Mitochondria and Bioenergetics Flashcards
1
Q
In eukaryotes, where do most organisms tend to get their energy sources from?
A
- Sugar
- Fatty acids
- Amino Acids
*
2
Q
Where do plants tend to utilise their energy from?
A
Sunlight
3
Q
Where do prokaryotes tend to utilise their energy from?
A
- Methane
- Alcohols
- Nitrates
- Sulphur compounds
4
Q
Label this mitochondria
A
5
Q
How was the mitochondria thought to have come about?
What features support this event happening?
A
Formed via an Endosymbiotic event
Some main features of the mitochondria that support this are;
- Double membrane
- cDNA
- Mitochondrial specific transcription/ translation which is independent to the nuclear genome
6
Q
What size is mitochondrial DNA?
A
16 kbp
7
Q
What does the mitochondrial DNA encode?
A
- 13 respiratory chain proteins (proteins responsible for the electrochemical gradient)
- rRNA: large and small ribosomal subunits
- tRNA to support translation
8
Q
Why is it thought that the mitochondria is derived from ancestor of Rickettsia prowazekii?
A
- single event
- genes found in all mitochondria
9
Q
Plasmodium - protozoan
Schizosaccharomycees - yeast
Prophyra – red algae
Acanthamoeba – amoeba
Marchantia - moss
Reclinomonas – protozoan
A
10
Q
What is electron microscope tomography?
A
A technique for obtaining 3D structures of sub-cellular macro-molecular objects. It uses a transmission electron microscope to collect that data
11
Q
What main reactions occur in the mitochondrial matrix?
What do these both produce and what is this product useful for?
A
The TCA cycle and Beta-oxidation of fatty acids
Both of these reactions produce NADH. There are high energy electrons that are associated with NADH and these are used to generate an electron gradient
12
Q
Name some other reactions that also occur in the mitochondrial matrix?
A
- The urea cycle
- Amino acid synthesis
- Mitochondrial protein synthesis
13
Q
What is the main event that occurs in the urea cycle?
A
Highly toxic ammonia into urea which can be excreted
14
Q
The TCA cycle produces biosynthetic precursors, what processes does the TCA cycle provide starting materials for?
A
- Amino acids
- Porphyrins (haem, chlorophyll)
- Purines
- Pyrimidines
15
Q
Complete the TCA cycle
A
16
Q
What part of the mitochondrial membrane contains the most protein?
A
The inner mitochondrial membrane
17
Q
What part of the mitochondrial membrane contains the most lipids?
A
The Golgi
18
Q
What is the IMM and OMM both poor in?
A
sterols
19
Q
What is the IMM rich in as opposed to the OMM?
A
CL (Cardiolipin)
20
Q
What structure is this?
A
Cardiolipin
21
Q
What are the features of the Cardiolipin head group?
A
- Glycerol: Bridges two phosphatidic acids
- Double negative charge: this makes it an anionic lipid (functional cytochrome C)
22
Q
What are the features of the Cardiolipin acyl chains?
A
- four acyl chains per lipid
- chains occupy a large volume in the lipid bilayer
23
Q
What does the OMM form a link between?
What does the OMM allow?
A
The OMM is an interface between the cell and endosymbiosis (the leading evolutionary theory of the origin of eukaryotic cells from prokaryotic organisms)
The OMM allows free passage of substrates for energy generation
24
Q
What processes is the OMM involved in?
A
Apoptosis (the death of cells which occurs as a normal and controlled part of an organisms growth or development)
and
Mitophagy (the selective degradation of mitochondria by autophagy (consumption of the bodys own tissue as a metabolic process occuring in starvation and certain diseases))
25
OMM proteins are known as porins what does this mean?
They are beta barrel proteins
26
Tell me about the permeability of a porins membrane?
Have a highly permeable membrane
27
Are porins viewed as analogous to bacteria outer membrane?
What makes porins different?
yes they are somewhat comparable in certain respects
However...
* Lipid composition is different to OMM in bacteria which are rich in lipid polysaccharide
28
The OMM is rich in porin like proteins, but what else does it also possess?
Helical membrane proteins
29
What do porins play an important role in?
Transporting metabolites across the OMM
30
Name an OMM protein?
Voltage dependent anion channels (VDAC)
31
What does VDAC provide?
A low energy barrier to allow the exchange of ATP/ADP across the OMM
32
What are VDAC proteins most abundant in?
Porin like proteins
33
What are VDAC proteins composed of?
How are VDAC proteins arranged?
25 beta strands
They are arranged in the barrel like structure
34
Why is VDACs strucutre unusual?
Most bacterial porins possess an even number of beta-strands where as VDAC has an odd number of 25
35
What does the outside of the VDAC barrel interface with?
The lipid bilayer
36
What does the inside of the VDAC barrel provide a channel for and why is this the case?
The inside of the barrel provides a channel through which substrates can pass
In this case it is pore lined with **positive charge** (the blue part) accounting for ATP/ADP (anion) selectivity as it provides a selective channel for negatively charged molecules to pass through
37
Contact sites between OMM and IMM are rich in what?
VDAC
38
Although VDAC is a voltage gated channel what does it rely on?
It is reliant on the movement of the helix embedded in the center of the beta-barrel, it is unclear if this has any functional significance
39
What does the OMM provide mechanical links for?
* other organelles (e.g. ER endosomes)
* cytoskeleton
40
what cellular processes does the OMM regulate?
* Apoptosis (BH3 family- Bax/Bcl2)
* Mitophagy
41
What do these light micrographs show?
**(A)** a light micrograph of chains of elongated mitochondria in a living mammalian cell in culture. The cell was stained with a fluorescent dye (rhodamine 123) that specifically labels mitochondria in living cells
**(B)** An immunofluorescence micrograph of the same cell stained (after fixation) with fluorescent antibodies that bind to microtubules. Note that the mitochondria tend to be aligned along microtubules. (Courtesy of Lan Bo Chen.)
suggests that mitochondria tend to be where microtubules are
42
Label this image of a mitochondria
43
What do mitochondria use to move along in a cell?
What is this known as?
Microtubules
This is known as mitochondrial trafficking
44
What is Mitochondrial trafficking?
A specific mechanism of communication may exist in the ATP and Ca2+ demanding regions.
45
The movement of mitochondria is driven by what?
The motor proteins **Kinesin** and **dynein**
Both of these proteins have motor units which are similar to how myosin works
46
Tell me the role of the motor protein **Kinesin?**
It drives **anterograde transport,** this means that it drives things away from the nucleus and towards the +ve end of the strand
47
Tell me the role of the motor protein **dynein?**
It does **retrograde transport** which means that it moves things towards the nucleus
48
In neurones are a majority of neurones stationary or in antero/retro grade transport?
In neurones it appears that 70% are stationary whilst 15% are undergoing antero/retrograde transport
49
Label this diagram to support the components of mitochondrial trafficking
50
The motor complexes kinesin and dynein share strucutral homology with what?
the myosin headgroup and act in a similar manner to the complex along the track.
51
How is kinesin and dynein linked to the mitochondria?
They are linked to the mitochondria via **miro** (RhoT1/T2) and the adaptor protein **Milton** (TRAK1/2).
52
What is **miro?**
An OMM protein that anchors the mitochondria to the complex
Its an integral OMM protein
53
Whats **Milton?**
An adaptor protein whcih basically links it to the motor proteins
54
What are EF hands responsible for?
Ca2+ binding
55
Where do mitochondria remain immobile?
In areas with high levels of Ca2+ (something that is usually attributed to metabolically active regions of the cell)
56
What are the two mechansims in which mitochondria can be anchored?
1. The interaction of **OMM associated myosin,** linking to the actin network within the cell
2. The interaction of **Synaptophilin** with the microtubule
57
In addition, the motor complex can be disassociated from the microtubules. This can be done via what 2 ways?
1. The **Ca2+** dependent manner, where the binding of Ca2+ to the EF hands in Miro causes a conformational change in Miro which results in the disassociation of kinesin from the microtubule
2. Finally, the disassociation of the mitochondria from the complex. This arises due to signaling through the **PINK1/parkin pathway** (genes linked to hereditary forms of Parkinson’s disease), which results in the **ubiquitination** (enzymatic post-translational modification in which a ubiquitin protein is attached to a substrate protein) **of Miro** and **degradation**. This pathway is linked to the clearance of mitochondria exhibiting poor electrochemical potentials and targets them for autophagy. PINK1 accumulates on the membrane surface- this recruits Parkin
58
Images for the 4 types of mitochondrial trafficking
59
What are EF hands?
Its a helix-loop-helix structural domain or motif found in a large family of Ca2+ binding proteins
60
What are the 4 ways to stop mitochondria trafficking?
If any of these processes can be reversed then explain how
1. **Myosin** on mitochondria can inhibit their transport by tethering mitochondria to the axonal actin cytoskeleton
2. **Syntaphilin** is present on stationary axonal mitochondria and anchors them through interactions with microtubules
3. **Ca2+ binding to miro** causes a rearrangement of the complex. The motor protein domain of kinesin directly binds to Miro and is blocked from binding to microtubules. When cytosolic Ca2+ is lowered, this rearrangment can be reversed to permit continued kinesin-powered movement
4. The **PINK1/Parkin pathway** causes an irreversible dissociation of the motors from the mitochondrial surface by causing Miro to be degraded. Miro degradation by the proteasome is triggered by PINK1 phosphorylation of Miro and Parkin ubiquitination of Miro
61
what are mitochondrial contact sites?
Physical linkages between two membranes
62
What is the importance of mitochondrial contact sites?
* exchance of lipids between organelles
* mediate Ca2+ signalling from ER to mitochondria
Contact points are important for communication between mitochondria and different organelles
63
What occurs at the IMM?
What is in abundance here?
Its the site of energy generation
It is rich in proteins involved in respiratory chain
64
Why has the structure of the IMM evolved to the way it is?
The structure has evolved to optimise the role-restricted diffusion, localisation of reactions
65
what does the IMM contain and give examples?
It contains transporters to move substrates out into the cell e.g. ATP and Acetyl-CoA
66
Whats the role of the electron transport chain?
To transfer high energy electrons from donor to terminal receptor (O2)
67
Whats the role of F1F0- ATP synthase?
It couples H+ gradient to ATP synthesis
68
In the F1F0-ATP synthase what is the F1 and F0 components and what are they responsible for?
**F1:** is the water soluble head and is responsible for ATP biosynthesis
**F0:** is the transmembrane domain and it couples proton transport to catalytic cycle. it has 2 half channels and proton transport link to rotation
69
The ATP/ADP shuttle is also known as what?
The adenine nucleotide translocator
70
Tell me the strucutres which this shuttle contains?
RRRMMM nucleotide binding motif
2 x 6 TMD/ dimer
71
What does ATP binding in the matrix trigger?
conformational change
72
Why are IMM so complex?
It is complex as it needs a large surface area as need lots of ETC to generate an electron chemical gradient
73
In ATPase the subunit e promotes dimerisation which is essential for what?
Curvature
74
What does MICOS stand for?
Mitochondrial contact site and cristase organising system
75
What does ATPase dimerisation drive?
membrane curvature
76
What does the MICOS complex drive?
It drives membrane invagination
(Mix60∆ large lamellar structure)
77
Most of the components of MICOS have been determined using genetic knockouts that reveal what?
* knocking out Mic60, produces long lamellar like structures
* Knocking out ATPase, long lamellar linking sides of mitochondria
78
What does Mic60 form and what does the size depend on?
Mic60 forms a large complex and the size depends on species
79
The interaction between the proteins of MICOS and Mic60 is dependent on what?
The presence of the lipid cardiolipid
80
What does MICOS have and what is their name?
two distinct complexes called **Mic10** and **Mic60**
81
What is **Mic10** responsible for?
Tell me about its features?
what does it require to work?
membrane sculpting
Features:
* 2 TMD with TMD Gly motif and +ve loop
* forms large oligomeric complexes and oligomerisation drives curvatures in the membrane
It requires Cardiolipin to work
82
What does **Mic60** form and have interactions with ?
* Mic60 forms contact sites with OMM
* forms the core of the complex
* Forms contacts with OMM (interactions with VDAC, TOM/TIM and SAM)
* Forms contact sites between the IMM and OMM and seems to fscilitate the importation of proteins by aligning components of the IMM and OMM
VDAC/TOM (transport OM)
SAM (sorting and assembly complex)
83
The disruption of what interactions disrupts what formation?
Cristae formation
84
What is MICOS responsible for?
localisation activites in IMM and OMM
85
Why does the IMM have such elaborate geometry?
To get more membrane as the cristae provides a large surface area for the enzymes
86
What localisation reactions occurs at the IMM?
* ETC localised on cristae
* efficient substrate transport
* H+ gradient localised
* Restricts membrane proteins diffusion
87
When energy is converted from 2H2 + O2 --\> H2O, what two methods can this be done via and explain each one?
**1. Direct combustion**
* H2 + 1/2 O2 --\> H2O
* during the conversion there is an explosive release of heat energy
* its difficult to harness energy via this method
**2. Biological oxidation**
* H2 seperates into H+ and electrons
* 2H+ + 2e-
* the e- energy is harnessed and converted to a stored form
* the 2e- binds with 1/2 O2 and 2H+ to form water
88
In the 1950s/60s it was thought that direct combustion was the method that was used to harness energy from catabolism of sugar that would be linked to ATP, However this was not the case, the solution to this problem was proposed by Mitchel who suggested what?
**The Chemiosmotic theory**
89
When was the Nobel prize awarded to Mitchell for this work with the chemiosmotic theory?
1978
90
What are the two links to the chemiosmotic theory?
Give examples of each of these links
**1. The use of high energy electrons to generate and electrochemical gradient**
* Mitochondria: High energy electrons from oxidation of food
* Chloroplasts: harvesting light
**2. Utilise this electrochemical gradient to**
* power molecular motors that drive ATP biosynthesis (ATPases)
* Drive transport of molecules against their concentration gradients
91
Is the Chemiosmotic theory an important mechanism?
Yes, it is used across all organisms, eukaryotes, prokaryotes etc.
92
In mitochondria, how is the electrochemical gradient generared?
give the expression and its units
from protons
93
whats the expressions for a chemical electrochemical gradient ?
What is it?
chemical (∆pH)
difference in concentration of H+ across the bilayer
94
Whats the expression for an electrical gradient?
What is it?
Electrical (∆psi)
seperation of charge across the bilayer
95
In bioenergetics, how is the following often defined?
The proton motive force ∆p with units of mV
96
How many complex does the ETC have and why are they unique?
The ETC has 4 complexes (I, II, III, IV)
They are unique as they are rich in redox centres
97
What is the name for each of the ETC complexes?
Complex I/ **NADH/UQ oxidoreductase**
Complex II/ **succinate dehydrogenase**
Complex III/ **Ubiquinone/ Cyt C oxidoreductase**
Complex IV/ **Cytochrome c oxidase**
98
What is Em a measure of?
The tendancy of a chemical species to acquire electrons and thereby be reduced
Em denotes the potential at which the compound is hald oxidised and half reduced
99
What does a large and low Em represent?
Large Em : high affinity for electrons
Low Em: low affinity for electrons
100
What is ∆Em related to?
∆G
101
What is the equation which relates Em to ∆G?
What does each part in the equation represent?
∆G= -zF∆E
∆G= Gibbs free energy
z= change in charge
F= faradays constant
∆E= change in internal energy of a system
102
Will electrons move to sites with larger or smaller Em's?
electrons will move to sites with larger Em
103
If we know that: ∆G= -zF∆E then...
1. rewrite the equation for free energy in our system for a given mass action ratio?
2. In terms of the electrochemical potential
104
What is generally used as the source for electrons in the transport chain?
NAD+/NADH
105
When NAD+ is converted to NADH, what is required?
What is the ∆Em value?
What is this molecules used to shuttle around?
106
Tell me what is needed in the redox centre Flavin?
Why is this expected to be more favourable to electrons compared to NAD+/NADH?
107
What is needed for the redox centre ubiquinone?
What is the ∆Em value?
What type of chain is it and what does this mean to its binding?
108
Why does Ubiquinone tend to tether to the mitochondrial membrane?
Its hydrophobic
109
What is UQ used for?
To transport electrons from different complexes
110
Using what you know about the Em values for NADH, Flavins and UQ, place them in order of most favourable to least favourable to electrons
Most favourable
UQ
Flavin
NADH
Least favourable
111
How many electrons do FeS centres carry?
They carry 1 electron, irrespective of the number of Fe atoms
112
How are the Fe atoms linked in a FeS centre?
Whats their Em value?
Linked via either...
1. Cys
2. Cys/ His
113
How many Fe atoms can you have in a FeS centre?
2 or 4 Fe atoms
114
Does the redox potential vary in a FeS centre?
yes
115
Would electrons be transferred from UQ to FeS and why?
Yes as it is more favourable as the Em is larger again
116
What are the different cytochromes?
What do they all have in common?
Tell me about their Em values?
117
The ETC is an ordered series of redox centres, how are these ordered?
according to their potential
118
As electrons make small jumps from one redox centre to the next what happens?
The complexes of the ETC couple this to the transport of protons
119
When as electron make the small jumps for one redox centre to the next, the complexes of the ETC, couple this to the transport of protons.
How does this coupling occur?
Couple loss of energy in each stage to transport of protons from one side of bilayer to another
120
**Complex I**
What does the NADH bind to at the start of the process?
What does the UQ bind to?
NADH binds close to **FMN**
UQ binds close to **Q region**
121
**Complex I**
How many Nqo groups are there?
16
122
**Complex I**
Tell me about N6a involvment in electron transfer?
Has a low potential and is a distance from other centres so it likely the site for control
123
**Complex I**
What is the role of N1a in electron transfer?
Its off pathway but its conserves- supress ROS
124
**Complex I**
Why does NADH bind to the FMN region?
For effective hydride transfer
125
**Complex I**
What is the main start and end for electrons ?
FMN --\> Q region
126
**Complex I**
When electrons shuttle from FMN to Q what do they move via?
FeS redox centres
127
**Complex I**
How many FeS redox centres are present?
9
128
**Complex I**
Whats the role of N7?
Ensuring that electrons don't disappear to destroy mitochondrial membrane
129
**Complex I**
The following shows the transfer of electrons from FMN to Q via the FeS centres, how must electrons transfer between FeS centres as the distances between then is so large?
The electrons move via **Quantum tunnelling**
130
**Complex I**
Draw a diagram to explain quantum tunnelling
The electrons tunnel through energy barrier rather than going over it
Makes the rate across the redox centres relatively efficient
131
**Complex I**
Whats the distance of UQ binding sites from the bilayer?
15Å
132
**Complex I**
Why isn't UQ found in the bilayer?
Charged species aren't favourable in the lipid bilayer
133
**Complex I**
The tunnel in which UQ is found in has what polarity?
hydrophilic
134
**Complex I**
What forms the binding site for UQ and what does this suggest?
Nqo 4,6,7,8 form the binding site
Hydrophilic in nature
suggests that it guides the headgroup
135
**Complex I**
The location where the UQ binds has binding sites for what?
UQ head and isoprenoid chain
136
The protons in the UQ binding site come from what amino acids?
Tyr and His
137
**Complex I**
What's the thought reason that the UQ is distal from the membrane?
Its distal from the membrane as its thought that if it has the 2- charge and it was located within the bilayer, energetically this would be unfavourable
138
**Complex I**
If a protein controls quinone protonation, what can the charge be used to drive?
Conformational changes and only after they are completed will the quinone be protonated and released
139
**Complex I**
In proton pumping, what is used as the **antiporters** in this process?
Nqo 8/12/13/14
140
Whats an antiporter?
An antiporter (also called exchanger or counter-transporter) is a cotransporter and integral membrane protein involved in secondary active transport of two or more different molecules or ions across a phospholipid membrane such as the plasma membrane in opposite directions, one into the cell and one out of the c
141
**Complex I: Proton pumping**
Of the 14 conserved transmembrane helices (TMH), how many form the functional core?
10 TMH
142
**Complex I: Proton pumping**
2x5 TMH are related by what?
2x screw axis symmetry
143
**Complex I: Proton pumping**
What have the proton pumping complexes evolved from?
What do there contribute?
The proton pumping complex have evolved from a Na+/H+ antiporter- and it is these that contribute the half channels. There is no evidence for Na+ being pumped anymore though
144
**Complex I: Proton pumping**
What is each half channel lined with and occupied with?
Each half channel is lined with polar residues and occupied with water
145
**Complex I: Proton pumping**
The half channels require a gate, what group can be used?
Needs a gate – a group with pKa that can be modulated – this is donated by a key Lys residue – strange as pKa modulated by nearby Glu – this is probably a historic relic, mimicking the original antiporter from which it came
146
**Complex I: proton pumping**
Tell me about the antiporter Nqo8?
* Highly charged for an integral membrane protein
* Tm5 discontinuous and thought Glu residues contribute to transport of protons
* forms E channel
147
**Complex I: proton pumping**
Tell me about the general movement of electrons in this process?
Transfer of electrons across half channels as the negative groups repel quinone which causes a conformational change and move electrons from Nqo 14 --\> 13 --\> 12
148
**Complex I: proton pumping**
What are the transverse helices one Nqo12 used for?
To stabilise the complex
coordinate conformational change
149
**Complex I: proton pumping**
What is N2/UQ2- role?
Provides energy for pumping
Repulsion with acidic patch
150
**Complex I: proton pumping**
Whats the likely site of energy required for conformational change?
N2/UQ reductions
151
**Complex I: proton pumping**
What may UQ2- interact with to initiate a conformational change?
The centre of Glu/Asp
152
**Complex I: proton pumping**
Whats the role of antiporters?
They function cooperatively with each other by pushing one another into the other conformation
153
**Complex II**
What does complex II have as its cofactors?
Flavins
154
What shape is complex I and what is its shape?
Complex I is a large, L-shaped multisubunit protein complex located on the innermembrane of the mitochondria
155
What does Complex I accept?
It accepts the high energy electrons from NADH molecules
156
In Complex I what does the NADH molecule donate?
The two electrons onto an acceptor group found on the vertical component of complex I called FMN (Flavin mononucleotide)
157
In Comple I, what is FMN reduced to when electrons bind to it?
FMN is reduced to the FMNH2 form
158
Whats the main overall process that occurs in complex I?
The electrons move along a series of FeS groups and are ultimately transferred to coenzyme Q (ubiquinone).
159
What happens when the electrons arrive at Q in complex I?
The ubiquinone uptakes 2 protons from the matrix, therby transforming into the fully reduced ubiquinol (QH2)
160
As the electrons move through a series of clusters of FeS, what does the complex use the electrical work to do in complex I?
to pump 4 H+ ions out of the matrix and into the intermembrane space
161
What is complex II and what does it contain?
Complex II is a protein complex that contains succinate dehydrogenase, which functions in the citric acid cycle
162
What happens in complex II?
It converts succinate into fumarate and generate FADH2
163
In complex II, when the FADH2 is produced what happens to it?
It remains attached to the complex and gives off the 2 electrons to a series of Fe-S clusters that ultimately transfer them to ubiquinone
164
does complex II pump protons?
NO
165
Complex II
166
In complex III, what is oxidised and what is reduced?
Ubiquinone is oxidised
Cytochrome C is reduced
167
What is the function of complex III?
To catalyse the transfer of electrons from ubiquinol to cytochrome c
168
What are the important structures in complex III?
What do they contain?
1. Cytochrome c1 (contains 1 haem group)
2. Cytochrome b (contains 2 haem groups)
3. Rieske Centre (2Fe-2S groups)
169
In complex III what is meant by the **Q cycle?**
The process which electrons travel from the QH2 to cytochrome C
170
What does the Q cycle in complex III begin with?
begins when the first QH2 binds to complex III, upon binding the two electrons follow different paths
171
In complex III, what are the 2 paths that the electrons go on?
1. one electrons moves onto the 2Fe-2S group of the **Rieske centre** and then is transferred on the haem group of Cytochrome c1. Its then picked up by cytochrome C which diffuses away and travels to complex IV. This process leaves a UQ radical at the Qp site. 2H+ are then moved onto the p face when the second QH2 binds
2. The second electron moves passes through cytochromes bI and bH to a UQ at the second UQ site (QN).
This results in the formation of UQ at the Qp site, and proceeds with a similar cycle, releasing a further 2 protons to the P face
This time the radical in the UQ site gets further reduced to UQ2- at which point picks up 2 H+ from the N face
172
What happens in complex III, when a second QH2 attaches onto complex III?
The second QH2 transfers a second pair of electrons through the same pathways as before except now a ubiquinol is generated at the end. this pathway also pumps 2H+ and reduces a second cytochrome C
173
In conclusion, what happens overall in complex III?
* two QH2 are oxidised into Q, releasing 4H+
* one Q is reduced into QH2 (reducing step)
* two cytochrome c molecules are reduced
174
Where does complex III contain the UQ binding sites?
On opposite sides of the bilayer P and N
175
When UQH2 in QN is formed where can it be released?
Into the membrane to enter the UQ pool, where it can be oxidised again
176
in total whats used/ produced in complex III?
In total 2UQH2 molecules are used to generate one Cytochrome c and a further 1 UQH2
This is coupled to the pumping of a net 2 protons
177
Why electrons take different paths in complex III
178
What size is cytochrome c?
Whats bound to it?
Its small: 12.5 kDa with a bound haem c
179
Whats the role of cytochrome c?
To shuttle electrons between complex III and complex IV, but its release following disruption of the OMM is one of the first steps associated with apoptosis
180
What is cytochrome's C surface rich in?
positive charges and a small pocket exists that provides access tot he haem
181
What are the interactions between electron donor and acceptor complex thought to be mediated by in cytochrome C?
Charge/charge interactions
182
In complex III the distance between cyt c and haem of complex III is as small as what?
What does this promote?
Thought to be as small as 9Å
This promotes a rapid transfer of electrons
183
What do the positive charged on the surface of cytochrome c also provide?
An anchor to the membrane surface
184
What is the IMM rich in?
The binding of the positively charged protein to the negatively charged bilayer surface ensures what?
CL and anionic lipid
As discussed previously, the IMM is rich in CL, and anionic lipid. The binding of the positively charged protein to the negatively charged bilayer surface ensure that diffusion within the plane of the membrane is preferred, ensuring rapid transit of the electrons between the complexes.
185
Whats the function of complex IV?
The function is to transfer the electrons from reduced cytochrome C molecules to oxygen
186
What does complex IV contain?
* two haem groups (haem a and haem a3)
* three copper atoms (CuA/CuA) and CuB
187
Tell me the main overall steps that occur in complex IV
188
Tell me about the number of electrons that enter from cytochrome c?
4 electrons enter one at a time from cytochrome c
189
Cytochrome C binds via weak electrostatic interactions which favours what?
The rapid exchange of cyt c
190
Transport of electrons to the a3/CuB center in complex IV
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What does a CuA center undergo?
undergoes 1 e- reductions
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The transfer of protons is mediated by what?
water filled channels which are lined with polar amino acids
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What are the 3 channels close to the reaction centre in complex IV?
D channel Asp D91 - E242
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Whats the K channel in complex IV and where does it terminate close to?
K319- channel terminates close to Tyr crosslinked to farnesyl group of haem
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**Complex IV**
Where does the reduction of O2 occur?
What does it require?
At the binuclear centre (Cyt a3 / CuB)
It requires a conserved Tyr
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**Complex IV**
In the reduction of O2, where does the O2 bind to and what is it split between ?
O2 binds to Cyt a3
Split between Cyt a3 and CuB
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**Complex IV: reduction of O2**
What is it reduced by?
Reduced by e- from Cyt c (P face), whilst picking up H+ from N face
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**Complex IV: reduction of O2**
What do we need to be careful of whilst doing this?
Make sure that the reactive species are not released
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In a diagram if you had black and blue protons, what does this resemble?
Black protons: used for reduction of O2
Blue protons: translocated
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**Complex IV: reduction of O2**
Where does the reduction of molecular oxygen occur?
At a binuclear center composed of CuB and Cyt a3, together with a conserved Tyr
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**Complex IV: reduction of O2**
How is ∆ phi made?
4e- are taken from the p face and 4H+ are taken from the N face which gives rise to ∆ phi
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The stages of oxygen reduction in complex IV
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**Complex IV: pumping protons**
What is the K channel lined with?
protons
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**Complex IV: pumping protons**
What is the D channel proposed to be involved in?
The transport of protons
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**Complex IV: pumping protons**
What are the channels involved with this?
K and D
There is evidence of a H channel however there is no concrete evidence for why this channel exists, but definitely K and D
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**ATP generation: F1F0-ATPase**
What is the role of F1 and F0?
F1: Site of ATP synthesis (a/b subunit)
F0: motor unit
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**ATP generation: F1F0-ATPase**
Its couple driven by what?
couple ∆p driven protein rotation from ATP synthesis
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Where is there coupling between F1 and F0 in the central stalk?
gamma, delta and epilson
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Label this F1F0-ATPase
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**ATP generation: F1F0-ATPase**
Are there several classes of ATPase's?
yes: V, P etc.
211
ETC chain focusses on the ATPase found in mitochondria
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**ATP generation: F1F0-ATPase**
How big are the complexes (depending on the organism)?
550-1600 kDa
213
**ATP generation: F1F0-ATPase**
In the F1 headgroup, what does it contain and where are these located?
Contains 3-alpha and 3-beta subunits
Sites located in beta subunit with Arg contributed from the alpha
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**ATP generation: F1F0-ATPase**
The F0 generates what?
Has an alpha unit surrounded by what?
F0 generates rotational motion
generates torque
a-subunit, surrounded by a ring of 8-17 c-subunits
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What does the Cryo-Em structure of F1F0-ATPase allow?
* single particle analysis
* improved detectors
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**Structure of F1F0-ATPase**
What does the central stak do?
Transmits motion from the motor F0 domain to the F1 ATPase domain
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**Structure of F1F0-ATPase**
Tell me about the structure of the peripheral stalk?
A helical bundle
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**Structure of F1F0-ATPase**
What does the OSCP do?
Links the stalk to the alpa subunit of F1 but with sufficient flexibility to permit the alpha subunit to undergo the motion driven by the central stalk
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**F0 Motor: utilising ∆p**
What are the 2 subunits that it is composed of?
1. a: **Stator** (outside of motor and bit that doesn't move), provide half channels for translocation of proton across the bilayer
2. c: **rotor** (bit that moves), 8 copies in mitochondria
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**F0 Motor: utilising ∆p**
Whats the role of the F0 motor?
couple movement of protons across the bilayer to the rotation of the central stalk
221
In the motor assembly, tell me about its structure?
* c subunit: each has 2 transmembrane domains which associate together to form donut like structure
* conserved Glu give hint to functions
222
**The proton conduction pathway**
Where are two half channels formed?
Where do these channels face?
What happens further up the channel?
Two half channels are formed in the **a subunit**
One is facing the **crista lumen** and one is facing the **matrix**
Protons are free to move up the channel but get blocked halfway across
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**The proton conduction pathway**
What are the proton channels blocked by?
R239
224
**The proton conduction pathway**
For protons to move past the channel blockage, what is this facilitated by?
The **c-motor ring**
225
**The proton conduction pathway**
How does the c-motor ring work in helping to transport protons past the blockage?
* Occurs through proton protonation of a conserved Gly residue
* When Glu is protonated, the absence of charge allows it to rotate through the membrane
* That is until it meets R239, conserved in all a subunits, which forces the release of the proton and allows its exit into the matrix
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**The proton conduction pathway**
Whats the driving force for this motion?
What is this force sufficient enough to do?
∆p which applies a force on the deprotonated Glu in the bilayer
This force is sufficient enough to drive the rotation of the c-subunit ring
227
Whats one of the good things when working with Cryo-Em?
You are only studying a single molecule
when analysing thousands of images, it is possible to pick up small variations which would be lost in the crystal structures.
228
What would we notice if we were looking at the EM structures of ATPase?
That the central stalk processes around the rotor axis, as we see this asymmetry has function consequences when we look at how it interacts with the F1 domain
229
**Transmission to F1 domain via the central stalk**
interestingly what do each of these states show and what is this consistent with?
Each of these states show a 120˚ rotation this would be consistent with rotation between the difference a/b dimers
each different ˚ needs different number of protons to rotate (3 conformational states)
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**F1 domain: the catalytic cycle**
Tell me about what it contains?
3 alpha/beta domains
* 1 ATP
* 1 ADP + Pi
* 1 empty
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**F1 domain: the catalytic cycle**
What are the conformational changes initiated by?
interaction with the gamma subunit
232
**F1 domain: the catalytic cycle**
Tell me about the similarity between the alpha and beta subunits?
have similar conformation but only 20% similarity
233
**F1 domain: the catalytic cycle**
Tell me about the N-terminal domain structure?
Tell me about the C-terminal domain structure?
N-terminal domain: 6 beta strands
C-terminal domain: either helical and sheet
234
**F1 domain: the catalytic cycle**
Whats the GXXXXGK(T/S) motif and where is it found?
Walker motif binds nucleotide and is found in the P-loop
235
**F1 domain: the catalytic cycle**
What do the alpha/beta subunit become distorted by?
The passage of gamma subunits
236
**F1 domain: the catalytic cycle**
What do each of the alpha/beta pairs host?
a ATP BS. can be ATP, ADP+ Pi, empty (all crystallised)
237
What does the gamma-subunit distort?
The nucleotide binding site
238
What does the gamma subunit interact with?
**DELSEED motif**, interactions made with gamma when in ATP bound state
239
How does the gamma-subunit distort the nucleotide binding site?
* large changes in **P-loop**
* suggests that these deformations in the binding site drive ATP formation
* upon further counter clockwise rotatio, ATP is released
240
**Coupling rotation to ATP synthesis**
As the gamma subunit rotates around it, what does it cause?
the movement of the other subunit around it
241
**Coupling rotation to ATP synthesis**
What does Cryo-EM analysis show?
That the central stalk can adopt 1 of 3 key states
242
**Three site alternating binding mechanism**
The rotation of gamma-subunit converts what?
What does this allow?
Tell me about the events?
* Convert ATP site to open site, allowing release of ATP
* Converts ADP/Pi into a tight binding site
This allows:
* the binding of ADP/Pi to the new empty site
* conversion of ADP/Pi into ATP
The events are cooperative, coordinated action between 6 subunits. Alone none of them would work. Generate 3 molecules of ATP.
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