å from ferric (iii) superoxide. In the potential energy surface scan, the nitric oxide approaches the distal oxygen in ferric superoxide, and a nitrogen-oxygen bond begins to form to eventually give ferric peroxynitrile. At the end of the scan, the oxygen-oxygen bond in ferric peroxynitrile breaks and neutral nitrogen dioxide is cleaved.
The energy of each step of the potential energy surface scan was recorded and converted from Hartree to kcalmol-1. The graph shows that as the distance between nitric oxide and Ferric Superoxide decreases from 2.4Å, the energy increases slightly until nitric oxide is 2.0Å from ferric Superoxide when ferric peroxynitrile is formed. At a distance of 1.4Å, the system is at an intermediate state: nitrogen dioxide is cleaved from ferric peroxynitrile as the oxygen-oxygen bond was broken.
At 1.0Å nitrogen-oxygen bond in nitrogen dioxide is very short and the molecule is distant from the haem group. The high energy implies that the short bond length of nitrogen-oxygen bond is not a stable conformation of nitrogen dioxide.
The energy barrier for this reaction is therefore 16.444kcalmol-1. Exact figures extracted and used to plot this graph can be found in the Appendix (A3).
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0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.60.00
10.0020.0030.0040.0050.0060.0070.0080.0090.00
Graph To Show Energy/kcalmol-1 Against Distance/Å Between Nitric Oxide and Ferric
Superoxide
Distance/Å
Ener
gy/k
calm
ol-1
FIGURE 11 SHOWS FORMATION OF FERRIC PEROXYNITRILE
FIGURE 10 SHOWS FERRIC SUPEROXIDE AND NITRIC OXIDE
IN INITIAL STATE
FIGURE 9 SHOWS FORMATION OF NEUTRAL NITROGEN DIOXIDE AND
FERROUS OXIDE
Using the data collected from spin2012.exe, spin density was plotted against distance between ferric superoxide and nitric oxide. The exact figures can be found in the Appendix (A4).
The most important groups are Group 2 (iron in haem group), Group 5 (distal oxygen of ferric superoxide) and Group 6 (nitric oxide) as these are the groups mainly affected by and involved in the reaction. The charge of the whole system has also been noted.
Iron ion spin decreases slightly as nitric oxide approaches ferric superoxide but increases slightly as ferric peroxynitrile is formed. Spin density of the iron ion then decreases as nitrogen dioxide is cleaved from ferric peroxynitrile.
For the distal oxygen, spin density increases as nitric oxide approaches ferric superoxide and decreases as ferric peroxynitrile is formed. The spin density of the distal oxygen increases slightly as nitrogen dioxide is cleaved from ferric peroxynitrile.
Spin density of nitric oxide decreases significantly as nitric oxide approaches ferric superoxide and begins to increase only as ferric peroxynitrile is formed. Nitrogen dioxide is cleaved from ferric peroxynitrile so the spin density of nitric oxide decreases, but spin density increases again as distance between nitrogen dioxide and the haem group increases.
For the whole system, total spin density remains the same throughout the process.
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0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
Graph To Show How Spin Density Varies With Distance/Å Between Nitric Oxide and Ferric Superoxide
Group 1 Spin Group 2 SpinGroup 3 SpinGroup 4 SpinGroup 5 SpinGroup 6 SpinWhole System
Distance/Å
Spin
Den
sity
Using the data collected from spin2012.exe, charge density was plotted against distance between ferric superoxide and nitric oxide. The exact figures can be found in the Appendix (A5).
The most important groups are Group 2 (iron in haem group), Group 5 (distal oxygen of ferric superoxide) and Group 6 (nitric oxide) as these are the groups mainly affected by and involved in the reaction. The charge of the whole system has also been noted.
For the whole system, as nitric oxide approaches ferric superoxide the charge decreases rapidly until ferric peroxynitrile is formed. The charge of the whole system increases slightly as nitrogen dioxide is cleaved, and then remains the same for the rest of the scan.
Group 2 charge decreases slightly as nitric oxide approaches ferric superoxide and forms ferric peroxynitrile. Charge continues to decrease as nitrogen dioxide is cleaved from ferric peroxynitrile.
For Group 5, the charge increase slightly as nitric oxide approaches ferric superoxide. The charge decreases slightly as ferric peroxynitrile is formed and then increases again as nitrogen dioxide is cleaved from ferric peroxynitrile.
Charge density for Group 6 shows a similar pattern: as nitric oxide approaches ferric superoxide, the charge decreases. However charge increases as ferric peroxynitrile is formed. This is because another nitrogen-oxygen is being formed between ferric superoxide and nitric oxide. The charge then decrease as nitrogen dioxide is cleaved from ferric peroxynitrile.
The charge density of Group 5 and 6 at distance 1.0Å are both almost at 0, suggesting that neutral nitrogen dioxide has been formed.
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0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6
-1.20000
-1.00000
-0.80000
-0.60000
-0.40000
-0.20000
0.00000
0.20000
0.40000
0.60000
Graph To Show How Charge Density Varies With Distance/Å Between Nitric Ox-ide and Ferric Superoxide
Group 1 Charge Group 2 ChargeGroup 3 ChargeGroup 4 ChargeGroup 5 ChargeGroup 6 ChargeWhole System
Distance/Å
Char
ge D
ensit
y
2.QM Calculations on L-Tryptophan and Nitrogen Dioxide L-Tryptophan and Neutral Nitrogen Dioxide The potential energy surface scan of L-tryptophan and neutral nitrogen dioxide that neutral nitrogen dioxide bonds with an aromatic carbon (C11) on L-tryptophan.
The energy of each step of the potential energy surface scan was recorded recorded and converted from Hartree to kcalmol-1. The graph shows that as the distance between neutral nitrogen dioxide and L-tryptophan decreases from 4.1Å to 3.6Å, the energy of the system remains the same. The energy decreases from 3.6Å to an energy minimum at 3.4Å indicating an intermediate at 3.4Å. At the intermediate point, hydrogen bonding appears to form between neutral nitrogen dioxide and amine group of L-tryptophan. The energy increases as neutral nitrogen dioxide approaches the carbon in the aromatic ring. The energy reaches a maximum at 1.8Å implying a transition state: nitrogen dioxide forms a bond with the carbon (C11), breaking the aromaticity of L-tryptophan.
The energy barrier for the reaction and therefore the energy of the transition state for the nitration of L-tryptophan is therefore 13.015kcalmol-1. Exact data can be found in the Appendix (A6).
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1.5 2 2.5 3 3.5 4 4.50.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Graph To Show How Energy/kcalmol-1 Varies With Distance/Å Between L-Tryptophan And
Neutral Nitrogen Dioxide
Distance/Å
Ener
gy/k
calm
ol-1
FIGURE 14 SHOWS L-TRYPTOPHAN AND
NEUTRAL NITROGEN DIOXIDE
FIGURE 12 SHOWS THE INTERMEDIATE STATE: H-BOND
FORMATION WIT L-TRYPTOPHAN WITH NEUTRAL NITROGEN
DIOXIDE
FIGURE 13 SHOWS THE TRANSITION STATE: NITROGEN DIOXIDE IS FORMING A BOND AT AROMATIC CARBON ON L-
TRYPTOPHAN
Using the data collected from spin2012.exe, spin density was plotted against distance between L-tryptophan and neutral nitrogen dioxide. The exact figures can be found in the Appendix (A7).
The most important groups are Group 5 (aromatic carbons) and Group 11 (neutral nitrogen dioxide) as these are the groups mainly affected by and involved in the reaction. The charge of the whole system has also been noted.
The spin density of the whole system remains at 1 until 2.2Å where the spin density drops briefly, which is where nitrogen dioxide bonds to aromatic carbon (C11) and breaks aromaticity of the benzene ring.
The spin density of the aromatic carbons increases steadily as neutral nitrogen dioxide approaches L-tryptophan and binds to the aromatic carbon (C11),
For neutral nitrogen dioxide spin density decreases as neutral nitrogen dioxide approaches L-tryptophan and forms a hydrogen bond. The spin density drops rapidly at 2.2Å, when nitrogen dioxide binds to aromatic carbon (C11). However spin density increases again at 2.0Å to fall again to 0 spin density by the end of the scan.
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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
-0.20000
0.00000
0.20000
0.40000
0.60000
0.80000
1.00000
1.20000
Graph To Show How Spin Density Varies With Distance/Å Between L-Tryptophan and Neutral Nitrogen Dioxide
Group 1 Spin
Group 2 Spin
Group 3 Spin
Group 4 Spin
Group 5 Spin
Group 6 Spin
Group 7 Spin
Group 8 Spin
Group 9 Spin
Group 10 Spin
Group 11 Spin
Whole System
Distance/Å
Spin
Den
sity
Using the data collected from spin2012.exe, charge density was plotted against distance between L-tryptophan and neutral nitrogen dioxide. The exact figures can be found in the Appendix (A8).
The most important groups are Group 5 (aromatic carbons) and Group 11 (neutral nitrogen dioxide) as these are the groups mainly affected by and involved in the reaction. The charge of the whole system has also been noted.
For the whole system, charge density remains at 0 throughout the potential energy surface scan.
Aromatic carbons initially have negative charge density, which remains when neutral nitrogen dioxide approaches L-tryptophan and forms a hydrogen bond to nitrogen (Group 1) at 3.5Å. Charge density of aromatic carbons becomes slightly more positive as neutral nitrogen dioxide approaches and binds to an aromatic carbon (C11) at a distance of 2.2Å. This bonding interaction breaks aromaticity of the benzene ring thus making the charge density of the normally electron rich benzene more positive.
Neutral nitrogen dioxide initially has zero charge density, which decreases as neutral nitrogen dioxide approaches the aromatic ring in L-tryptophan. The charge density of neutral nitrogen dioxide remains constant at a negative value for the remainder of the scan.
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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5
-1.5
-1
-0.5
0
0.5
1
1.5
2
Graph To Show How Charge Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide
Group 1
Group 2
Group 3
Group 4
Group 5
Group 6
Group 7
Group 8
Group 9
Group 10
Group 11
Whole SystemDistance/Å
Char
ge D
ensit
y
FIGURE 15 SHOWS FINAL GEOMETRY FROM SCAN
BETWEEN L-TRYPTOPHAN AND POSITIVE NITROGEN
DIOXIDE
FIGURE 16 SHOWS INTERMEDIATE STATE
BETWEEN L-TRYPTOPHAN AND POSITIVE NITROGEN
DIOXIDE
FIGURE 18 SHOWS TRANSITION STATE
BETWEEN L-TRYPTOPHAN AND POSITIVE NITROGEN
DIOXIDE
FIGURE 17 SHOWS OPTIMISED GEOMETRY
OF L-TRYPTOPHAN AND POSITIVE NITROGEN
DIOXIDE
L-Tryptophan and Positive Nitrogen DioxideThe optimised geometry of L-tryptophan and positive nitrogen dioxide showed positive nitrogen dioxide attaching to the negatively charged carbonyl group of L-tryptophan. A potential energy surface scan was carried out and the positive form of nitrogen dioxide remains attached to the carbonyl group of L-tryptophan. This is because the oxygen in the carbonyl is negatively charged, while nitrogen dioxide is positively charged and this electrostatic attraction is too strong to be broken. No further analysis of spin and charge density of L-tryptophan and positive nitrogen dioxide were carried out.
The energy of each step of the potential energy surface scan was recorded recorded and converted from Hartree to kcalmol-1.
Evaluating just the graph of how energy varies with distance, it appears as though there is a reaction between positive nitrogen dioxide and L-tryptophan as there is a presence of a transition state at 3.8Å, an intermediate state at 3.6Å, and then another possible transition state with greater energy at 3.0Å. This is similar to what was found in the reaction between neutral nitrogen dioxide and L-tryptophan.
However upon visualisation, it can be seen that at all these distances, positive nitrogen dioxide does not approach the aromatic group of L-tryptophan and no nitration mechanism occurs. Exact figures can be found in the Appendix (A9).
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2.5 3 3.5 4 4.5 5 5.5 60.00
0.50
1.00
1.50
2.00
2.50
3.00
Graph To Show How Energy/kcalmol-1 Varies With Distance/Å Between L-Tryptophan And
Positive Nitrogen Dioxide
Distance/Å
Ener
gy/k
calm
ol-1
ConclusionThe main purpose of this study was to find the mechanism of nitration of L-tryptophan with the haem group in P450 TxtE enzyme.
As can be seen from the results, ferric superoxide containing a radical oxygen species is the catalyst for the reaction. Ferric superoxide binds to nitric oxide to form ferric peroxynitrile. Neutral nitrogen dioxide is then cleaved from ferric peroxynitrile. Neutral nitrogen dioxide then nitrates L-tryptophan at an aromatic carbon (C11).
Neutral nitrogen dioxide is used in the nitration mechanism of L-tryptophan, which is unexpected as in organic synthesis the reactive species in an aromatic electrophilic substitution reaction is positive nitrogen dioxide.
Using a completed ONIOM calculation, further studies can be carried out using molecular dynamics simulations to find compatibilities of the haem group with organic solvents [9] and whether other aromatic substrates can also be nitrated using TxtE.
28 | P a g e
FIGURE 19 SHOWS MECHANISM OF ACTION FOR NITRATION OF L-TRYPTOPHAN WITH P450
TXTE [12]
Appendix
A1: Some Linux Commands
A2: Some Errors Encountered Setting Up ONIOM
A3: Table to Show Energy/kcalmol-1 Against Distance/Å between Nitric Oxide and Ferric Superoxide
A4: Table To Show How Spin Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide
A5: Table To Show How Charge Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide
A6: Table To Show How Energy/kcalmol-1 Varies with Distance/Å between L- Tryptophan and Neutral Nitrogen Dioxide
A7: Tables To Show How Spin Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide
A8: Tables To Show How Charge Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide
A9: Table to Show Energy/kcalmol-1 Against Distance/Å between Positive Nitrogen Dioxide and L-Tryptophan
29 | P a g e
A1: Some Linux Commands [16] [17]
Command Resultls Lists all files in directoryls-a List all files in directory including hidden filesmkdir Make new directorycd /nameofdirectory/ Change directorycd . Print current directorycd .. Print parent directorypwd Print working directory~ Refer to home directorycp *.file new.file Copy file.* to newfile.*rmdir nameofdirectory Remove directoryclear Clear window of previous commandscat file.* Read only of text fileless file.* Writes file content page at a timehead file.* Writes first 10 lines of filetail file.* Writes last 10 lines of fileless word file.* Searches for a word within fileg09boon *.com Run calculation in any input file on Gaussian09wxyz *.log View results from calculation outputxyzlast.exe *.log Writes last geometry from *.log to h.xyzxyzlow13.exe *.log Writes lowest geometry from *.log to h.xyzxyzlowforce.exe *.log Writes lowest force geometry from *.log to h.xyzvi *.com or vi *.log Opens VIM text editor in terminal
Some Commands Within VIM editor on Linux TerminalCommand Resulti Allows insertion of characters into the filer Allows characters to be replaced within the file:/word Searches for instances of “word” in the file/word Searches for instances of “word” in the file:/11,6437s/ 0 /-1 /g Between lines 11 and 6437 find the pattern “ 0 “
and replace with “-1 “:/11,6437s/ 0 /-1 /gc Between lines 11 and 6437 find the pattern “ 0 “
and replace with “-1 “ confirming each change
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A2: Some Errors Encountered Setting Up ONIOM
MM micro iterations failed
Checked connectivity of all residues
Optimising particular residues at a time 1-32, 33-69 but cannot do this as structure will not optimise to correct saddle point [5].
Increasing processors/memory
iop(4/33=3) long computational time and large memory required
scf=qc and scf=xqc with opt(Cartesian) calculation terminates with error l999 . cannot use Cartesian when freezing co-ordinates.
creating a checkpoint file and restarting from checkpoint no data in checkpoint so cannot restart from here.
nomicro requires a large amount of memory ~7666MW
opt(redundant) MM failure / sometimes appears to work… very long computational time / requires large memory and processors
opt(GEDIIS) MM failure
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A3: Table to Show Energy/kcalmol-1 Against Distance/Å between Nitric Oxide and Ferric Superoxide
Step Number Distance/Å Energy Energy-EnergyRC Energy / kcalmol-1
1 2.4 -1790.60675 0.01832 11.495522 2.2 -1790.60226 0.02280 14.309923 2.0 -1790.60134 0.02373 14.888294 1.8 -1790.61261 0.01245 7.815005 1.6 -1790.62293 0.00214 1.342876 1.4 -1790.62507 0.00000 0.000007 1.2 -1790.59886 0.02620 16.443898 1.0 -1790.49466 0.13040 81.82849
A4: Table To Show How Spin Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide
Step Number
Distance/Å Group 1 Spin
Group 2 Spin
Group 3 Spin
Group 4 Spin
Group 5 Spin
Group 6 Spin
Whole System
1 2.4 0.02119 1.11841 -0.45500 -0.08323 -0.66735 1.06597 0.999992 2.2 0.01651 1.03926 0.43447 -0.06725 0.63892 -1.06192 0.999993 2.0 0.00830 1.04123 0.42630 -0.07356 0.60093 -1.00020 1.003004 1.8 0.01334 1.02198 0.01162 -0.08067 -0.10684 0.14056 0.999995 1.6 0.01643 1.01425 0.04675 -0.07829 -0.01518 0.01603 0.999996 1.4 0.01578 1.01508 0.05117 -0.07832 -0.00763 0.00392 1.000007 1.2 0.01682 1.01620 0.05371 -0.07941 -0.00858 0.00126 1.000008 1.0 0.06517 0.69669 -0.67126 -0.07235 0.20820 0.77355 1.00000
A5: Table To Show How Charge Density Varies With Distance/Å Between Neutral Nitric Oxide and Ferric Superoxide
Step Number
Distance/Å Group 1 Charge
Group 2 Charge
Group 3 Charge
Group 4 Charge
Group 5 Charge
Group 6 Charge
Whole System
1 2.4 -0.21142 0.47885 -0.24682 -0.07172 -0.18363 -0.11982 -0.354562 2.2 -0.17943 0.46532 -0.02498 -0.69659 -0.17635 -0.16316 -0.775193 2.0 -0.17093 0.46245 -0.25190 -0.69219 -0.16219 -0.18479 -0.999554 1.8 -0.21987 0.45645 -0.32462 -0.73867 -0.19237 0.01907 -1.000015 1.6 -0.20778 0.44326 -0.32930 -0.72702 -0.18724 0.00808 -1.000006 1.4 -0.19295 0.43090 -0.32013 -0.70807 -0.18019 -0.02956 -1.000007 1.2 -0.17678 0.40764 -0.30577 -0.68113 -0.13553 -0.10843 -1.000008 1.0 -0.29188 0.41925 -0.40854 -0.71593 -0.01974 0.01683 -1.00001
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A6: Table To Show How Energy/kcalmol-1 Varies with Distance/Å between L- Tryptophan and Neutral Nitrogen Dioxide
Step Number Distance/Å Energy/au Energy - EnergyRC Energy/kcalmol-1
1 4.09435 -891.4484 0.0023 1.4691
2 3.99435 -891.4484 0.0024 1.4759
3 3.89435 -891.4483 0.0024 1.4986
4 3.79435 -891.4483 0.0025 1.5385
5 3.69435 -891.4483 0.0025 1.5382
6 3.59435 -891.4483 0.0025 1.5400
7 3.49435 -891.4490 0.0018 1.1121
8 3.39435 -891.4507 0.0000 0.0000
9 3.29435 -891.4507 0.0000 0.0052
10 3.19435 -891.4507 0.0001 0.0407
11 3.09435 -891.4505 0.0002 0.1542
12 2.99435 -891.4502 0.0006 0.3508
13 2.89435 -891.4497 0.0010 0.6416
14 2.79435 -891.4491 0.0017 1.0458
15 2.69435 -891.4482 0.0026 1.6044
16 2.59435 -891.4470 0.0038 2.3549
17 2.49435 -891.4455 0.0053 3.3100
18 2.39435 -891.4437 0.0070 4.3900
19 2.29435 -891.4413 0.0095 5.9332
20 2.19435 -891.4384 0.0124 7.7684
21 2.09435 -891.4353 0.0155 9.7047
22 1.99435 -891.4325 0.0182 11.4193
23 1.89435 -891.4307 0.0200 12.5727
24 1.79435 -891.4300 0.0207 13.0152
25 1.69435 -891.4301 0.0207 12.9771
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A7: Tables To Show How Spin Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide
Distance/Å Group 1 Spin Group 2 Spin Group 3 Spin Group 4 Spin Group 5 Spin
4.0 0.00000 0.00007 0.00000 0.00000 -0.000013.8 0.00000 0.00010 0.00000 -0.00010 0.000513.6 0.00000 0.00016 0.00001 -0.00005 0.003123.4 0.00001 0.00009 0.00002 -0.00012 0.010053.2 0.00027 -0.00006 0.00005 0.00000 0.032773.0 0.00010 0.00018 0.00000 -0.00020 0.062122.8 0.00017 0.00108 0.00053 -0.00116 0.122142.6 0.00007 0.00159 0.00069 -0.00178 0.201392.4 -0.00012 0.00200 0.00073 -0.00199 0.291172.2 -0.00021 0.00218 0.00050 -0.00209 0.425722.0 -0.00020 0.00217 0.00014 -0.00162 0.613331.8 -0.00001 0.00165 -0.00014 -0.00016 0.808031.6 0.00024 0.00089 -0.00027 0.00164 0.929281.4 0.00038 0.00044 -0.00032 0.00288 0.979581.2 0.00036 0.00035 -0.00032 0.00343 1.004541.0 0.00019 0.00050 -0.00026 0.00342 1.02987
Group 6 Spin
Group 7 Spin
Group 8 Spin
Group 9 Spin
Group 10 Spin
Group 11 Spin
Whole System
0.00000 0.00000 -0.00066 -0.00003 0.00000 1.00062 0.999990.00001 0.00000 -0.00075 -0.00003 0.00000 1.00017 0.999910.00002 0.00002 -0.00082 -0.00007 0.00001 0.99759 0.999990.00002 0.00005 -0.00091 -0.00023 0.00003 0.99099 1.000000.00000 0.00002 -0.00152 -0.00019 -0.00207 0.97074 1.000010.00003 0.00019 -0.00288 -0.00024 -0.00175 0.94246 1.000010.00017 0.00080 -0.00496 -0.00056 -0.00209 0.88386 0.999980.00031 0.00136 -0.00790 -0.00052 -0.00206 0.80684 0.999990.00044 0.00175 -0.01098 -0.00043 -0.00158 0.71901 1.000000.00047 0.00214 -0.01516 -0.00032 -0.00090 0.05877 0.471100.00041 0.00219 -0.02068 -0.00025 0.00022 0.40430 1.000010.00026 0.00146 -0.02340 -0.00021 0.00138 0.21116 1.000020.00008 0.00036 -0.01942 -0.00014 0.00200 0.08534 1.00000-0.00003 -0.00038 -0.01539 -0.00002 0.00226 0.03061 1.00001-0.00007 -0.00069 -0.01651 0.00006 0.00190 0.00695 1.00000-0.00007 -0.00061 -0.02599 0.00007 0.00098 -0.00810 1.00000
34 | P a g e
A8: Tables To Show How Charge Density Varies With Distance/Å Between L-Tryptophan And Neutral Nitrogen Dioxide
Distance/Å Group 1 Charge
Group 2 Charge
Group 3 Charge
Group 4 Charge
Group 5 Charge
4.0 -0.73511 -0.09594 -0.03716 -0.37538 -1.021403.8 -0.73465 -0.09637 -0.03610 -0.37535 -1.021253.6 -0.73450 -0.09643 -0.03581 -0.37586 -1.018793.4 -0.73525 -0.09607 -0.03519 -0.37733 -1.014613.2 -0.75226 -0.09917 -0.03445 -0.37318 -1.004033.0 -0.75080 -0.10045 -0.03402 -0.37246 -0.991262.8 -0.75607 -0.10471 -0.02994 -0.37029 -0.972312.6 -0.75710 -0.10851 -0.02878 -0.36849 -0.940302.4 -0.75496 -0.11032 -0.02712 -0.36942 -0.903592.2 -0.75435 -0.11184 -0.02716 -0.36928 -0.862802.0 -0.75294 -0.11346 -0.02682 -0.36918 -0.835561.8 -0.74992 -0.11221 -0.02598 -0.37070 -0.837981.6 -0.74669 -0.11126 -0.02644 -0.37063 -0.851851.4 -0.74441 -0.11019 -0.02594 -0.37113 -0.863921.2 -0.74356 -0.11358 -0.02713 -0.37131 -0.885021.0 -0.74656 -0.12580 -0.02773 -0.36612 -0.92613
Group 6 Charge
Group 7 Charge
Group 8 Charge
Group 9 Charge
Group 10 Charge
Group 11 Charge
Whole System
-0.63803 0.35285 1.11371 0.19461 1.23060 0.01125 0.00000-0.63923 0.35293 1.11335 0.19456 1.23064 0.01147 0.00000-0.63957 0.35350 1.11586 0.19390 1.23091 0.00679 0.00000-0.63893 0.35465 1.12111 0.19104 1.23171 -0.00113 0.00000-0.64100 0.35097 1.13193 0.19282 1.23149 -0.00311 0.00001-0.64084 0.35162 1.14241 0.19384 1.23261 -0.03064 0.00001-0.64154 0.35415 1.16784 0.19478 1.23867 -0.08059 -0.00001-0.64187 0.35646 1.19522 0.19573 1.24182 -0.14419 -0.00001-0.64234 0.35922 1.22170 0.19708 1.24251 -0.21324 -0.00048-0.64232 0.36206 1.25191 0.19748 1.24418 -0.28788 0.00000-0.64242 0.36427 1.27097 0.19786 1.24588 -0.33858 0.00002-0.64178 0.36489 1.27059 0.19768 1.24571 -0.34028 0.00002-0.64049 0.36456 1.27018 0.19692 1.24345 -0.32776 -0.00001-0.64070 0.36565 1.28342 0.19547 1.24012 -0.32837 0.00000-0.64065 0.36643 1.31801 0.19612 1.24129 -0.34059 0.00001-0.64226 0.36813 1.39640 0.19638 1.24337 -0.36968 0.00000
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A9: Table to Show Energy/kcalmol-1 Against Distance/Å between Positive Nitrogen Dioxide and L-Tryptophan
Step Number Distance/Å Energy/au Energy - EnergyRC Energy/kcalmol-1
1 5.8094 -891.25970829 0.00008146 0.051116924
2 5.6094 -891.25965264 0.00013711 0.086037828
3 5.4094 -891.25977672 0.00001303 0.008176449
4 5.2094 -891.25978975 0.00000000 0.000000000
5 5.0094 -891.25976709 0.00002266 0.014219365
6 4.8094 -891.25956753 0.00022222 0.139445161
7 4.6094 -891.25923404 0.00055571 0.348713304
8 4.4094 -891.25887284 0.00091691 0.575369736
9 4.2094 -891.25848399 0.00130576 0.819376805
10 4.0094 -891.25808690 0.00170285 1.068554552
11 3.8094 -891.25780519 0.00198456 1.245330253
12 3.6094 -891.25959603 0.00019372 0.121561140
13 3.4094 -891.25913882 0.00065093 0.408464759
14 3.2094 -891.25795004 0.00183971 1.154435502
15 3.0094 -891.25552272 0.00426703 2.677601862
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