Download Cytochrome c Oxidase dysfunction in cancer

Cytochrome c Oxidase dysfunction in
cancer
Exploring the molecular mechanisms
Ida Namslauer
©Ida Namslauer, Stockholm 2012
ISBN 978-91-7447-424-4 (pages 1-65)
Printed in Sweden by Universitetsservice US-AB, Stockholm 2012
Distributor: Department of Biochemistry and Biophysics, Stockholm University
List of publications
This thesis is based on the following publications, which will be referred to
by their Roman numerals:
I.
Laura S Busenlehner, Gisela Brändén, Ida Namslauer, Peter
Brzezinski and Richard N Armstrong. Structural elements involved in
proton translocation by Cytochrome c Oxidase as revealed by
backbone amide hydrogen-deuterium exchange of the E286H mutant.
Biochemistry 2008, 47:73-83.
II.
Ida Namslauer, Huyn-Ju Lee, Robert B Gennis and Peter Brzezinski.
A pathogenic mutation in Cytochrome c Oxidase results in impaired
proton pumping while retaining O2-reduction activity. Biochimica et
Biophysica Acta 2010, 1797:550-556.
III. Ida Namslauer and Peter Brzezinski. A mitochondrial DNA mutation
linked to colon cancer results in proton leaks in Cytochrome c Oxidase.
Proceedings of the National Academy of Sciences of the United States
of America 2009, 106:3402-3407.
IV. Ida Namslauer, Marina S Dietz and Peter Brzezinski. Functional
effects of mutations in Cytochrome c Oxidase related to prostate
cancer. Biochimica et Biophysica Acta 2011, 1807:1336-1341.
Additional publication
Suman Chakrabarty, Ida Namslauer, Peter Brzezinski and Arieh
Warshel. Exploration of the Cytochrome c Oxidase pathway puzzle
and examination of the origin of elusive mutational effects. Biochimica
et Biophysica Acta 2011, 1807:413-426.
Contents
Introduction ..................................................................................................9
Metabolism and oxidative phosphorylation ..........................................................9
Oxidative phosphorylation................................................................................10
The mitochondrial respiratory chain...............................................................12
Cytochrome c Oxidase..............................................................................13
Structure and function of R. sphaeroides CytcO................................................14
General structural features ..............................................................................14
Function ...............................................................................................................15
Proton pumping in CytcO..................................................................................17
Methods for studying CytcO function .............................................................18
Functionally important structural features of CytcO .........................................19
The D pathway for proton uptake ...................................................................19
Glu286: an internal H+ donor/acceptor .........................................................20
Structural changes in the D pathway affect Glu286....................................21
The proton exit route beyond Glu286 ............................................................22
The mammalian CytcO ...........................................................................................24
Similarities between the mammalian and the R. sphaeroides CytcO.......24
The additional subunits of the mammalian CytcO .......................................26
An additional proton transfer pathway...........................................................27
The aim of this thesis ...............................................................................28
Modeling mtDNA mutations in the R. sphaeroides CytcO ................29
Mitochondrial DNA mutations ..........................................................................29
Model systems of mtDNA mutations....................................................................30
Transmitochondrial hybrid cell lines ...............................................................31
Yeast models.......................................................................................................31
Mouse models .....................................................................................................32
The relevance of the R. sphaeroides CytcO model ......................................33
CytcO amino-acid substitutions in cancer............................................35
Reports on CytcO defects in cancer................................................................35
Functional characterization of the pathogenic substitutions ...........................37
Effects on the CytcO catalytic activity............................................................38
Effects on proton pumping ...............................................................................40
The results correlate between mammalian and R. sphaeroides CytcO ....42
Comments on the results .................................................................................43
The relevance of CytcO substitutions found in cancer ......................44
Energy metabolism of cancer cells .................................................................45
Oxidative stress and apoptosis in cancer ......................................................46
The mitochondrial electrochemical gradient in cancer cells .......................47
Concluding remarks and future perspectives......................................49
Sammanfattning på svenska ..................................................................50
Acknowledgements ...................................................................................52
References ..................................................................................................54
Introduction
Mutations in the mitochondrial DNA (mtDNA) have been found in
connection to various types of human cancer. Since the mtDNA encode
several polypeptides of the respiratory-chain enzymes, mtDNA mutations
often affect the function of oxidative phosphorylation. Some of the identified
mutations cause amino-acid substitutions in the enzyme Cytochrome c
Oxidase (CytcO). CytcO is an essential enzyme in oxidative phosphorylation
and it is found in almost all eukaryotic organisms as well as in many
bacterial and archaeal species. Although amino-acid substitutions in CytcO
have been found in human cancer cells, the impact of the substitutions on the
function of the enzyme has in most cases not been verified.
We have investigated, on a molecular level, the functional effects of
substitutions in CytcO related to colon- and prostate cancer. For these
studies the well-characterized bacterial CytcO from the model organism
Rhodobacter sphaeroides was used. The studies have provided detailed
insights into how the function of CytcO is affected by the mtDNA mutations
originally reported in connection to cancer.
In this thesis the relevance of using a bacterial model system when
investigating molecular mechanisms of diseases found in humans is
discussed. Further, possible links between a defectively functioning CytcO
and the development of cancer are proposed. First, however, there is some
background information on oxidative phosphorylation and on the structure
and function of both R. sphaeroides and mammalian CytcO.
Metabolism and oxidative phosphorylation
Every living cell needs energy. The source of energy that is used by a cell
varies depending on for example the cell type and its environment. Humans,
like all mammals and other animals, use chemical energy derived from food.
In every cell the energy needs to be converted in order to be accessible for
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energy-requiring processes such as growth, organization, transport and
reproduction. Metabolism is a set of chemical reactions involved in the
uptake, conversion, digestion and excretion of energy-containing nutrients.
The key players of metabolism, catalyzing the chemical reactions, are
enzymes. In eukaryotic cells, mitochondria are the cellular compartments
(organelles) that play a key role in energy metabolism. Mitochondria contain
the enzyme complexes of metabolic processes such as the citric acid cycle,
the fatty acid oxidation and oxidative phosphorylation. Therefore, most of
the adenosine triphosphate (ATP), which is the molecule essential for many
energy-requiring reactions in the cell, is generated in mitochondria.
Mitochondria also contain their own genetic material; mitochondrial DNA
(mtDNA).
Oxidative phosphorylation
The process of producing ATP using energy released by oxidation of
nutrient molecules is called oxidative phosphorylation. In this process,
electrons (derived from nutrients) are transferred from electron donors to
electron acceptors in redox reactions. The reactions are catalyzed by a
number of membrane-bound enzyme complexes constituting the respiratory
chain (figure 1). The flow of electrons from an electron donor having a low
midpoint potential to an electron acceptor with a higher midpoint potential is
exergonic, releasing free energy. The energy is used by the enzyme
complexes to translocate protons across the inner mitochondrial membrane
in eukaryotes or the plasma membrane in bacteria and archaea. Thus, the
energy is transiently stored in the electrochemical proton gradient. The
exergonic flow of protons down the gradient is allowed through the enzyme
ATP synthase in which the proton flow is coupled to the phosphorylation of
adenosine diphosphate (ADP), producing ATP. The ATP is then transported
out of the mitochondrion to be used in various energy-requiring processes in
the cell.
Compared to glycolysis or lactic-acid fermentation, oxidative
phosphorylation is a highly efficient way of utilizing chemically stored
energy and is a process that is used by almost all aerobic organisms. In
mammalian cells with functional mitochondria it has been estimated that
approximately 90 % of the total cellular ATP is produced by oxidative
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phosphorylation [1]. However, oxidative phosphorylation is also involved in
the production of potentially harmful reactive oxygen species (ROS). During
electron transfer between the complexes of the respiratory chain a small
amount of ROS is constantly being formed, mainly in respiratory chain
complexes I and III [2-4]. Normally, cells have ways of eliminating ROS.
Several enzymes, such as superoxide dismutase, peroxidases and catalase,
are involved in ROS detoxification. In addition, small antioxidant molecules
such as ascorbic acid and tocopherols are important in reducing the amount
of ROS in cells. Although ROS have been suggested to be important in
certain cell-signaling processes, an excess production of ROS (oxidative
stress) is harmful to cells, leading to damage of DNA, proteins and lipids.
Oxidative stress has been linked to the normal aging process as well as to
diseases such as cancer and Alzheimer’s disease [5-8]
Figure 1. The enzyme complexes of the mitochondrial respiratory chain; NADH
dehydrogenase (I), succinate dehydrogenase (II), the bc1 complex (III) and CytcO
(IV), together with ATP synthase. Electrons are provided through the oxidation of
NADH and succinate. Quinone (Q) and cytochrome c (cyt c) are electron carriers.
Complexes I, III and IV contribute to the establishment of the electrochemical proton gradient by translocating protons from the negative side of the membrane to the
positive. Protons flow down the gradient through ATP synthase, resulting in the
production of ATP from ADP and inorganic phosphate (Pi ). Electron transfers are
indicated by yellow arrows and protons by red arrows. The position of the membrane is indicated in yellow.
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The mitochondrial respiratory chain
There is a general organization of the mitochondrial respiratory chain in
most eukaryotes. The mammalian respiratory chain consists of four enzyme
complexes; NADH dehydrogenase, succinate dehydrogenase, the bc1
complex and CytcO (figure 1). All four enzymes contain redox-active
cofactors involved in the transfer of electrons from an electron donor to an
acceptor. The electron donors of the respiratory chain are NADH and
FADH2. Quinone and cytochrome c act as electron carriers, transferring
electrons from one enzyme complex to another. The final electron acceptor
of the mammalian respiratory chain is molecular oxygen, which is reduced
to water at the catalytic site of CytcO. In mammalian cells the protein
subunits of NADH dehydrogenase, the bc1 complex, CytcO and ATP
synthase are encoded both in the nuclear genome and in the mtDNA.
Traditionally, it was believed that the enzyme complexes diffuse freely in
the membrane and that electron transfer would occur upon random collisions
[9]. A more recent suggestion is that the respiratory-chain complexes form
large supercomplexes. Such supercomplexes have been found in mammals
as well as in yeast and bacteria [10-12] (figure 2). An organization of the
enzymes into larger complexes has been suggested to allow greater structural
stability of the individual enzymes, efficient substrate channeling and a
minimum formation of ROS, as compared to a scenario where the individual
enzymes are located at some distance from each other.
Figure 2. Cryo-EM 3D map of a purified supercomplex from bovine heart mitochondria, with the docked x-ray crystal structures of complex I (blue), complex III
(red) and complex IV (green). The figure is modified from [10] and is reprinted with
permission.
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Cytochrome c Oxidase
Cytochrome c Oxidase (CytcO) is the terminal oxidase of the respiratory
chain in mitochondria and many bacteria and archaea. CytcO was discovered
in the 1930’s as a member of the respiratory chain. It was mainly the work of
Otto Warburg and David Keilin that established its role in metabolism [13,
14]. In the 1970’s and 1980’s the knowledge on CytcO was greatly increased
and in 1977 it was shown to be a proton pump [15].
Figure 3. The structure of the bacterial CytcO from R. sphaeroides (PDB 1M56).
The four subunits are shown in different colours: Subunit I in yellow, subunit II in
magenta, subunit III in orange and subunit IV in pink. The heme groups; heme a and
heme a3, are shown in green and the cupper cofactors as orange spheres. The black
line indicate the approximate position of the lipid bilayer. The figure was prepared
using the PyMOL software.
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About 15 years ago, the first x-ray crystal structures of CytcO were
published. These were the mammalian CytcO from bovine heart
mitochondria [16, 17] and the bacterial CytcO from Paracoccus
denitrificans [18]. A few years later, the R. sphaeroides CytcO x-ray crystal
structure was determined [19]. The following chapter describes features of
the bacterial CytcO from R. sphaeroides. Then, there is a comparison
between the bacterial and the mammalian enzyme.
Structure and function of R. sphaeroides CytcO
General structural features
The available 3D-structures of the R. sphaeroides CytcO have revealed an
enzyme complex containing four protein subunits [19] (figure 3). Subunit I
harbors three of the four redox-active cofactors; heme a and the heme a3-CuB
catalytic site. In subunit I, two proton transfer pathways leading from the
negatively charged (N) side of the membrane to the vicinity of the catalytic
site have been identified [19, 20]. Subunit II contains two transmembrane αhelices and an extramembrane β-barrel domain where the additional copper
cofactor; CuA resides. The cytochrome c-binding site is presumably found in
subunit II [21, 22]. The interaction between reduced cytochrome c and
CytcO occurs through hydrophobic as well as electrostatic interactions
between positively charged Lysine residues on cytochrome c and negatively
charged residues on CytcO. Subunit III consists of seven membranespanning α-helices. Although subunit III does not contain any redox-active
cofactors it is essential for the function of the enzyme. Removal of subunit
III results in defective proton pumping [23, 24]. There is also experimental
evidence showing that removal of subunit III leads to turnover-induced
inactivation of the enzyme [25]. Subunit IV of the R. sphaeroides CytcO
consists of one single transmembrane α-helix. It is connected to subunits IIII and held in position only via lipid molecules [19].
14
Figure 4. Electron and proton transfers to the R. sphaeroides CytcO catalytic site.
The heme and cupper cofactors are shown in green and orange, respectively. Electrons are transferred via CuA and heme a to the heme a3-CuB catalytic site. Oxygen
binds to the reduced heme a3 iron. Protons are transferred via specific pathways to
the catalytic site. For the reduction of one oxygen molecule, four protons and four
electrons are required. In addition, one proton per electron transferred to the catalytic site is pumped through the enzyme. Subunit I of the CytcO is in yellow and
subunit II in pink. The figure was prepared using the PDB entry 1M56 and the PyMOL software.
Function
CytcO catalyses the reduction of oxygen to water. For this reaction to take
place electrons, protons and oxygen need to reach the catalytic site (figure
4). The electrons are donated one-by-one from reduced cytochrome c and are
transferred via the redox-active cofactors CuA and heme a to the CytcO
catalytic site [26]. Oxygen binds to the reduced heme a3 iron at the catalytic
site. The electron transfer is coupled to proton uptake to the catalytic site and
oxygen is reduced to water [27, 28]. Part of the energy available from the
exergonic reduction of oxygen is used by the enzyme to pump four protons
per oxygen molecule from the N side of the membrane to the positively
charged (P) side, contributing to the formation of the electrochemical
gradient [15].
Since the electrons needed for oxygen reduction are transferred one-byone to CytcO during turnover, the catalytic site cycles through a number of
15
partly reduced intermediate states, identified spectroscopically by their
different redox states and ligand-binding properties [29]. These are
illustrated schematically in the CytcO catalytic cycle (figure 5). In brief,
oxygen binds to the two-electron reduced (R2) catalytic site. Breakage of the
O-O bond forms the P2 intermediate. The next intermediate, F3, is formed
when an electron and a proton is transferred to the catalytic site. The F3
intermediate decays into the fully oxidized O 4(0) state when another electron
and a proton is transferred to the catalytic site. Subsequent transfer of two
protons and two electrons results in formation of water and reduction of the
catalytic site. Protons are pumped upon formation of the F3, O4(0) and R2
intermediate states. If the CytcO is preloaded with four electrons (all
cofactors are reduced) the oxidative part of the catalytic cycle (R2 to O4(0))
can be studied spectroscopically using the flow-flash technique [30]. In that
case, there is electron transfer from heme a to the catalytic site upon
formation of the P3 intermediate. Thus, the electron and proton transfer to the
catalytic site required for formation of the F3 intermediate are separated in
time and can be studied individually.
16
Figure 5. (a). A schematic illustration of the catalytic cycle of O2 reduction in
CytcO. The redox intermediate states (reduced; R, peroxy; P, ferryl; F and oxidized;
O) are shown as well as the events taking place in the transitions between the states.
The digits in superscript indicate the number of electrons present at the catalytic
site. (b). The sequence of events observed when starting with the fully reduced
CytcO, as in the flow-flash absorption spectroscopy. The enzyme cofactors are
shown as spheres and the colours indicate an oxidized (grey) or a reduced (red)
cofactor. The catalytic cycle is further described in the text.
Proton pumping in CytcO
For proton pumping to be achieved, there are some basic requirements that
need to be fulfilled. First, there has to be a proton pathway all the way
through the enzyme. This pathway can, however, not be open to both sides
of the membrane simultaneously since that would result in proton leaks and
disruption of the electrochemical gradient. Thus, there has to be a gating
mechanism ensuring that the proton transfer is unidirectional. The gating
mechanism could, for example, be one or several amino-acid residues
alternating between two different positions. There are also suggestions for an
acceptor site for pumped protons in CytcO. This acceptor site (proton
loading site) is a yet unidentified protonatable group situated above the
17
hemes. Most probably, there is a connection between the proton gate and the
proton loading site, so that the proton loading site changes its protonation
state depending on the position of the gate. Such a mechanism implies that
the pKa of the proton loading site is high when there is a connection to the N
side and low when there is a connection to the P side [31, 32]. Experimental
evidence indicate that the proton loading site is located in the area near the
propionates of heme a and heme a3 [33, 34].
In CytcO, the energy needed for the pumping of protons against the
electrochemical gradient is provided by the exergonic reduction of oxygen.
As soon as the electrons and protons needed for the reaction reach the
catalytic site, the free energy is lost. Therefore, the events that drive proton
pumping must occur before that. There are several ways in which this could
be accomplished: The proton to be pumped could be transferred to the
proton loading site before the electron and substrate proton reach the
catalytic site or the transfer of the substrate proton to the catalytic site is
coupled to a reaction (e.g. a structural change) that conserves the free
energy. Then the transfer of the pumped proton would be triggered by the
structural change. The mechanism for proton pumping in CytcO is still not
fully revealed, but is reviewed in [31, 35].
Methods for studying CytcO function
The function of CytcO has been studied for several decades. Optical
absorption spectroscopy, together with other spectroscopic techniques, such
as Fourier Transform Infrared Spectroscopy (FTIR) and Electron
Paramagnetic Resonance Spectroscopy (EPR), are valuable tools for
functional studies of CytcO [32, 36-38]. Site-directed mutagenesis of the
bacterial CytcO allows substitution of specific amino-acid residues. In
combination with spectroscopic techniques, mutational studies have offered
insights into CytcO function and provided information on what parts of the
protein are involved in specific processes. The atomic resolution 3D
structures of CytcO are of great importance for the interpretation of results
from functional studies since they can be used to link the function of the
enzyme to the structure. Lately, computational tools such as electrostatic
calculations and molecular-dynamics simulations have been used, especially
for the mammalian CytcO, to investigate processes such as shifts in pKa
18
values of titratable groups and small structural changes taking place within
the enzyme [39-41]. Quantum chemistry tools are also used and are valuable
for evaluating whether a proposed mechanistic event is energetically
favorable or even possible [42]. In general, results from computer
simulations and calculations agree with experimental findings.
Functionally important structural features of CytcO
Functional studies, using site-directed mutagenesis, as well as investigations
of the x-ray crystal structures have identified specific regions of CytcO that
are important for the function of the enzyme. Those are, for example, two
proton uptake pathways in subunit I, a highly conserved Glutamate residue
(Glu286) close to the catalytic site and a region close to the heme groups,
towards the positive side of the membrane, involved in the exit of pumped
protons [19, 20, 33, 34, 43-45]. Most of the amino-acid residue substitutions
investigated in the studies presented in this thesis are localized to the abovementioned areas, which are described in more detail below.
The D pathway for proton uptake
Since protons are charged they need specific transport pathways in a protein
interior. Such pathways consist of water molecules and polar amino-acid
residues, forming a hydrogen-bonded chain along which protons can travel.
In the R. sphaeroides CytcO, two input pathways for protons have been
identified [19, 20]. One of them is called the D pathway, named after an
Aspartic acid residue (Asp132) closest to the entrance. This pathway is used
for the transfer of protons needed for the reduction of oxygen as well as for
the pumped protons. The D pathway stretches along a distance of ~24 Å
from the N side of the membrane to the Glutamate residue (Glu286) near the
catalytic site (figure 6). According to the x-ray crystal structure ~10 water
molecules are found within the pathway. These molecules are hydrogen
bonded to a number of amino-acid residues along the path [19, 46]. Using
site-directed mutagenesis, it has been shown that these residues are
important for the efficient transfer of protons through the D pathway [20, 4750]. In addition to the D pathway, there is a second proton-uptake pathway
19
called the K pathway, which is suggested to be used for proton uptake during
reduction of the catalytic site [30].
Figure 6. The D pathway is used for delivery of protons to the catalytic site and for
the uptake of pumped protons. It consists of approximately 10 water molecules
(shown as red spheres) and several polar amino-acid residues forming a hydrogenbonded chain from the N side of the membrane to the Glutamic acid residue
(Glu286) near the catalytic site. The cluster of water molecules and the two Arginine
residues (Arg481 and Arg482), located above the heme groups, are involved in the
transfer of pumped protons. The heme groups are in green and the cupper cofactors
as orange spheres. The CytcO subunit I is shown in yellow and subunit II in pink.
The figure was prepared using the 1M56 PDB entry and the PymMOL software.
Glu286: an internal H+ donor/acceptor
The Glu286 residue is situated close to the catalytic site (figure 6). In wildtype CytcO, proton pumping and proton transfer to the catalytic site takes
place upon formation of the F and O intermediates, as well as upon reduction
20
of the oxidized CytcO [32] (figure 5). Site-directed mutagenesis studies of
the R. sphaeroides CytcO in combination with spectroscopic techniques
have shown that the substitution of Glu286 to Glutamine, Alanine or
Histidine impairs proton transfer to the catalytic site as well as proton
pumping [43, 51] (paper I). The Glu286 residue is considered to be a
branching point, from where protons can be transferred either to the catalytic
site or to a proton loading site. In addition, Glu286 (or water molecules
around this residue) is suggested to function as a transient donor/acceptor for
protons. It is believed that protons can be transferred from Glu286 to the
catalytic site and that Glu286 is then quickly reprotonated from the N side.
Results from investigations of the internal proton transfer from Glu286 to the
catalytic site indicate that the pKa of Glu286 is 9.4 [52].
There are suggestions that the Glu286 residue can adopt two different
conformations and that the structural change of Glu286 is involved in
directing protons to the catalytic site and to the proton loading site [53]. This
mechanism was based on information obtained from CytcO x-ray crystal
structures. A comparison of the wild-type and the Glu286Gln CytcO crystal
structures showed that the Glu286 side chain adopted different
conformations in the two structures [19]. This conformational change of
Glu286 also conferred structural changes in the region around the hemes and
two conserved Arginine residues. A proton pumping mechanism involving
the conformational change of Glu286 was proposed [53]. More recently, a
conformational change of the equivalent Glutamate residue was observed in
the crystal structure of P. denitrificans CytcO harboring an amino-acid
substitution in the D pathway [54]. In this case, the proton pumping was
abolished and it was suggested that the reason for uncoupling was the altered
conformation of the Glutamate side chain resulting from the amino-acid
substitution in the D pathway.
Structural changes in the D pathway affect Glu286
Experimental evidence show that substitutions of specific amino-acid
residues in the D pathway result in both uncoupling of proton pumping and a
changed apparent pKa of the Glu286 residue [48, 50], but with retained
oxygen reduction activity. From these results, it was suggested that the
amino-acid substitutions change the hydrogen-bonded network around the
21
Glu286 residue. As a result, protons would still be transferred to the catalytic
site but not to the proton loading site. In addition, molecular-dynamics
studies based on x-ray crystal structures of CytcO have identified a cluster of
water molecules hydrogen-bonded to two Serine residues (Ser200 and
Ser201) in the D pathway [40, 55]. This region was suggested to be a
“proton trap” allowing fast protonation of Glu286, which is consistent with
the high pKa of Glu286. To further investigate the interactions between
amino-acid residues and water molecules in the D pathway and their role in
the proton-pumping mechanism, the Serine residues were substituted for
Valines. Absorption spectroscopy studies of the CytcO variants showed that
the substitutions resulted in a decreased steady-state activity, lowered
stoichiometry of proton pumping and slowed proton transfer from Glu286 to
the catalytic site [47]. The rate of the internal proton transfer to the catalytic
site was independent on the pH and the pKa of Glu286 was suggested to
increase to >12 in the Ser200Val/Ser201Val CytcO.
The proton exit route beyond Glu286
A specific pathway for pumped protons from Glu286 towards the P side of
the membrane has not been identified. However, there is a hydrogen-bonded
network observed in the area above the heme groups [19, 33, 34, 56] (figure
6). This region contains water molecules and two Arginine residues (Arg481
and Arg482) that interact electrostatically with the D-propionates of heme a
and heme a3. Spectroscopic studies have shown that the Arg481 and 482
residues are important for the function of CytcO [33, 34, 56]. Based on the
experimental data on CytcO in which the Arg481 was exchanged for a
Lysine, it was suggested that the proton loading site is the heme a3 Dpropionate [33]. In another study, though, the identity of the proton loading
site was suggested to be the heme a A-propionate [34]. Although a specific
group acting as a proton loading site has not been identified, the area around
the heme propionates and the Arginines are likely to be involved in the
proton pumping activity.
In paper I, structural elements involved in proton pumping were
investigated using amide hydrogen/deuterium exchange followed by mass
spectrometry. The study lead to the suggestion that a region of CytcO
involved in gating of pumped protons resides in a loop consisting of amino22
acid residues 169-175 (the gating loop) [57]. With the R. sphaeroides wildtype CytcO, the gating loop showed a different amide H/D exchange pattern
in the F intermediate of the CytcO catalytic cycle compared to the P and O
intermediates, indicating that a conformational change takes place upon
formation of the F intermediate. Since proton pumping occurs before as well
as after formation of the F intermediate, a conformational change of the 169175 loop was suggested to be involved in proton gating, ensuring
unidirectional proton transfer. With the CytcO in which the Glu286 was
substituted for a Histidine, the amide H/D exchange pattern of the gating
loop was similar in all intermediate states of the catalytic cycle (paper I). In
addition, a peptide of residues 320-340, suggested to be part of a proton exit
pathway, showed dramatically decreased deuterium incorporation with the
Glu286His CytcO compared to the wild-type enzyme. The Glu286His
CytcO is unable to pump protons. Presumably, the Glu286His substitution
prevents conformational changes required for proton pumping. Since the
substitution of the Glu286 residue leads to conformational changes in the
fairly distant proton gating region of the enzyme, the results from paper I
indicate that there is a connection, probably formed by hydrogen bonds,
between Glu286 and the suggested proton gating region.
23
The mammalian CytcO
Now, the structure and function of the bacterial CytcO from R. sphaeroides
have been described. Since in these studies, the R. sphaeroides CytcO has
been used as a model system for investigations of amino-acid residue
substitutions originally identified in the human enzyme, the mammalian
CytcO from bovine heart mitochondria (figure 7) will now be described in
terms of structure and function. Overall, the mammalian and the R.
sphaeroides CytcOs are functionally as well as structurally similar.
Figure 7. The structure of the mammalian CytcO from bovine heart mitochondria
(PDB entry 10CC). The mammalian CytcO consists of 13 subunits illustrated in
different colours. The figure was prepared using the PyMOL software.
Similarities between the mammalian and the R. sphaeroides
CytcO
The amino-acid sequence identities of the R. sphaeroides CytcO compared
to the mammalian enzyme are 52 % (subunit I), 39 % (subunit II) and 49 %
(subunit III) [58], which is considered a high sequence identity. These
numbers are comparable to subunit I of P. denitrificans NADH
24
dehydrogenase, having a 64 % sequence identity to the mammalian
counterpart [59]. The ATP synthase from E. coli is, with a 70 % amino-acid
sequence identity, even slightly more similar to the mammalian enzyme
[60]. The structures of subunits I, II and III of the mammalian CytcO, which
are considered to be the catalytically active core of the enzyme, are very
similar to the equivalent subunits of R. sphaeroides CytcO [61]. A structural
alignment of subunits I and II of R. sphaeroides and mammalian CytcO is
shown in figure 8. The only clear differences between the structures are at
the N- and C-termini and in a loop consisting of residues 64-89 (R.
sphaeroides amino-acid numbering).
The functionally important parts of the R. sphaeroides CytcO discussed
previously are also found in the mammalian enzyme. The D pathway is
present in the mammalian CytcO and is believed to be used for transfer of
protons from the N side to the catalytic site [17]. The Glu286 residue and the
two Arginines (Arg481 and 481) close to heme a are conserved between the
R. sphaeroides and the mammalian CytcO. Due to the difficulties of using
site-directed mutagenesis to introduce amino-acid substitutions in the
mammalian CytcO, functional mechanisms are often investigated using
computational methods. Results from several in silico studies, including
molecular dynamics simulations and electrostatic calculations, suggest a
crucial role of Glu242 (Glu286 in the R. sphaeroides enzyme) in the
mammalian CytcO [39, 40, 62]. The area above the heme groups, important
for gating and exit of pumped protons, has also been investigated using
computational studies [41, 63]. Based on simulations showing dissociation
of a hydrogen-bonded network near the heme a3 propionate upon reduction
of heme a, it was suggested that the proton loading site is localized to the
area of the heme a3 propionates.
Experimental studies indicate that neither the spectral properties nor the
function of the mammalian CytcO differ considerably from the R.
sphaeroides enzyme [58]. Electron transfer that takes place during the single
turnover reaction of fully reduced to oxidized CytcO from R. sphaeroides
and bovine heart mitochondria have been evaluated and compared using
absorption spectroscopy techniques [64]. The rates of the electron-transfer
events in the bacterial and the mammalian CytcO are comparable and the
25
same intermediates of the catalytic cycle (figure 5) are formed with similar
rates [65].
Figure 8. An alignment of subunits I and II of the bovine (PDB 10CC) and the R.
sphaeroides (PDB 1M56) CytcO. The R. sphaeroides enzyme is shown in magenta
and the mammalian CytcO in blue. The figure was prepared using the PyMOL software.
The additional subunits of the mammalian CytcO
The mammalian CytcO is a large enzyme complex consiting of 13 subunits
(figure 7). The nuclear-encoded subunits (IV, Va, Vb, VIa, VIb, VIc, VIIa,
VIIb, VIIc and VIII) are translated in the cytosol and transported into the
mitochondrial inner membrane. These are not present in the bacterial CytcO.
Considering the functional similarities between the mammalian CytcO and
26
the four-subunit R. sphaeroides CytcO, the function of the additional 10
subunits is elusive. It has been suggested that these subunits stabilize and/or
regulate the mitochondrial CytcO. In a recent study, small interfering RNA
(siRNA) depletion of subunit Vb resulted in decreased activity and assembly
of CytcO as well as in a decreased membrane potential and increased ROS
formation [66]. Other studies have focused on the regulatory roles of the
nuclear encoded subunits and suggest that there are several ATP/ADP
binding sites in the mammalian CytcO, allowing allosteric regulation of the
enzymatic activity [67].
An additional proton transfer pathway
Apart from the two proton-conducting D and K pathways found in both the
bacterial and the mammalian CytcO, in the mitochondrial protein an
additional proton pathway, the H pathway, has been identified based on the
existence of a hydrogen-bonded network observed in the x-ray crystal
structure [17]. Mutational studies of the bovine CytcO confirmed the
importance of the H pathway for maintaining proton pumping [68, 69]. The
mutations were made using an expression system constructed in HeLa cells.
The system produces a bovine-human hybrid enzyme, which could
complicate the interpretation of the results. Moreover, there is no evidence
for an H pathway in the bacterial CytcO. Several of the amino-acid residues
found in the mitochondrial CytcO H pathway are not conserved in the R.
sphaeroides enzyme and substituting the corresponding H pathway aminoacid residues in the bacterial CytcO had no effect on the proton pumping
activity [70, 71]. In the yeast (Saccharomyces cerevisiae) CytcO structure,
which is a homology model based on the bovine x-ray crystal structure, there
are hydrophilic amino-acid residues identified in the area of the H pathway.
However, amino-acid residues of the D and the K pathway are conserved to
a higher extent than residues of the H pathway [72].
27
The aim of this thesis
Despite the large number of reports on mtDNA mutations associated with
cancer, the functional significance of the amino-acid substitutions caused by
the mutations remains elusive in the majority of cases. In order to understand
disease mechanisms and develop therapeutic approaches it is important to
uncover the functional characteristics resulting from potentially pathogenic
mtDNA mutations. The aim of my studies has been to characterize, on a
molecular level, a number of phenotypes of CytcO found in connection to
cancer. On the basis of the results, the remaining part of this thesis addresses
the following issues:
1. The relevance of using CytcO from the bacterial model R. sphaeroides for
functional studies on amino-acid residue substitutions found in human
disease.
2. The possible consequences of the substitutions for the development or
progression of cancer.
28
Modeling mtDNA mutations in the R.
sphaeroides CytcO
Mitochondrial DNA mutations
The human mtDNA is folded into a double-stranded, circular molecule of
approximately 16600 basepairs and contains 37 genes. 13 genes encode
polypeptides of the electron transport chain of which seven are subunits for
NADH dehydrogenase, one for the bc1 complex, three for CytcO and two for
ATP synthase. The other genes encode 22 tRNA molecules and two
ribosomal RNA molecules (figure 9).
Figure 9. A schematic illustration of the human mtDNA; a circular molecule of
approximately 16600 basepairs. The mtDNA encodes mRNA for cyt b, NADH dehydrogenase, CytcO and ATP synthase as well as tRNA and rRNA. The D loop (displacement loop) is a non-coding segment and is part of the control region of the
mtDNA molecule.
29
The mammalian mtDNA molecule lacks introns and thus has a high
proportion of coding DNA. Moreover, the mtDNA has a higher mutation
rate compared to the nuclear genome [73, 74]. This is, at least partly,
probably due to its close proximity to the generation sites of ROS. Many
mtDNA alterations are neutral polymorphisms [75]. However, in the late
1980’s the first pathogenic mtDNA mutations were identified [76, 77]. Since
then, there has been extensive research on mtDNA and several hundreds of
potentially pathogenic mutations have been identified [78].
Mutations in the mtDNA have been found in a large number of different
diseases. The so-called mitochondrial diseases such as Leigh syndrome,
LHON (Leber’s hereditary optical neuropathy) and mitochondrial myopathy
are all severe disorders affecting the function of oxidative phosphorylation,
resulting in insufficient synthesis of ATP [78, 79]. More recently, mtDNA
mutations have been found in cancer, neurodegenerative diseases and normal
aging [5-8, 79, 80]. One of the first reports on mtDNA mutations in
connection to cancer is from 1998, when Polyak et al identified a number of
mtDNA mutations in human colorectal tumors [81]. Since then, there have
been more than 300 reports on human cancers containing mutations in
coding parts of the mtDNA [82].
Model systems of mtDNA mutations
Although several mtDNA mutations are observed in cancer cells, there is a
lack of experimental data, investigating the phenotype resulting from the
mutations and how it could be linked to the disease. This has resulted in
discussions on the actual functional relevance of the mtDNA mutations [8385]. To determine the effects of a mutation and understand how the aminoacid residue substitution resulting from a mutation could be involved in
disease mechanisms, functional studies have to be made. This requires some
kind of model system. There are various models available, enabling
observations of how different features and cellular processes are affected by
an amino-acid substitution. Three model systems that are frequently used to
investigate the functional effects of mtDNA mutations are
transmitochondrial hybrid cell lines, yeast cells and mouse models. I will
describe these briefly and discuss what information can be obtained using
30
the different systems. For my studies, I chose to model mtDNA mutations in
CytcO from the bacterium R. sphaeroides. The relevance of using this
bacterial model to investigate mtDNA mutations found in humans will also
be discussed.
A summary of some advantages and shortcomings of the different models
used for studies on mtDNA mutations are listed in Table 1.
Transmitochondrial hybrid cell lines
Transmitochondrial hybrid cell lines or cybrids, are obtained by fusing
enucleated cytoplasts, derived from patient cell lines, with immortalized
human cell lines devoid of mtDNA. The first transmitochondrial cybrids
were described in 1989 [86]. Since then, cybrids have been used for studies
of potentially pathogenic human mtDNA point mutations [87, 88].
Transmitochondrial cybrids is an in vitro system with the advantage that the
phenotype resulting from a mtDNA mutation can be studied against a fixed
nuclear background. The model allows a certain phenotype to be assayed
regarding for example oxygen consumption rates, activities of the
respiratory-chain enzymes, ROS production and apoptosis. Further, there are
studies where the transmitochondrial cybrids containing a mtDNA mutation
have been transferred to mice in order to investigate the impact of the
mutation on tumor development or metastasis [89, 90]. However, the use of
cybrids is not uncontroversial. There are several cases where in vivo
phenotypes could not be replicated in an in vitro cybrid system [91, 92].
Yeast models
Yeast models have been used in several cases to study the effects of diseaserelated mtDNA mutations [93-95]. Introduction of mutations into the
mtDNA of yeast (such as S. cerevisiae) is possible [72]. The respiratorychain complexes of yeast show a high similarity in both structure and
function to the mammalian counterparts. The use of yeast cells enables
investigations of the impact of a mutation on for example cellular growth
rate, respiratory function, activities of the individual respiratory chain
enzymes and ROS production. In addition, the protein with the amino-acid
substitution resulting from the mutation can be purified. In that way a more
31
detailed analysis of the function of the protein can be obtained. The ability of
S. cerevisiae to survive without a functional respiratory chain makes it
possible to study the effects of mtDNA mutations that result in severe
respiratory defects. As S. cerevisiae lacks NADH dehydrogenase, studies on
mutations affecting complex I have been made using the obligate aerobic
yeast Yarrowia lipolytica, that contain a respiratory chain including NADH
dehydrogenase [96].
Mouse models
Using mouse models for studies on mtDNA mutations allows genetic
manipulations such as depletion or deletion of genes. Introduction of specific
point mutations in the mouse mtDNA is not easily accomplished. However,
the development of transmitochondrial mice models has enabled in vivo
studies of the impact of mtDNA point mutations [97]. Mammalian in vivo
models is the only alternative when studying certain aspects of mtDNA
mutation pathogenesis, such as how one specific mutation can result in
multiple different phenotypes, why a deficiency of a specific enzyme is
observed in several different diseases or why some tissues are more affected
by a mutation than others. Using mice it is also possible to address questions
concerning the onset and severity of a disease.
32
Advantages
Shortcomings
Purified
bacterial enzyme
• Genetic modifications easily accomplished
• Well-defined system
• Relatively cheap & quick
• Molecular level studies
• Prokaryotic system
lacking several subunits
• No cellular context
Yeast cells
• Eukaryotic system
• Genetic modifications possible
• Genetic modifications
more difficult compared to
bacteria
Cybrids
• Allows comparisons of phenotypes against a
fixed nuclear background
• Mammalian system
• Experimentally difficult
Animals
• Mammalian system
• Cellular context
• Organismal level
• Genetic modifications
difficult
• Expensive
• Ethical issues
Table 1. Some advantages and shortcomings of the different model systems that can
be used when studying functional effects of mtDNA mutations.
The relevance of the R. sphaeroides CytcO model
Traditionally, CytcO has been purified from bovine heart mitochondria, S.
cerevisiae and the bacterial species P. denitrificans and R. sphaeroides.
Since the beginning of the 1990’s the R. sphaeroides CytcO has been used in
a large number of both structural and functional studies. Several
investigations have thoroughly evaluated the R. sphaeroides CytcO as a
model for the eukaryotic enzyme and found that it is functionally
homologous to the mammalian CytcO [58, 64, 65]. Functional studies using
R. sphaeroides CytcO have been of great importance for the elucidation of
for example the proton-transfer pathways and the proton-pumping
mechanism. Although some pieces of information are still missing, today we
have a good picture of the functional mechanisms of R. sphaeroides CytcO.
The increasing number of x-ray crystal structures, provide valuable
information for the interpretations of experimental results. Bacterial CytcO
has previously been used for studies on functional effects of pathogenic
mutations [93, 98]. These investigations provided detailed analyses of the
effects of mtDNA mutations found in mitochondrial diseases.
33
Similar to other bacterial models, R. sphaeroides is experimentally easy
and fairly cheap to work with. Methods for both expression and purification
of R. sphaeroides CytcO have been developed and work satisfactory.
Further, the use of R. sphaeroides CytcO enables site-directed mutagenesis
studies. As mentioned previously, S. cerevisiae CytcO has also been used to
study effects of pathogenic mtDNA mutations. However, genetic
modifications of S. cerevisiae are more difficult and time-consuming
compared to a bacterial organism. In spite of the fact that yeast is a
eukaryotic organism, the amino-acid sequence of subunits I-III of S.
cerevisiae and R. sphaeroides CytcO has in fact a similar degree of
homology to the mammalian enzyme [19, 99]. Further, the number of
functional studies made with the R. sphaeroides CytcO greatly outnumbers
the ones where yeast has been used. Naturally, a highly validated model is
more attractive to use. Experimental results are easier to interpret and
evaluate if there are already a large number of studies made using that
specific model.
The various models for studying mtDNA mutations enable investigation of
different functional characteristics of the phenotype. In vivo and also in vitro
models, such as purified mitochondria or transmitochondrial cybrids,
provide a cellular context that is absent when using purified proteins. On the
other hand, the use of purified proteins enables molecular-level studies and
can provide information that is impossible to obtain using other model
systems. Detailed information on the functional effects of amino-acid
substitutions found in pathogenic conditions is valuable since it leads to
elucidation of possible disease mechanisms.
34
CytcO amino-acid substitutions in cancer
Reports on CytcO defects in cancer
Amino-acid substitutions in CytcO have been found for example in prostate
cancer, pancreatic cancer and ovarian cancer [90, 100-102]. In addition,
substitutions in CytcO have been reported in normal colonic crypt stem cells
in colon cancer patients [103, 104]. Greaves et al identified the mutation
6277A>G in the CytcO subunit I gene. The mutation, resulting in the aminoacid substitution Gly125Asp, was found in colonic crypt stem cells showing
a CytcO deficiency. The authors suggested that the substitution was highly
likely to be the cause for the CytcO deficiency, but no further studies were
made to confirm this. In another study, Pye et al identified a missense
mutation in the gene encoding subunit I of CytcO in respiratory-deficient
transmitochondrial cybrids [104]. The mutation resulted in a Tyr19His
amino-acid substitution. The transmitochondrial cybrids were produced
using normal human colon cells from patients treated for adenocarcinoma.
Studies on mitochondria isolated from the cybrids showed a decrease in the
CytcO activity. In 2005, there was a report on a number of mutations in the
gene encoding subunit I of CytcO found in prostate cancer cells [90]. The
amino-acid substitutions resulting from the mutations were located to
different regions of subunit I. Some of the substitutions affected highly
conserved amino-acid residues. The functional effects of the substitutions
were, however, not investigated.
The background to my studies is the reports on CytcO substitutions found
in patients with colon cancer and prostate cancer [90, 103, 104] and the aim
of the work was to elucidate if and/or how the functional mechanisms of the
enzyme was affected by the substitutions. All substitutions were found in
subunit I of CytcO (figure 10). The equivalent mutations were produced in
the subunit I gene of R. sphaeroides CytcO and the enzyme containing the
35
corresponding amino-acid substitutions was purified. The Gly125 and the
Tyr19 residues that were affected in the colon cancer patients, correspond to
Gly171 and Tyr33, respectively in the R. sphaeroides CytcO. Among the
amino-acid substitutions found in CytcO of prostate cancer cells, Asn11Ser,
Ala122Thr, Ala341Ser and Val380Ile were chosen for the study. In R.
sphaeroides CytcO these residues correspond to Asn25, Ser168, Ala384 and
Val423. For the functional studies, various spectroscopic techniques were
used. Thereby we could examine, at a molecular level, how the amino-acid
substitutions affected the individual electron- and proton transfer events of
the CytcO catalytic cycle as well as the proton pumping activity of the
enzyme.
36
Figure 10. Subunit I (yellow) and II (pink) of R. sphaeroides CytcO. The amino-acid
residues that were substituted in colon- and prostate cancer are indicated in blue.
The red spheres are the oxygen atoms of the water molecules in the D pathway. The
Arginine residues Arg481 and Arg482 are shown in cyan. The arrows indicate the
proton-transfer routes. The figure was prepared using the 1M56 PDB entry and the
PyMOL software
Functional characterization
substitutions
of
the
pathogenic
None of the amino-acid substitutions in subunit I of CytcO that were studied
resulted in a complete loss of enzymatic activity. However, with the
Gly171Asp and the Tyr33His CytcO, a decreased catalytic activity was
observed. Moreover, in these two cases specific steps of the catalytic cycle
were slowed compared to the wild-type enzyme. Investigations of the
37
proton-pumping activity showed that in four out of six of the CytcO variants
the proton pumping was lost or clearly defective. The results from papers IIV are summarized in Table 2.
Wild-type
Steadystate
activity
(% of
wt)
Proton
pumping
(number
of H+/e-)
Proton
leak
Rate of
O2
binding
(µs)
Rate of P
formation
(µs)
Rate of F
formation
(µs)
Rate of O
formation
(ms)
100
0.6
No
10
50
100
1
Glu286His
1
0
No
10
80
10000
1200
Gly171Asp
30
0.35
Yes
10
50
100
1
Tyr33His
40
0
No
10
50
600
4
Asn25Ser
80
0.25
No
10
50
100
1
Ser168Thr
50
0.4
Slight
10
50
100
1
Ala384Ser
90
0.6
Slight
10
50
100
1
Val423Ile
100
0.6
Slight
10
50
100
1
Table 2. Summary of the results from papers I-IV. For details on the different catalytic intermediates (P, F and O) see figure 5 and page 16.
Effects on the CytcO catalytic activity
The Tyr33 residue is situated in the D pathway. Substitution of the Tyr33 for
a Histidine residue resulted in slowed proton uptake through the D pathway
(paper II) and a steady-state activity of approximately 40 % of that of the
wild-type CytcO. The rate of internal proton transfer from the Glu286
residue to the catalytic site was also slowed with the Tyr33His CytcO
compared to the wild-type enzyme. We suggest that the Tyr33His aminoacid substitution results in a structural change of the hydrogen-bonded
network around the Glu286 residue and that this structural change affects the
proton delivery to the catalytic site as well as to the pump site. A similar
effect was observed with the CytcO in which the D pathway residues Ser200
and Ser201 were substituted to Valines [47]. The Tyr33 residue is located
next to the Ser200 and Ser201 residues (figure 11).
The Gly171 residue is situated close to heme a. It is part of the putative
proton gating loop above heme a (figure 11). With the Gly171Asp CytcO the
reduction of heme a was significantly slowed compared to the wild-type
enzyme, which caused a decrease of the steady-state catalytic activity to
38
around 30 % of the wild-type activity (paper III). Probably, the Gly171Asp
substitution results in a lowered midpoint potential of heme a. This effect
has been observed before upon the insertion of a negatively charged aminoacid residue close to heme a [38].
The four amino-acid substitutions that were originally found in human
prostate cancer cells, showed steady-state turnover activities between 50 and
80 % of the wild-type (paper IV). At neutral pH, no effects on the rate of
heme oxidation were observed. However, when the heme oxidation was
monitored at pH 10, there was a decrease in the rate of the slowest phase of
the heme oxidation in all CytcO variants. Since the substituted amino acids
are located at different positions in subunit I of CytcO, we explain the slower
rate of heme oxidation as originating from a structural destabilization of the
protein, occurring only at high pH. Most likely there are small changes in the
electron equilibrium of the redox cofactors in the structurally modified
CytcOs at pH 10.
39
Figure 11. The structure around the heme groups of subunit I of R. sphaeroides
CytcO. The two Arginine residues (Arg481 and 482) are shown in cyan and the
Gly171 residue in blue. The loop consisting of residues 169-175, that is presumably
involved in the gating mechanism, is shown in blue. The oxygen atoms of water
molecules in the D pathway and in the area above the hemes are shown as red
spheres. The polar contacts in the cluster of water molecules surrounding the
Ser200, Ser201 and Tyr33 amino-acid residues is indicated as black dotted lines.
The figure was prepared using PDB entry 1M56 and the PyMOL software.
Effects on proton pumping
Most of the disease-related substitutions in CytcO induced changes in the
proton-pumping activity. With the Tyr33His CytcO, there was a complete
loss of the proton-pumping activity (paper II). This could in part be due to
the slowed proton-transfer rate through the D pathway, as has been observed
before
with
the
Asp132Asn,
Asp132Ala,
Ser197Asp
and
Ser200Val/Ser201Val CytcOs [20, 47, 49]. There are, however, examples of
amino-acid residue substitutions in the D pathway that result in lost proton
pumping but a normal proton-transfer rate through the D pathway [48, 52,
105]. In these cases, changes in the structure around the Glu286 and
associated shifts in the pKa of the Glu286 side chain have been proposed to
40
lead to uncoupling of proton pumping. With the Tyr33His CytcO, there is
indeed a shift in the pKa of the Glu286 residue compared to the wild-type
CytcO. Presumably, also in this case, the amino-acid substitution results in a
change in the structure of water molecules around the Glu286 residue,
leading to loss of proton pumping.
With the Gly171Asp CytcO, there was a putative proton leak through the
enzyme (paper III). The residue is part of a peptide, consisting of residues
169-175, suggested to be involved in the gating of pumped protons [57]. A
suggested conformational change of the gating loop could control the
formation of a putative proton-exit path. Exchange of the Gly171 to an
Aspartic acid-residue could probably result in a structural change of the
gating loop allowing a proton back leak. Preliminary results from H/D
exchange studies indicate that with the Gly171Asp CytcO the loop
consisting of residues 158-172 is highly accessible to deuterium exchange at
all catalytic intermediate states (Busenlehner, Namslauer et al, unpublished).
This is different from the results obtained with the wild-type CytcO, with
which there was decreased deuterium incorporation in the F intermediate of
the catalytic cycle. Moreover, the Gly171 residue is situated close to the
Arg481 residue, that is suggested to be involved in the exit of pumped
protons [33, 56]. Taken together, data from paper III and from the
unpublished study by Busenlehner, Namslauer et al. indicate that the
structure and the function of the proton gating region is indeed affected by
the Gly171Asp substitution.
With the Asn25Ser, Ser168Thr, Ala384Ser and Val423Ile CytcOs proton
pumping was detected in all cases (paper IV), although the Asn25Ser CytcO
showed somewhat less proton pumping compared to the wild-type CytcO.
With the other three there were indications of a slight proton leak, although
not at all as prominent as with the Gly171Asp CytcO. As the Ser168 is part
of the putative gating loop and also close to Arg481 and Arg482, the effect
on proton pumping could in this case be explained in terms of a slight
structural change in the proton-gating region. Ala384 and Val423 are both
found in the interior of subunit I (figure 10). There were indications of a
slight proton leak also with these structurally modified CytcOs. The Val423
residue is close to heme a3; with a distance of about 7 Å to the heme a3 iron.
Ala384 is further away from both the catalytic site and Glu286.
41
Nevertheless, as evident from the slight tendency of a proton leak, both
Val423 and Ala384 are probably close enough to the structural parts of the
enzyme involved in gating of the pumped protons.
The results correlate between eukaryotic and R. sphaeroides
CytcO
With the Gly171Asp and the Tyr33His CytcO, found in colon cancer
patients, cellular and transmitochondrial cybrid studies suggested that the
amino-acid substitutions caused a CytcO deficiency and a lowered
respiratory function, respectively [103, 104]. My studies provided detailed
information on how the substitutions affected the CytcO functional
mechanisms (paper II, paper III). The observed effects confirmed the
pathogenic properties of the substitutions and lead to a deeper understanding
of the phenotypes.
In an unpublished study, we investigated the effects of a mutation in the
CytcO subunit I gene that was previously linked to a mitochondrial disease.
In the mammalian CytcO the mutation resulted in the substitution of a
Leucine residue to an Isoleucine (Leu240Ile) [106]. In the R. sphaeroides
CytcO Leu240 corresponds to Leu196, which is situated next to Ser197 in
the D pathway (figure 6). Previously published and unpublished cellular
studies indicated ambiguities concerning the relevance of the substitution for
the respiratory deficiency. [106](Taylor R W, personal communication). Our
studies showed that the Leu196Ile substitution had no effects on the CytcO
function (Namslauer I et al, unpublished). These results, using the R.
sphaeroides CytcO, correlated well with the results from previous
investigation of the equivalent substitution in S. cerevisiae CytcO [93]. In
that study, Bratton et al. investigated the functional properties of several
CytcO substitutions in yeast and bacteria. In the cases where the same
substitution was studied both in yeast and in R. sphaeroides, the results were
similar.
When the phenotypes of the mtDNA mutations found in prostate cancer
cells were characterized, in most cases no functional studies had been made
previously [90]. Thus, it is not possible to correlate the results to any
functional properties observed in mammalian cells. It should be mentioned,
though, that the Ser168Thr substitution (corresponding to the Ala122Thr
42
substitution in mammalian CytcO) has, apart from prostate cancer cells, also
been identified in breast cancer [100]. In this study, a 50 % decrease in the
CytcO steady-state activity was observed due to the substitution. This
corresponds well to the slight reduction in enzymatic activity that was
observed with the R. sphaeroides Ser168Thr CytcO (paper IV).
Comments on the results
To conclude, these studies have lead to a deep understanding of how the
cancer-related substitutions in CytcO affect the function of the enzyme on a
molecular level. This was accomplished using CytcO from the highly
validated model organism R. sphaeroides and various absorption
spectroscopy techniques. In several cases, the substitutions had large effects
on the enzymatic function. Considering the similarities between the bacterial
and the mammalian enzyme, the effects would most probably be similar in
the human CytcO. In cases where the phenotype resulting from a mtDNA
mutation has been investigated not only in R. sphaeroides but in CytcO from
other species as well, the results are similar.
In addition to elucidating possible disease mechanisms, this work has
contributed to the general knowledge of the function of CytcO, especially
concerning the D pathway and the putative gating loop. The results with the
Tyr33His CytcO agree with the observation that D pathway substitutions can
lead to hydrogen-bond reorganizations around Glu286. Further, the study
supports the suggestions that Glu286 is involved in directing protons to
different sites and that changes in the structure around Glu286 result in
uncoupling of proton pumping. The investigations of the Gly171Asp CytcO
propose that there is a proton-gating mechanism also in the vicinity of that
residue. Perhaps a conformational change in this region takes place at certain
steps of the CytcO catalytic cycle, making sure that protons can only be
transferred in one direction.
The rest of this thesis deals with the question whether the functional
impairments of CytcO that were observed could be involved in the
development or progression of cancer.
43
The relevance of CytcO substitutions found in
cancer
As numerous mtDNA mutations have been observed in cancer, the effects of
the mutations might somehow be involved in disease mechanisms. By
modeling the mtDNA mutations found in colon and prostate cancer in the R.
sphaeroides CytcO and using spectroscopy techniques to study the
enzymatic activity, the effects of the amino-acid substitutions on the
molecular function of CytcO have been ascertained. In several cases, the
substitutions lead to a decreased catalytic activity and a defective protonpumping activity. Now; could these functional defects be involved in the
onset or progress of the disease in which they were detected? And in that
case, what could be the connection between the defects in CytcO and
cancer?
Analysis of tumor cells has revealed that mitochondrial functions,
including the respiratory chain activity, are impaired in cancer cells as
compared to normal cells [107-111]. Respiratory chain dysfunction could be
involved in many other abnormalities, that are frequently observed in cancer
cells, such as an increased dependency on glycolysis for ATP synthesis,
decreased apoptosis and increased ROS production [112, 113]. Some
examples of these and how they could be linked to each other are illustrated
in figure 12.
44
Figure 12. A schematic figure showing various effects of respiratory-chain dysfunction and how these could be linked to each other The figure is based on information
obtained from [112-117].
Energy metabolism of cancer cells
Cancer cells, to a much higher extent than normal cells, rely on glycolysis
for production of ATP [107]. This phenomenon is called “aerobic
glycolysis”, or the Warburg effect, and has been known for over 50 years to
be a common feature of tumor cells [118]. The mechanisms of metabolic
reprogramming are complex, involving activation or deactivation of a large
number of genes [108]. Even today the reason for the upregulation of
glycolysis is not fully understood and it remains controversial if the
metabolic shift is a cause or a consequence of cancer. Nevertheless, by
relying on glycolysis instead of oxidative phosphorylation, cancer cells are
able to survive in conditions of fluctuating oxygen tension. Although the
increased glycolytic activity is not necessarily accompanied by a decreased
respiratory chain activity, mtDNA mutations causing defects in oxidative
phosphorylation could contribute to the increased glycolysis observed in
cancer cells. Such a link has been observed for example in [116], where
inhibition of respiratory-chain activity by oligomycin triggered an increase
in glycolysis in cancer cells.
The amino-acid substitutions found in colon cancer resulted in a lowered
CytcO activity as compared to the wild-type enzyme (paper II and III). A
lowered CytcO catalytic activity could lead to a decreased rate of electron
45
flux through the entire respiratory chain and thus insufficient amounts of
ATP being produced. In addition, with the Tyr33His CytcO the oxygen
reduction was uncoupled from proton pumping (paper II). Thus, the energy
from the chemical reaction at the catalytic site is not conserved to the same
extent as in the wild-type CytcO, resulting in an even less energy-efficient
respiratory chain. Also in the Gly171Asp CytcO, the putative proton leak
(paper III) could dissipate the electrochemical gradient and thereby reduce
the energy-efficiency of the respiratory chain. A decreased synthesis of ATP
through oxidative phosphorylation might however not be as devastating for a
cancer cell as for a normal cell, due to the metabolic shift occurring in
tumors. In fact, a defective respiratory-chain activity could contribute to
initiating the metabolic reprogramming that occurs in cancer cells. If the
metabolic shift is a prerequisite for a normal cell to develop into a cancer
cell, then a decreased respiratory-chain activity could be involved in the
onset of the disease.
Oxidative stress and apoptosis in cancer
Oxidative stress has been one of the suggestions for how respiratory-chain
defects contribute to the development of cancer as well as to aging and
neurodegeneration. This was first explained in the so-called mitochondrial
free radical theory, proposing that mitochondria-derived ROS cause aging
due to oxidative damage to macromolecules [119]. This theory has been
further developed to suggest a ROS vicious cycle, where oxidative damage
to mitochondria themselves results in even faster rates of ROS production
[120]. Today, it is well known that ROS could have harmful effects on
DNA, lipids and amino acids. In addition, ROS can activate apoptosis [121].
An increased ROS production has been shown to result from decreased
respiratory-chain activity as well as increased mitochondrial membrane
potential [113, 115, 122].
There are several studies where ROS production, caused by mtDNA
mutations in cancer, has been investigated [89, 90, 117]. Petros et al., found
that an amino-acid substitution in ATP synthase enhanced cancer cell
growth. The same substitution caused a decreased ATP-synthase activity and
an increased ROS production [114]. Further, mutations in the NADH
dehydrogenase subunit 6 gene result in decreased complex I activity and
46
overproduction of ROS. These functional effects were indeed coupled to
tumor cell metastasis by Ishikawa et al [89]. An additional study on the role
of complex I substitutions in cancer showed that increased tumor growth was
linked to changes in ROS generation and apoptosis [117].
Normally, ROS are not formed from the reduction of oxygen at the
catalytic site of CytcO [123]. However, several studies have shown that
chemical inhibition of CytcO activity could in fact lead to increased ROS
formation from other respiratory-chain complexes. An increased ROS
production due to CytcO inhibition by azide has been observed in both
mammalian cells and submitochondrial particles [2, 124, 125]. Use of
cyanide and NO to inhibit CytcO also results in an increased ROS formation
[126, 127]. In these cases the suggested mechanism is that the inhibition of
CytcO causes an increased leak of electrons, and hence ROS formation, from
complexes I and/or III of the respiratory chain. Taking this into account, it is
possible that the decreased CytcO activity resulting from the Gly171Asp and
Tyr33His substitutions (paper II and III) could lead to an increased
mitochondrial ROS production. Moreover, the slowed rate of intramolecular
electron transfer observed in both the Gly171Asp and the Tyr33His CytcO,
result in accumulation of partly reduced intermediate states of the enzyme.
This could increase the probability of electron leakage and hence ROS
production also from CytcO itself.
The mitochondrial electrochemical gradient in cancer cells
The mitochondrial electrochemical gradient formed by the chemiosmotic
coupling is essential, not only for ATP synthesis, but also for other processes
such as ion, metabolite and protein trafficking. It constitutes an electrical as
well as a pH difference across the membrane. The electrical component (the
mitochondrial membrane potential) can be monitored in absolute scale and
has a value around 200 mV measured in isolated mitochondria [128, 129].
However, the value of the membrane potential varies depending on for
example cell type, metabolic state and age of the organism [130, 131].
Alterations of the mitochondrial membrane potential have been observed in
cancer cells [132, 133]. Heerdt et al found that an increased mitochondrial
membrane potential of mammalian cells was beneficial to tumor progression
47
as well as to the initiation of angiogenesis and escape from apoptosis
occurring in cancer cells.
Interestingly, mitochondrial uncoupling has recently been observed in
cancer cells [134]. In this case, an increase in expression of the
mitochondrial uncoupling protein-2 (UCP-2) in cancer cells resulted in a
decreased membrane potential. Further, overexpression of UCP-2 in cancer
cells results in a lowered production of ROS, inhibition of apoptosis and an
onset of the metabolic reprogramming [135-139]. Using the chemical
mitochondrial uncoupler FCCP, a similar effect on ROS production was
observed. Thus, it is suggested that a benefit of mitochondrial uncoupling in
cancer cells is a minimimal production of ROS [140, 141].
With the Gly171Asp and also to some extent with the Ser168Thr CytcO,
a proton leak was observed (paper III and IV). This would diminish the
energy efficiency of the respiratory chain, but could also influence other
mitochondrial processes such as ion transport. To keep the electrochemical
gradient unchanged upon the introduction of a specific proton leak, the
relative fraction of the membrane potential would presumably have to
increase. An increased membrane potential has been linked to increased
ROS production [115]. On the other hand, as pointed out above,
mitochondrial uncoupling has also been observed in cancer and is believed
to be beneficial for the tumor cells.
48
Concluding remarks and future perspectives
By using the R. sphaeroides bacterial model system, amino-acid
substitutions in CytcO originally found in cancer, have been investigated.
The studies have provided molecular insights into how the substitutions
affected the function of the enzyme. The results revealed functional
characteristics that could be involved in cancer mechanisms. However, since
cancer is caused by a number of different mutations, often affecting tumor
suppressor genes or oncogenes, it is unlikely that the mtDNA mutations
investigated here are the sole cause of the disease. It could be, though, that
the mtDNA mutations contribute to the carcinogenic process by impairing
the function of the respiratory chain. Respiratory chain dysfunction has
previously been observed in cancer cells and it is a feature also found in
aging and neurodegenerative disorders. Since both cancer and
neurodegenerative diseases are much more prevalent at a higher age, it is
possible that there are common mechanisms involved. On the basis of what
has been observed concerning cancer and mitochondria, it is possible that the
functional properties of the CytcO variants found in colon and prostate
cancer provide the conditions required for the development of a normal cell
into a cancer cell.
However, much is still uncertain regarding mtDNA mutations and the
metabolic changes occurring in cancer cells. To further investigate the
relevance of specific amino-acid substitutions for the development of a
disease, a combination of studies using various model systems would be
ideal. Studies on yeast cells or transmitochondrial cybrids would provide
information regarding certain parameters such as ROS formation and
apoptosis. By combining several different model systems a clear picture of
the phenotype of a specific pathogenic mutation could be obtained. This
would be useful information not only for the identification of possible
disease mechanisms, but also for the development of future alternative
therapeutic treatments.
49
Sammanfattning på svenska
Genom vår ämnesomsättning (metabolism), bryts maten vi äter ned i allt
mindre delar. Maten används dels till att bygga upp våra kroppar (muskler,
fettreserver o s v), men även till att ge oss den energi som behövs för att
driva alla processer som sker i kroppen, inte minst muskelarbete och
tankeverksamhet. De delar av maten som omsätts till energi är elektronerna i
näringsämnenas molekyler. Efter att maten har passerat ett antal metabola
processer hamnar elektronerna i ett protein som heter Cytokrom c Oxidas
(CytcO). CytcO är ett enzym, alltså ett protein som katalyserar en kemisk
reaktion. Den reaktion som CytcO katalyserar, där elektronerna behövs, är
omvandlingen av syre till vatten. Denna reaktion sker konstant i våra celler
och är en förutsättning för att vi ska kunna tillgodogöra oss energin i maten.
I de allra flesta av oss, fungerar CytcO som det ska. Men förändringar i
strukturen av CytcO har hittats i bland annat coloncancer och prostatacancer.
Vad som däremot inte har studerats är om, och i så fall hur, dessa
förändringar påverkar funktionen av CytcO. Detta är viktigt att veta för att
kunna utreda om förändringarna i CytcO är inblandade i uppkomsten av
cancer.
I vårt arbete har vi använt CytcO från en bakterie för att studera
effekterna av de förändringar i enzymet som hittats i colon- och
prostatacancer. Bakterierna har tillverkat det förändrade enzymet, som sedan
renats fram från bakteriecellerna. En biokemisk metod som kallas optisk
spektroskopi har sedan använts för att studera enzymets funktion på
molekylär nivå, vilket betyder att man kan följa till exempel hur elektroner
och protoner rör sig i enzymet.
Resultaten av dessa studier har publicerats i artiklarna II-IV som ligger till
grund för denna avhandling. Sex olika förändringar i CytcO har studerats
och resultaten visar att i två av fallen har förändringarna stora effekter på
CytcO’s funktion. I de andra fallen har endast små effekter kunnat
50
upptäckas. Artikel I handlar även den om effekterna av en förändring i
CytcO. I detta fall har däremot inte förändringen hittats i relation till någon
sjukdom, utan har konstruerats i syftet att studera den normala funktionen av
enzymet. Två viktiga aspekter av mitt arbete, som diskuteras i denna
avhandling, är:
1. Relevansen av att använda ett bakteriellt modellsystem för att studera
effekterna av förändringar i CytcO som hittats i mänskliga celler.
2. Om, och i så fall hur, de funktionella förändringarna i CytcO skulle kunna
leda till cancer.
Slutsatserna som dras är att det bakteriella CytcO vi använt för att studera
förändringar i enzymet är ett relevant modellsystem. Anledningarna till det
är att både strukturen och funktionen av det bakteriella CytcO i hög grad
stämmer överens med det mänskliga. Det bakteriella enzymet har använts i
snart 20 år i olika studier och är alltså ett mycket validerat system. Vidare
kan förändringar i CytcO produceras relativt enkelt i bakterieceller och
genom att isolera enzymet från bakterierna kan funktionen studeras på
detaljnivå. I några fall har de förändringar vi undersökt med hjälp av
bakteriellt CytcO även studerats i andra system, till exempel jästceller och
mänskliga celler och i dessa fall stämmer resultaten bra överens med våra.
Vad gäller kopplingarna mellan förändringarna i CytcO och cancer, kan jag
bara spekulera och jämföra resultaten av mina studier med vad som tidigare
visats. Jämfört med normala celler, har cancerceller en förändrad
metabolism. Ett sämre fungerande CytcO leder till en förändrad metabolism,
vilket skulle kunna vara en möjlig koppling till cancer. Jag hoppas och tror
att resultaten som presenteras i denna avhandling, kan bidra till att vi på
längre sikt får en tydligare bild av mekanismerna bakom uppkomsten av
cancer.
51
Acknowledgements
During my years (which are quite many) at this department, I have met so
many great people. Thank you all for making this department such a nice
place.
Some people have been more involved in my work than others and I want to
give them a special thanks.
Peter, du delar alltid med dig av din kunskap och kan förklara alla små
knepiga detaljer på ett så enkelt sätt. Men framför allt har du ett fantastiskt
engegemang och en entusiasm som är väldigt smittsam.
Tack för all hjälp och stöd.
Pia, du har tid för vilka frågor man än kan tänkas ha och kan alltid svara på
dem. Dessutom är du bra på att ställa frågor själv, vilket är väldigt värdefullt.
Tack för all hjälp, både teoretisk och praktisk.
I want to thank all my present and past co-workers for all the help, inspiration, nice chats, laughs, fun coffee-breaks and the good atmosphere that you:
Ann-Louise, Linda, Rosa, Hyun-Ju, Christoph, Gustav, Irina, Emelie,
Joachim, Maria, Davinia, Henrik, Josy, Lina, Håkan, Ullis, Peter L.,
Kristina, Gwen, Gisela, Magnus and Alessandro have provided during my
years as a PhD student. It has been great fun working with you.
Thanks also to my fantastic project worker Marina Dietz. Your work was
very much appreciated.
Ett tack också till Elzbieta, min biträdande handledare, för att du intresserat
dig för mitt projekt och för att du alltid är glad och pratsam.
This is a large department, run by many people. Thank you Stefan, Ann,
Maria, Lotta, Håkan T., Peter N., Torbjörn, Haidi, Lina and all others
for doing such a good job.
Alla vänner och bekanta utanför denna betongkoloss som är min
arbetsplats, tack för att ni ger mig andrum från naturvetenskapen. Ni är
verkligen sköna att umgås med.
52
Mona och Rolf, mamma och pappa alltså; er hjälpsamhet och omsorg är helt
otrolig och mycket uppskattad. Tro aldrig nåt annat. Jag hoppas att jag blir
likadan mot mina barn och barnbarn. Tack för allt.
Carl, Tina, Noah och Leo, det är alltid kul att träffa er. Tack för att ni är så
mysiga och roliga att vara med.
Claudia, Anna, Tommy, Anton och Elin, det är väldigt skönt att komma
till er på Ekenäs. Tack för gästfrihet, trevligt sällskap och fina växter.
Märta och Manne, nu när ni finns så vill jag inte leva en enda dag utan er.
Ni gör mitt liv så roligt, intressant och livligt och jag älskar er helt otroligt
mycket.
Andreas, det liv vi har tillsammans är det bästa och skönaste jag kan tänka
mig. Du är helt enkelt underbar.
53
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