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Enzyme models of chloroperoxidase and catalase

Claude, Cécile. Enzyme models of chloroperoxidase and catalase. 2001, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6278

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Abstract

As early as the 17th century it was recognized that one component in air is essential for life.
This extremely simple molecule later known as dioxygen or commonly oxygen was
discovered by Joseph Priestley and Carl Wilhelm in 1771-72 by thermal decomposition of
mercury oxide and potassium nitrate respectively. During the next three years Lavoisier
worked on the characterization of the new element composing dioxygen. In 1775 he named it
oxygen due to its presence in numerous acids (oxy = acid, gen = generate). Lavoisier also
established the role of dioxygen in the respiratory process. The fundamental equation
dioxygen=life was formulated for aerobic systems.
Nearly two centuries later, in 1929, the electronic structure of dioxygen was solved by Linus
Pauling using the theory of molecular orbitals. Pauling could explain the paramagnetism of
dioxygen (cM=0.994 T-1) as well as the interatomic distance (d=1.207�) and the dissociation
energy (Ediss=117.36 kcal mol-1) which were until then poorly understood by chemists. This
state is defined as the triplet state and depicted in term of molecular orbital in figure 1. The pioneering theory of Pauling could also predict the two metastable states of dioxygen
where the molecule becomes diamagnetic. Both states are known as D and S singlet states
(figure 2) and differ from the orbitals filling: the 1Sy
+ state has the two electrons paired in the
p*
2py orbital whereas the 1Dy has still on electron in each p* orbital but with reversed spin. The
latter state has a life time of 45 Min. and an O-O bond distance of 1.223�. These singlet states are important for the reactivity of dioxygen in particular in the generation
of ozone and radical species. These radicals are responsible for the formation of hydrogen
peroxide, a highly oxidizing substance discovered by Thenard in 1818. Accumulation of
hydrogen peroxide in the cell is extremely dangerous as it destroys the protein structure. The
relationship between oxygen, hydrogen peroxide and the respiratory processes has been more
deeply understood during the 20th century. For example, myoglobin which binds oxygen was
the first protein of which the structure was solved by X-Ray diffraction in 1960 [1]. This is
showing the interests and efforts developed to establish how dioxygen is used by aerobic
organisms. In the second part of the 20th century the progress of medicine and biology has provided a
better understanding of the importance of dioxygen and led to the discovery of numerous
enzymes which are related to dioxygen.
Although dioxygen is the basic source of energy for aerobic systems, its consumption
produces radicals such as superoxide ion (O2
-) in cells. This highly reactive radical is very
toxic and implied in numerous pathologies. Fortunately a “dioxygen cycle” (figure 3) exists
by which oxygen [3O2] can be activated or recycled such that toxicity problems are prevented. The close dependency of hydrogen peroxide (H2O2) and dioxygen reflects the complexity of
the nature despite the use of very simple molecule for ensuring life. Moreover, hydrogen
peroxide which is known as a cell killer due to its high oxidizing power is required as a
substrate for many enzymes. A real paradox exists: hydrogen peroxide and the superoxide ion
have to be present in living organisms but their concentration has to be controlled accurately
so that they are only short living in cells. It was recognized early that metal containing species, in particular iron complexes, were
involved in these processes. Rapidly the idea was accepted that a relatively simple unit is
responsible for the transport and activation of dioxygen. Küster postulated a tetrapyrrolic
structure [2] termed porphyrin. Its synthesis by Hans Fischer [3] established that its iron
complex known as heme was able to bind dioxygen. Such heme units were shown to be
essential for activating oxygen for oxidation reactions.
There are now seven hemes known which differ from each other by the nature of the
substituents on the b-pyrrole positions. The most common heme is heme b or hematin (figure
4). Several hundreds of heme b proteins are nowadays known which have different functions
such as electron transfer, oxygen or nitric oxide transport or oxidation as shown in table 1. As
all these proteins share exactly the same metal complex, it has been pointed out that the
protein environment around heme b plays a decisive role in determining the function of the
protein:
1) The amino acid residues maintain the heme unit bound to the protein
2) One amino acid residue coordinates axially to the iron in all heme proteins.
The nature of this axial coordination is thought to be the most important factor which
determines the enzyme’s activity. Therefore heme proteins are often classified according to
the nature of the axial ligand (table 1). Finally the hydrophobic or polar character of the
amino acid residues on the side opposite to the axial ligand (= distal side) tunes the specificity
for a particular substrates and aids the formation of catalytic intermediates. The best known heme proteins are the globins, hemoglobin and myoglobin which are
essential blood constituents. Peroxidases have also been the centre of intense studies as they
were using hydrogen peroxide for oxidation in living organisms and have the same axial
ligand, the nitrogen from histidine, as the globins. Less attention has been paid to Catalases
which have a tyrosinate axial, similar to peroxidases. Finally heme-thiolate proteins are
subject of special attention due to the unusual presence of a thiolate coordinating to the iron. Several heme models of the active site of the enzymes Chloroperoxidase and Catalase have
been synthesized in order to study the influence of the axial ligand and peripherical meso
heme substituents on the formation and characterization of catalytic intermediates. Each metal-free porphyrin was thoroughly characterized by NMR spectroscopy prior to the
introduction of the iron atom to form the desired active site model.
Each heme model was then characterized by UV-Vis, NMR, an EPR spectroscopy. Following
results were observed: The attempts to obtain better models finally support the approach of bridged structures over
tailed models. Contrary to the conclusion of Nakamura et al. with hydrogen bonded stabilized
heme thiolate complex [122] we could not notice such an effect with the precursors 49 and 55.
Nevertheless the results obtained for metal free tailed porphyrin have permit to establish that
upon bridging the UV-Vis spectra are red shifted and that the conformation of the ortho amide
substituents on the meso aryl groups is changed.
For these reasons the future direction for having better binding constant is probably still to use
an ortho amide function on the aryl moiety. But instead of having a bulky substituent directly
linked to the carbonyl group of the amide this one should be anchored at a greater distance.
For example the replacement of the tert-butyl group by a furan already improved the binding
abilities of 11 [118].
Advisors:Woggon, Wolf-Dietrich
Committee Members:Kaden, Thomas Angelo Henry
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie
UniBasel Contributors:Woggon, Wolf-Dietrich
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6278
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:180
Language:English
Identification Number:
Last Modified:05 Apr 2018 17:31
Deposited On:13 Feb 2009 14:39

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