Theoretical studies on structure and mechanism of vanadium haloperoxidases [Elektronische Ressource] / von Masroor Ahmad Khan Bangesh
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Theoretical studies on structure and mechanism of vanadium haloperoxidases [Elektronische Ressource] / von Masroor Ahmad Khan Bangesh

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Masroor BangeshTHEORETICAL STUDIES ON STRUCTURE ANDMECHANISM OF VANADIUM HALOPEROXIDASESDISSERTATIONTheoretical Studies on Structure andMechanism of Vanadium HaloperoxidasesDISSERTATIONzur Erlangung des akademischen Grades doctor rerum naturalium(Dr. rer. nat.)vorgelegt dem Rat der Chemisch-Geowissenschaftlichen Fakult¨atder Friedrich-Schiller-Universit¨at Jenavon M.Phil. Masroor Ahmad Khan Bangeshgeboren am 09.03.1971in Balakot, PakistanGutachter1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Tag der o¨ffentlichen Verteidigung:Knowledge brings power. And along it, more control inthe hands of powerful. The level of destruction whichmodern militaries are able to inflict upon helplessnon-combatants is a testimony to this. This humble workis dedicated to the innocent victims of unethical use ofknowledge.AcknowledgementsGratitude is offered where it is due, and foremost is to my advisor Prof. Dr. Winfried Plasswhose continued support and guidance in all the matters scientific put me in a position topresent this work. Particular is the appreciation for his endless patience which helped mediscover my own eccentricities.I feel overwhelmingly indebted to BBG and all the members of our family for theirpatience during my long stay abroad. That I took so long to return is my failing.



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Published 01 January 2008
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Masroor Bangesh
DISSERTATIONTheoretical Studies on Structure and
Mechanism of Vanadium Haloperoxidases
zur Erlangung des akademischen Grades doctor rerum naturalium
(Dr. rer. nat.)
vorgelegt dem Rat der Chemisch-Geowissenschaftlichen Fakult¨at
der Friedrich-Schiller-Universit¨at Jena
von M.Phil. Masroor Ahmad Khan Bangesh
geboren am 09.03.1971
in Balakot, PakistanGutachter
1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Tag der o¨ffentlichen Verteidigung:Knowledge brings power. And along it, more control in
the hands of powerful. The level of destruction which
modern militaries are able to inflict upon helpless
non-combatants is a testimony to this. This humble work
is dedicated to the innocent victims of unethical use of
Gratitude is offered where it is due, and foremost is to my advisor Prof. Dr. Winfried Plass
whose continued support and guidance in all the matters scientific put me in a position to
present this work. Particular is the appreciation for his endless patience which helped me
discover my own eccentricities.
I feel overwhelmingly indebted to BBG and all the members of our family for their
patience during my long stay abroad. That I took so long to return is my failing. That
they still own me is their magnanimity.
Greatly acknowledged are the contributions of my colleague Dr. Axel Buchholz towards
my handling of matters of which I had little knowledge and no experience, i.e. living in
Germany, understanding thevogonic poetry of German officialdom and moving from Siegen
to Jena. Besides, his help in solving occasional computer hardware problems enabled my
research work to run smoothly.
Special thanks to my former Rumanian colleague Dr. Simona Nica for giving the sem-
blance of social existence to what was a secluded life in Siegen. During her stay in AG
Plass, she was always kind and helpful. The memorable conversations we had, covered
a wide range of topics from latest scandals (from anywhere) to Schopenhauer. Particu-
larly, her aversion towards agreeing on almost anything made the conversations much more
enjoyable (and explosive).
I am thankful to my many colleagues of past and present for maintaining a peaceful co-
existence with me in our office (in case of Arne Roth, Dr. Manjola Manka, Eike Spielberg
and Daniel Geibig), in the coffee-room (Anja Burkhardt and Lotte Neupert) and in the
group (rest of group). I am specially thankful to Daniel Geibig and Rainer Wilcken for
translating the abstract into German language. I highly regard Arne’s deep understanding
Aof the finer aspects of LT X and tea, and his hitchhiking experiences in the distant partsE
of the galaxy. His readiness to share these is even more appreciated.
At different stages of my stay in Jena I was lucky enough to have the friendship of
many wonderful people. My gratitude is to Dr. Altaf Ahmad, Dr. Mohammad Asim,
Dr. Aurangzeb Jathol, Dr. Ijaz Hussain, Dr. Kamran Mirza, Krishna, Dr. Shaid Raja,
Dr. Yogesh Tiwari, Waseem and Dr. Zafar Warraich for their nice company and light
cricket. Where applicable, their respective better-halves are thanked for the hospitality I
enjoyed at their homes. Special thanks to Dr. Jathol for the help he provided during my
viiI am grateful to Jameel Butt Sahib, Ch. Abdulbasit Sahib and Baljinder Singh Sahib,
for the undeserved level of love and respect I got from these extremely kind persons.
Maria, Benji, Carla, Sandra and Paulis are the loving friends I had the privilege to have
in Jena. I am thankful to them for being the nice company during many stressful times.
Duly acknowledged are excellent supercomputing facilities provided by John von Neu-
mann Institute for Computing (NIC) at the Forchungzentrum Ju¨lich as without these, the
numbers reported in this work would not have been obtained. Moreover, opportunities
to attend the extraordinary scientific workshops and schools organised by NIC were great
experience both in scientific and social terms.
After all is said and done, what should not remain unmentioned are the conducive re-
search and personal conditions. Former were made possible due to smooth working of com-
puting and other facilities in AG Plass. Later mainly because of uninterrupted provision
of food, beverages and shelter. Money for both came from Deutche Forchungsgemeinschaft
and Friedrich-Schiller Universit¨at Jena, which is duly acknowledged.
Computational implementation of density functional theory was used to investigate the
active site structure and haloperoxidative activity of vanadium containing haloperox-
idases (VHPO). These enzymes utilise hydrogen peroxide and halide as cosubstrates
and produce a reactive hypohalous species by two-electron oxidation of halide. Hypo-
halous species thus produced is responsible for halogenated organic substances found in
the habitat of VHPO carrying organisms. For instance, the halogenated organic com-
pounds isolated from sea water are attributed to haloperoxidative activity of VHPO
found in sea weeds. The following two reactions describe the chemical transformation
performed by VHPO,
− +X + H + H O −→ HOX + H O (A)2 2 2
− − 1X + 2H O −→ X + 2H O + O ( ) (B)2 2 2 2 g
i.e. either a hypohalous species is produced (Eq. A) which then goes on to halogenate
the available organic substrates. Or in the absence of organic substrates, net dispropor-
tionation of two hydrogen peroxide molecules into water and molecular oxygen (in first
excited state) is carried (Eq. B). VHPO enzymes are classified on the basis of whether
they can oxidise chloride in addition to bromide and iodide, in which case VHPO is
named vanadium chloroperxidase (VCPO). The VHPO which catalyse the oxidation
of only bromide and iodide are termed as vanadium bromoperoxidase (VBPO). The
x-ray crystal structure of both type of enzymes reveal an identical active site char-
acterised by a histidine residue coordinated to vanadate cofactor. Moreover, anionic
oxygen atoms of vanadate are found to receive hydrogen bonds from the side chains of
same set of amino acids. The VCPO sequence of these hydrogen bonding amino acids
is Lys353, Arg360, Ser402, Gly403, His404 and Arg490. A comparison of active sites
of VCPO and VBPO according to their x-ray crystal structures is shown in Fig. 1.1.
Several questions regarding the structure and mechanism of VHPO pertained at the
time when this investigation started. A summary of conclusions of this thesis is given
in the following paragraphs.
RestingstateofVHPO. The most fundamental question was regarding the geome-
try, electronic structure and protonation state of vanadate cofactor. In short, this could
be neatly framed in following queries: Does vanadate cofactor has trigonal bipyramidal
(TBP) geometry in the resting state of VHPO as was widely believed on the basis of
3+its appearance in x-ray crystal structure? And whether vanadium(V) exists as VO
+or VO in the native VHPO active site? Fortunately, apart from the x-ray crystal2
structure, the optical spectrum of active site had been successfully utilised to monitor
the enzyme turn-over. This provided an experimental hint about the electronic struc-
ture of vanadate cofactor. Time-dependent density functional theory (Chapter 2) was
utilised to calculate the optical excitations of a variety of optimised structures modelled
after the pentacoordinated vanadate cofactor (Chapter 3). These models (Fig. 3.1)
included, pentacoordinated vanadate in trigonal bipyramidal (TBP) geometries and
in monoanionic, neutral, and cationic protonation states. In addition, dianionic and
monoanionic TBP structures enclosed in protein matrix (comprised of above listed ac-
tive site residues), were also studied (Fig. 3.2). And finally, a pentacoordinated square
ixpyramidal geometry (SP) in monoanionic form was considered (Fig. 3.1). By comparing
calculated electronic excitation energies/oscillator strengths of different structures with
above mentioned experimental optical spectrum allowed us to conclude that vanadate
in the resting state active site exists in distorted TBP geometry with most probably in
monoanionic (diprotonated) protonation state.
Hydrogen bonding in VCPO and VBPO active sites. The confirmation of this
conclusion, to be described shortly, again came under different context. The unequal
catalytic performance of VCPO and VBPO in carrying chloroperoxidase activity ap-
peared to be greatly puzzling in the view of the fact that both enzyme systems share an
identical active site in terms of amino acid residues surrounding the vanadate cofactor.
We hypothesised that observed crystallographic positions of active site amino acid side
chains are dictated by secondary protein structure exterior to active site pocket. This
would subtly determine the variation in pattern of hydrogen bonding between anionic
oxygen atoms of vanadate and positively charged amino acid residues of active site. In
order to substantiate this hypothesis, it was necessary to study the active site hydrogen
bonding network in detail. The crystallographic positions of (non-hydrogen) atoms of
active site residues were taken as carriers of all the effects of outer protein environment,
and geometry of TBP vanadate was optimised in active site pockets of both VCPO and
VBPO, while preserving the crystallographically determined orientations of hydrogen
bonding residues of active site (Chapter 4). Furthermore, different variations on proto-
nation states of TBP vanadate were also tried. Additionally, similar optimisations were
done by utilising x-ray crystal structures of site mutated versions of VCPO enzyme in
which Arg360 and His404 residues are replaced.
The energies of hydrogen bonding interaction between vanadate and active site
residues in the structures obtained from above mentioned optimisations and some small
model hydrogen bonded complexes, were calculated with supermolecular approach. In-
dividual hydrogen bonds were also analysed with quantum theory of atoms in molecules
(QTAIM). The resulting conclusions (Chapter 4) are listed below.
• The monoanionic (diprotonated) vanadate TBP structures were found to be en-
ergetically most stable in active site pockets derived both from VCPO and VBPO
x-ray crystal structures (CPO1 andCPO2 in Fig. 4.9). This is the reconfirma-
tion of earlier summarised conclusions drawn from TD-DFT studies. Moreover,
the orientation of protonated oxygen atoms in active site pocket was also deter-
mined. It was found that apical oxygen atom of TBP vanadate structure and one
equatorial oxygen atom oriented towards Ser residue were protonated.
• The model in which neutral His404 receives hydrogen bond from apical OH group
of TBP vanadate was found to be unstable in comparison to the one in which
positively charged His404 donates a hydrogen bond to apical OH group (CPO1
vs. CPO2 in Fig. 4.9).
• The potential energy surface for proton movement along mentioned His404 Oap
hydrogen bond was found to be sensitive to hydrogen bonding on equatorial
oxyanionic sites of vanadate. The weakening of later resulted in proton trans-
fer from His404 to apical oxygen atom of TBP vanadate, which can be then easily
expelled as a water molecule as discussed in Chapter 5.
• The active site pocket derived from VCPO crystal structure was capable of stabil-
ising the vanadate in both dianionic and monoanionic protonation states (CPO1
and CPO3 in Fig. 4.9). On the other hand, VBPO derived active site pocket
was able to stabilise only monoanionic vanadate (BPO1 andBPO2 in Fig. 4.9)
• It was found that His404 and Lys353 have a similar level of hydrogen bonding
interaction with vanadate in active site model based on VCPO crystal struc-
ture, while the VBPO active site model showed a weakened interaction between