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BIOL2056 > Mitochondria and Bioenergetics > Flashcards

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
    *
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2
Q

Where do plants tend to utilise their energy from?

A

Sunlight

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3
Q

Where do prokaryotes tend to utilise their energy from?

A
  • Methane
  • Alcohols
  • Nitrates
  • Sulphur compounds
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4
Q

Label this mitochondria

A
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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
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6
Q

What size is mitochondrial DNA?

A

16 kbp

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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
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8
Q

Why is it thought that the mitochondria is derived from ancestor of Rickettsia prowazekii?

A
  • single event
  • genes found in all mitochondria
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9
Q

Plasmodium - protozoan

Schizosaccharomycees - yeast

Prophyra – red algae

Acanthamoeba – amoeba

Marchantia - moss

Reclinomonas – protozoan

A
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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

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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

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12
Q

Name some other reactions that also occur in the mitochondrial matrix?

A
  • The urea cycle
  • Amino acid synthesis
  • Mitochondrial protein synthesis
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13
Q

What is the main event that occurs in the urea cycle?

A

Highly toxic ammonia into urea which can be excreted

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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
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15
Q

Complete the TCA cycle

A
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16
Q

What part of the mitochondrial membrane contains the most protein?

A

The inner mitochondrial membrane

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17
Q

What part of the mitochondrial membrane contains the most lipids?

A

The Golgi

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18
Q

What is the IMM and OMM both poor in?

A

sterols

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19
Q

What is the IMM rich in as opposed to the OMM?

A

CL (Cardiolipin)

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20
Q

What structure is this?

A

Cardiolipin

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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)
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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
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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

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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))

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25
Q

OMM proteins are known as porins what does this mean?

A

They are beta barrel proteins

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26
Q

Tell me about the permeability of a porins membrane?

A

Have a highly permeable membrane

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27
Q

Are porins viewed as analogous to bacteria outer membrane?

What makes porins different?

A

yes they are somewhat comparable in certain respects

However…

  • Lipid composition is different to OMM in bacteria which are rich in lipid polysaccharide
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28
Q

The OMM is rich in porin like proteins, but what else does it also possess?

A

Helical membrane proteins

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29
Q

What do porins play an important role in?

A

Transporting metabolites across the OMM

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30
Q

Name an OMM protein?

A

Voltage dependent anion channels (VDAC)

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31
Q

What does VDAC provide?

A

A low energy barrier to allow the exchange of ATP/ADP across the OMM

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32
Q

What are VDAC proteins most abundant in?

A

Porin like proteins

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33
Q

What are VDAC proteins composed of?

How are VDAC proteins arranged?

A

25 beta strands

They are arranged in the barrel like structure

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34
Q

Why is VDACs strucutre unusual?

A

Most bacterial porins possess an even number of beta-strands where as VDAC has an odd number of 25

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35
Q

What does the outside of the VDAC barrel interface with?

A

The lipid bilayer

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36
Q

What does the inside of the VDAC barrel provide a channel for and why is this the case?

A

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

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37
Q

Contact sites between OMM and IMM are rich in what?

A

VDAC

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38
Q

Although VDAC is a voltage gated channel what does it rely on?

A

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

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39
Q

What does the OMM provide mechanical links for?

A
  • other organelles (e.g. ER endosomes)
  • cytoskeleton
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40
Q

what cellular processes does the OMM regulate?

A
  • Apoptosis (BH3 family- Bax/Bcl2)
  • Mitophagy
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41
Q

What do these light micrographs show?

A

(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

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42
Q

Label this image of a mitochondria

A
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43
Q

What do mitochondria use to move along in a cell?

What is this known as?

A

Microtubules

This is known as mitochondrial trafficking

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44
Q

What is Mitochondrial trafficking?

A

A specific mechanism of communication may exist in the ATP and Ca2+ demanding regions.

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45
Q

The movement of mitochondria is driven by what?

A

The motor proteins Kinesin and dynein

Both of these proteins have motor units which are similar to how myosin works

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46
Q

Tell me the role of the motor protein Kinesin?

A

It drives anterograde transport, this means that it drives things away from the nucleus and towards the +ve end of the strand

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47
Q

Tell me the role of the motor protein dynein?

A

It does retrograde transport which means that it moves things towards the nucleus

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48
Q

In neurones are a majority of neurones stationary or in antero/retro grade transport?

A

In neurones it appears that 70% are stationary whilst 15% are undergoing antero/retrograde transport

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49
Q

Label this diagram to support the components of mitochondrial trafficking

A
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50
Q

The motor complexes kinesin and dynein share strucutral homology with what?

A

the myosin headgroup and act in a similar manner to the complex along the track.

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51
Q

How is kinesin and dynein linked to the mitochondria?

A

They are linked to the mitochondria via miro (RhoT1/T2) and the adaptor protein Milton (TRAK1/2).

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52
Q

What is miro?

A

An OMM protein that anchors the mitochondria to the complex

Its an integral OMM protein

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53
Q

Whats Milton?

A

An adaptor protein whcih basically links it to the motor proteins

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54
Q

What are EF hands responsible for?

A

Ca2+ binding

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55
Q

Where do mitochondria remain immobile?

A

In areas with high levels of Ca2+ (something that is usually attributed to metabolically active regions of the cell)

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56
Q

What are the two mechansims in which mitochondria can be anchored?

A
  1. The interaction of OMM associated myosin, linking to the actin network within the cell
  2. The interaction of Synaptophilin with the microtubule
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57
Q

In addition, the motor complex can be disassociated from the microtubules. This can be done via what 2 ways?

A
  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
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58
Q

Images for the 4 types of mitochondrial trafficking

A
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59
Q

What are EF hands?

A

Its a helix-loop-helix structural domain or motif found in a large family of Ca2+ binding proteins

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60
Q

What are the 4 ways to stop mitochondria trafficking?

If any of these processes can be reversed then explain how

A
  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
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61
Q

what are mitochondrial contact sites?

A

Physical linkages between two membranes

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62
Q

What is the importance of mitochondrial contact sites?

A
  • exchance of lipids between organelles
  • mediate Ca2+ signalling from ER to mitochondria

Contact points are important for communication between mitochondria and different organelles

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63
Q

What occurs at the IMM?

What is in abundance here?

A

Its the site of energy generation

It is rich in proteins involved in respiratory chain

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64
Q

Why has the structure of the IMM evolved to the way it is?

A

The structure has evolved to optimise the role-restricted diffusion, localisation of reactions

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65
Q

what does the IMM contain and give examples?

A

It contains transporters to move substrates out into the cell e.g. ATP and Acetyl-CoA

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66
Q

Whats the role of the electron transport chain?

A

To transfer high energy electrons from donor to terminal receptor (O2)

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67
Q

Whats the role of F1F0- ATP synthase?

A

It couples H+ gradient to ATP synthesis

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68
Q

In the F1F0-ATP synthase what is the F1 and F0 components and what are they responsible for?

A

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

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69
Q

The ATP/ADP shuttle is also known as what?

A

The adenine nucleotide translocator

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70
Q

Tell me the strucutres which this shuttle contains?

A

RRRMMM nucleotide binding motif

2 x 6 TMD/ dimer

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71
Q

What does ATP binding in the matrix trigger?

A

conformational change

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72
Q

Why are IMM so complex?

A

It is complex as it needs a large surface area as need lots of ETC to generate an electron chemical gradient

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73
Q

In ATPase the subunit e promotes dimerisation which is essential for what?

A

Curvature

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74
Q

What does MICOS stand for?

A

Mitochondrial contact site and cristase organising system

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75
Q

What does ATPase dimerisation drive?

A

membrane curvature

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76
Q

What does the MICOS complex drive?

A

It drives membrane invagination

(Mix60∆ large lamellar structure)

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77
Q

Most of the components of MICOS have been determined using genetic knockouts that reveal what?

A
  • knocking out Mic60, produces long lamellar like structures
  • Knocking out ATPase, long lamellar linking sides of mitochondria
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78
Q

What does Mic60 form and what does the size depend on?

A

Mic60 forms a large complex and the size depends on species

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79
Q

The interaction between the proteins of MICOS and Mic60 is dependent on what?

A

The presence of the lipid cardiolipid

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80
Q

What does MICOS have and what is their name?

A

two distinct complexes called Mic10 and Mic60

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81
Q

What is Mic10 responsible for?

Tell me about its features?

what does it require to work?

A

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

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82
Q

What does Mic60 form and have interactions with ?

A
  • 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)

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83
Q

The disruption of what interactions disrupts what formation?

A

Cristae formation

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84
Q

What is MICOS responsible for?

A

localisation activites in IMM and OMM

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85
Q

Why does the IMM have such elaborate geometry?

A

To get more membrane as the cristae provides a large surface area for the enzymes

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86
Q

What localisation reactions occurs at the IMM?

A
  • ETC localised on cristae
  • efficient substrate transport
  • H+ gradient localised
  • Restricts membrane proteins diffusion
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87
Q

When energy is converted from 2H2 + O2 –> H2O, what two methods can this be done via and explain each one?

A

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
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88
Q

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?

A

The Chemiosmotic theory

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89
Q

When was the Nobel prize awarded to Mitchell for this work with the chemiosmotic theory?

A

1978

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90
Q

What are the two links to the chemiosmotic theory?

Give examples of each of these links

A

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
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91
Q

Is the Chemiosmotic theory an important mechanism?

A

Yes, it is used across all organisms, eukaryotes, prokaryotes etc.

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92
Q

In mitochondria, how is the electrochemical gradient generared?

give the expression and its units

A

from protons

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93
Q

whats the expressions for a chemical electrochemical gradient ?

What is it?

A

chemical (∆pH)

difference in concentration of H+ across the bilayer

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94
Q

Whats the expression for an electrical gradient?

What is it?

A

Electrical (∆psi)

seperation of charge across the bilayer

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95
Q

In bioenergetics, how is the following often defined?

A

The proton motive force ∆p with units of mV

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96
Q

How many complex does the ETC have and why are they unique?

A

The ETC has 4 complexes (I, II, III, IV)

They are unique as they are rich in redox centres

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97
Q

What is the name for each of the ETC complexes?

A

Complex I/ NADH/UQ oxidoreductase

Complex II/ succinate dehydrogenase

Complex III/ Ubiquinone/ Cyt C oxidoreductase

Complex IV/ Cytochrome c oxidase

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98
Q

What is Em a measure of?

A

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
Q

What does a large and low Em represent?

A

Large Em : high affinity for electrons

Low Em: low affinity for electrons

100
Q

What is ∆Em related to?

A

∆G

101
Q

What is the equation which relates Em to ∆G?

What does each part in the equation represent?

A

∆G= -zF∆E

∆G= Gibbs free energy

z= change in charge

F= faradays constant

∆E= change in internal energy of a system

102
Q

Will electrons move to sites with larger or smaller Em’s?

A

electrons will move to sites with larger Em

103
Q

If we know that: ∆G= -zF∆E then…
1. rewrite the equation for free energy in our system for a given mass action ratio?

  1. In terms of the electrochemical potential
A
104
Q

What is generally used as the source for electrons in the transport chain?

A

NAD+/NADH

105
Q

When NAD+ is converted to NADH, what is required?

What is the ∆Em value?

What is this molecules used to shuttle around?

A
106
Q

Tell me what is needed in the redox centre Flavin?

Why is this expected to be more favourable to electrons compared to NAD+/NADH?

A
107
Q

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?

A
108
Q

Why does Ubiquinone tend to tether to the mitochondrial membrane?

A

Its hydrophobic

109
Q

What is UQ used for?

A

To transport electrons from different complexes

110
Q

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

A

Most favourable

UQ

Flavin

NADH

Least favourable

111
Q

How many electrons do FeS centres carry?

A

They carry 1 electron, irrespective of the number of Fe atoms

112
Q

How are the Fe atoms linked in a FeS centre?

Whats their Em value?

A

Linked via either…

  1. Cys
  2. Cys/ His
113
Q

How many Fe atoms can you have in a FeS centre?

A

2 or 4 Fe atoms

114
Q

Does the redox potential vary in a FeS centre?

A

yes

115
Q

Would electrons be transferred from UQ to FeS and why?

A

Yes as it is more favourable as the Em is larger again

116
Q

What are the different cytochromes?

What do they all have in common?

Tell me about their Em values?

A
117
Q

The ETC is an ordered series of redox centres, how are these ordered?

A

according to their potential

118
Q

As electrons make small jumps from one redox centre to the next what happens?

A

The complexes of the ETC couple this to the transport of protons

119
Q

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?

A

Couple loss of energy in each stage to transport of protons from one side of bilayer to another

120
Q

Complex I

What does the NADH bind to at the start of the process?

What does the UQ bind to?

A

NADH binds close to FMN

UQ binds close to Q region

121
Q

​Complex I

How many Nqo groups are there?

A

16

122
Q

​Complex I

Tell me about N6a involvment in electron transfer?

A

Has a low potential and is a distance from other centres so it likely the site for control

123
Q

​Complex I

What is the role of N1a in electron transfer?

A

Its off pathway but its conserves- supress ROS

124
Q

​Complex I

Why does NADH bind to the FMN region?

A

For effective hydride transfer

125
Q

​Complex I

What is the main start and end for electrons ?

A

FMN –> Q region

126
Q

​Complex I

When electrons shuttle from FMN to Q what do they move via?

A

FeS redox centres

127
Q

​Complex I

How many FeS redox centres are present?

A

9

128
Q

​Complex I

Whats the role of N7?

A

Ensuring that electrons don’t disappear to destroy mitochondrial membrane

129
Q

​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?

A

The electrons move via Quantum tunnelling

130
Q

​Complex I

Draw a diagram to explain quantum tunnelling

A

The electrons tunnel through energy barrier rather than going over it

Makes the rate across the redox centres relatively efficient

131
Q

​Complex I

Whats the distance of UQ binding sites from the bilayer?

A

15Å

132
Q

​Complex I

Why isn’t UQ found in the bilayer?

A

Charged species aren’t favourable in the lipid bilayer

133
Q

​Complex I

The tunnel in which UQ is found in has what polarity?

A

hydrophilic

134
Q

​Complex I

What forms the binding site for UQ and what does this suggest?

A

Nqo 4,6,7,8 form the binding site

Hydrophilic in nature

suggests that it guides the headgroup

135
Q

​Complex I

The location where the UQ binds has binding sites for what?

A

UQ head and isoprenoid chain

136
Q

The protons in the UQ binding site come from what amino acids?

A

Tyr and His

137
Q

​Complex I

What’s the thought reason that the UQ is distal from the membrane?

A

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
Q

​Complex I

If a protein controls quinone protonation, what can the charge be used to drive?

A

Conformational changes and only after they are completed will the quinone be protonated and released

139
Q

Complex I

In proton pumping, what is used as the antiporters in this process?

A

Nqo 8/12/13/14

140
Q

Whats an antiporter?

A

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
Q

Complex I: Proton pumping

Of the 14 conserved transmembrane helices (TMH), how many form the functional core?

A

10 TMH

142
Q

Complex I: Proton pumping

2x5 TMH are related by what?

A

2x screw axis symmetry

143
Q

Complex I: Proton pumping

What have the proton pumping complexes evolved from?

What do there contribute?

A

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
Q

Complex I: Proton pumping

What is each half channel lined with and occupied with?

A

Each half channel is lined with polar residues and occupied with water

145
Q

Complex I: Proton pumping

The half channels require a gate, what group can be used?

A

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
Q

Complex I: proton pumping

Tell me about the antiporter Nqo8?

A
  • Highly charged for an integral membrane protein
  • Tm5 discontinuous and thought Glu residues contribute to transport of protons
  • forms E channel
147
Q

Complex I: proton pumping

Tell me about the general movement of electrons in this process?

A

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
Q

Complex I: proton pumping

What are the transverse helices one Nqo12 used for?

A

To stabilise the complex

coordinate conformational change

149
Q

Complex I: proton pumping

What is N2/UQ2- role?

A

Provides energy for pumping

Repulsion with acidic patch

150
Q

Complex I: proton pumping

Whats the likely site of energy required for conformational change?

A

N2/UQ reductions

151
Q

Complex I: proton pumping

What may UQ2- interact with to initiate a conformational change?

A

The centre of Glu/Asp

152
Q

Complex I: proton pumping

Whats the role of antiporters?

A

They function cooperatively with each other by pushing one another into the other conformation

153
Q

Complex II

What does complex II have as its cofactors?

A

Flavins

154
Q

What shape is complex I and what is its shape?

A

Complex I is a large, L-shaped multisubunit protein complex located on the innermembrane of the mitochondria

155
Q

What does Complex I accept?

A

It accepts the high energy electrons from NADH molecules

156
Q

In Complex I what does the NADH molecule donate?

A

The two electrons onto an acceptor group found on the vertical component of complex I called FMN (Flavin mononucleotide)

157
Q

In Comple I, what is FMN reduced to when electrons bind to it?

A

FMN is reduced to the FMNH2 form

158
Q

Whats the main overall process that occurs in complex I?

A

The electrons move along a series of FeS groups and are ultimately transferred to coenzyme Q (ubiquinone).

159
Q

What happens when the electrons arrive at Q in complex I?

A

The ubiquinone uptakes 2 protons from the matrix, therby transforming into the fully reduced ubiquinol (QH2)

160
Q

As the electrons move through a series of clusters of FeS, what does the complex use the electrical work to do in complex I?

A

to pump 4 H+ ions out of the matrix and into the intermembrane space

161
Q

What is complex II and what does it contain?

A

Complex II is a protein complex that contains succinate dehydrogenase, which functions in the citric acid cycle

162
Q

What happens in complex II?

A

It converts succinate into fumarate and generate FADH2

163
Q

In complex II, when the FADH2 is produced what happens to it?

A

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
Q

does complex II pump protons?

A

NO

165
Q

Complex II

A
166
Q

In complex III, what is oxidised and what is reduced?

A

Ubiquinone is oxidised

Cytochrome C is reduced

167
Q

What is the function of complex III?

A

To catalyse the transfer of electrons from ubiquinol to cytochrome c

168
Q

What are the important structures in complex III?

What do they contain?

A
  1. Cytochrome c1 (contains 1 haem group)
  2. Cytochrome b (contains 2 haem groups)
  3. Rieske Centre (2Fe-2S groups)
169
Q

In complex III what is meant by the Q cycle?

A

The process which electrons travel from the QH2 to cytochrome C

170
Q

What does the Q cycle in complex III begin with?

A

begins when the first QH2 binds to complex III, upon binding the two electrons follow different paths

171
Q

In complex III, what are the 2 paths that the electrons go on?

A
  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
Q

What happens in complex III, when a second QH2 attaches onto complex III?

A

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
Q

In conclusion, what happens overall in complex III?

A
  • two QH2 are oxidised into Q, releasing 4H+
  • one Q is reduced into QH2 (reducing step)
  • two cytochrome c molecules are reduced
174
Q

Where does complex III contain the UQ binding sites?

A

On opposite sides of the bilayer P and N

175
Q

When UQH2 in QN is formed where can it be released?

A

Into the membrane to enter the UQ pool, where it can be oxidised again

176
Q

in total whats used/ produced in complex III?

A

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
Q

Why electrons take different paths in complex III

A
178
Q

What size is cytochrome c?

Whats bound to it?

A

Its small: 12.5 kDa with a bound haem c

179
Q

Whats the role of cytochrome c?

A

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
Q

What is cytochrome’s C surface rich in?

A

positive charges and a small pocket exists that provides access tot he haem

181
Q

What are the interactions between electron donor and acceptor complex thought to be mediated by in cytochrome C?

A

Charge/charge interactions

182
Q

In complex III the distance between cyt c and haem of complex III is as small as what?

What does this promote?

A

Thought to be as small as 9Å

This promotes a rapid transfer of electrons

183
Q

What do the positive charged on the surface of cytochrome c also provide?

A

An anchor to the membrane surface

184
Q

What is the IMM rich in?

The binding of the positively charged protein to the negatively charged bilayer surface ensures what?

A

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
Q

Whats the function of complex IV?

A

The function is to transfer the electrons from reduced cytochrome C molecules to oxygen

186
Q

What does complex IV contain?

A
  • two haem groups (haem a and haem a3)
  • three copper atoms (CuA/CuA) and CuB
187
Q

Tell me the main overall steps that occur in complex IV

A
188
Q

Tell me about the number of electrons that enter from cytochrome c?

A

4 electrons enter one at a time from cytochrome c

189
Q

Cytochrome C binds via weak electrostatic interactions which favours what?

A

The rapid exchange of cyt c

190
Q

Transport of electrons to the a3/CuB center in complex IV

A
191
Q

What does a CuA center undergo?

A

undergoes 1 e- reductions

192
Q

The transfer of protons is mediated by what?

A

water filled channels which are lined with polar amino acids

193
Q

What are the 3 channels close to the reaction centre in complex IV?

A

D channel Asp D91 - E242

194
Q

Whats the K channel in complex IV and where does it terminate close to?

A

K319- channel terminates close to Tyr crosslinked to farnesyl group of haem

195
Q

Complex IV

Where does the reduction of O2 occur?

What does it require?

A

At the binuclear centre (Cyt a3 / CuB)

It requires a conserved Tyr

196
Q

Complex IV

In the reduction of O2, where does the O2 bind to and what is it split between ?

A

O2 binds to Cyt a3

Split between Cyt a3 and CuB

197
Q

Complex IV: reduction of O2

What is it reduced by?

A

Reduced by e- from Cyt c (P face), whilst picking up H+ from N face

198
Q

Complex IV: reduction of O2

What do we need to be careful of whilst doing this?

A

Make sure that the reactive species are not released

199
Q

In a diagram if you had black and blue protons, what does this resemble?

A

Black protons: used for reduction of O2

Blue protons: translocated

200
Q

Complex IV: reduction of O2

Where does the reduction of molecular oxygen occur?

A

At a binuclear center composed of CuB and Cyt a3, together with a conserved Tyr

201
Q

Complex IV: reduction of O2

How is ∆ phi made?

A

4e- are taken from the p face and 4H+ are taken from the N face which gives rise to ∆ phi

202
Q

The stages of oxygen reduction in complex IV

A
203
Q

Complex IV: pumping protons

What is the K channel lined with?

A

protons

204
Q

Complex IV: pumping protons

What is the D channel proposed to be involved in?

A

The transport of protons

205
Q

Complex IV: pumping protons

What are the channels involved with this?

A

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

206
Q

ATP generation: F1F0-ATPase

What is the role of F1 and F0?

A

F1: Site of ATP synthesis (a/b subunit)

F0: motor unit

207
Q

ATP generation: F1F0-ATPase

Its couple driven by what?

A

couple ∆p driven protein rotation from ATP synthesis

208
Q

Where is there coupling between F1 and F0 in the central stalk?

A

gamma, delta and epilson

209
Q

Label this F1F0-ATPase

A
210
Q

ATP generation: F1F0-ATPase

Are there several classes of ATPase’s?

A

yes: V, P etc.

211
Q

ETC chain focusses on the ATPase found in mitochondria

A
212
Q

ATP generation: F1F0-ATPase

How big are the complexes (depending on the organism)?

A

550-1600 kDa

213
Q

ATP generation: F1F0-ATPase

In the F1 headgroup, what does it contain and where are these located?

A

Contains 3-alpha and 3-beta subunits

Sites located in beta subunit with Arg contributed from the alpha

214
Q

ATP generation: F1F0-ATPase

The F0 generates what?

Has an alpha unit surrounded by what?

A

F0 generates rotational motion

generates torque

a-subunit, surrounded by a ring of 8-17 c-subunits

215
Q

What does the Cryo-Em structure of F1F0-ATPase allow?

A
  • single particle analysis
  • improved detectors
216
Q

Structure of F1F0-ATPase

What does the central stak do?

A

Transmits motion from the motor F0 domain to the F1 ATPase domain

217
Q

Structure of F1F0-ATPase

Tell me about the structure of the peripheral stalk?

A

A helical bundle

218
Q

Structure of F1F0-ATPase

What does the OSCP do?

A

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

219
Q

F0 Motor: utilising ∆p

What are the 2 subunits that it is composed of?

A
  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
220
Q

F0 Motor: utilising ∆p

Whats the role of the F0 motor?

A

couple movement of protons across the bilayer to the rotation of the central stalk

221
Q

In the motor assembly, tell me about its structure?

A
  • c subunit: each has 2 transmembrane domains which associate together to form donut like structure
  • conserved Glu give hint to functions
222
Q

The proton conduction pathway

Where are two half channels formed?

Where do these channels face?

What happens further up the channel?

A

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

223
Q

The proton conduction pathway

What are the proton channels blocked by?

A

R239

224
Q

The proton conduction pathway

For protons to move past the channel blockage, what is this facilitated by?

A

The c-motor ring

225
Q

The proton conduction pathway

How does the c-motor ring work in helping to transport protons past the blockage?

A
  • 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
226
Q

The proton conduction pathway

Whats the driving force for this motion?

What is this force sufficient enough to do?

A

∆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
Q

Whats one of the good things when working with Cryo-Em?

A

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
Q

What would we notice if we were looking at the EM structures of ATPase?

A

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
Q

Transmission to F1 domain via the central stalk

interestingly what do each of these states show and what is this consistent with?

A

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)

230
Q

F1 domain: the catalytic cycle

Tell me about what it contains?

A

3 alpha/beta domains

  • 1 ATP
  • 1 ADP + Pi
  • 1 empty
231
Q

F1 domain: the catalytic cycle

What are the conformational changes initiated by?

A

interaction with the gamma subunit

232
Q

F1 domain: the catalytic cycle

Tell me about the similarity between the alpha and beta subunits?

A

have similar conformation but only 20% similarity

233
Q

F1 domain: the catalytic cycle

Tell me about the N-terminal domain structure?

Tell me about the C-terminal domain structure?

A

N-terminal domain: 6 beta strands

C-terminal domain: either helical and sheet

234
Q

F1 domain: the catalytic cycle

Whats the GXXXXGK(T/S) motif and where is it found?

A

Walker motif binds nucleotide and is found in the P-loop

235
Q

F1 domain: the catalytic cycle

What do the alpha/beta subunit become distorted by?

A

The passage of gamma subunits

236
Q

F1 domain: the catalytic cycle

What do each of the alpha/beta pairs host?

A

a ATP BS. can be ATP, ADP+ Pi, empty (all crystallised)

237
Q

What does the gamma-subunit distort?

A

The nucleotide binding site

238
Q

What does the gamma subunit interact with?

A

DELSEED motif, interactions made with gamma when in ATP bound state

239
Q

How does the gamma-subunit distort the nucleotide binding site?

A
  • 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
Q

Coupling rotation to ATP synthesis

As the gamma subunit rotates around it, what does it cause?

A

the movement of the other subunit around it

241
Q

Coupling rotation to ATP synthesis

What does Cryo-EM analysis show?

A

That the central stalk can adopt 1 of 3 key states

242
Q

Three site alternating binding mechanism

The rotation of gamma-subunit converts what?

What does this allow?

Tell me about the events?

A
  • 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.

243
Q
A

BIOL2056 (7 decks)

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