Energy Transduction

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Non-photosynthetic bacteria

Back 1

P. denitrificans

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Photosynthetic bacteria

Back 2

R. sphaeroides - Purple bacteria

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bacteria combining sulphur oxidation and light

Back 3

chlorobium

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Hydrogen carriers (4)

Back 4

NAD, FMN/FAD, Quinone, Tyrosine (Y) residue

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Quinones (4) and where to find them

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UbiQ -mitochondria & bacteria

PlastoQ - chloroplasts

MenaQ - some bacteria

PhylloQ - some PS I

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Metal electron carriers (5)

Back 6

Fe-S centre

Fe3+ haem

Mg2+ chlorophyll

Cu centre

Mn Centre

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Distance between protein residues for electron tunneling

Back 7

14 A

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Rate of electron tunneling depends on (4)

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Distance between carriers

redox potential between donor and acceptor

The response of the donor and acceptor (and environments) in response to change in charge

Dialectric constant of the protein

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Proportions of the complexes (6) in the membrane

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1 cpx I : 2 cpx II : 3 cpx III : 6 cpx IV : 6 cyt c : 60 ubiQ

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Complexes feeding electrons into quinone (mitochondria) (6)

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Complex I

Complex II (succinate dehydrogenase from TCA cycle)

Glycerol-3-phosphate dehydrogenase (electrons from cytosol in some tissues)

ETF:Q oxidoreductase (electrons from fatty acid oxidation)

dihydroorotate dehydrogenase (pyrimidine biosynthesis)

Malate quinone oxidoreductase (instead of MDH in some organisms)

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Complex I other name (mitochondria)

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NDH-ubiquinone oxidoreductase

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The two components of L-shaped complex I

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•peripheral arm, containing FMN and 8 Fe-S centres

• membrane domain with cofactors

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Pathway of electron flow through complex I

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NADH → FMN→8Fe-S→Q

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Other name for Complex II (mitochondrial)

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Succinate dehydrogenase of the TCA

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Role of complex II

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oxidises succinate to fumarate and reduces Q to QH2

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Electron flow in Complex II (mitochondria)

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Succinate (sub A) → Fad (A) →3xFeS(B)→ubiQ (C&D)

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What does b haem do in subunits C&D of complex II?

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Maybe helps to reduce electron loss from complex II to molecular oxygen

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COmplex III also known as?

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cytochrome bc1 complex

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Evidence for the Q cycle (complex III of mitochondria) (7)

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•Electron paramagnetic resonance

•sequence data and crystal structure for Q binding sites

•crystal structures for b haems

•Stoichiometry of proton pumping

•Single turnover experiments in photosynthetic bacteria

•addition of oxidised cyt c in the presence of QH2 generates QH at o site, which reduces b cytochromes - oxidant-induced reduction

•Mutation in ISP hinge stops complex

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Other name for complex IV (mitochondria

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cytochrome c oxidase

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Electron flow through complex IV

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cyt c (reduced) → CuA→haem a→cyt a3 &CuB

Then

O2 + heme a3→2H2O

4H+ consumed and 4 more pumped from the matrix

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Stuff about respirasome (3)

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Contains complexes I III IV

Depends on P, whose synthesis is regulated by Hypoxia Inducible Factor (makes sense since presumably respirasome is more efficient)

Cardiolipin might be involved

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Evidence for the order of the comonents in mitochondrial ETC (3)

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Em values

studies of reoxidation of reduced chain

Inhibitor effects

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What's up with plant mitochondria? 2

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additional NADPH-Q oxidoreductase and

Alternative oxidase

Passes electrons from Q to oxygen, without proton pumping

Control of heat production

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What's up with non-photosynthetic bacteria's mitochondria

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electron donors: FeII or H2

elector acceptors: nitrate or fumarate

P. denitrificans ETC like mitochondria

E. coli has diferent quinol oxidase (containing cytochromes b, o or d) depending on the growth conditions

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Energy coupling in photosynthesis, 4 steps

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1. Light harvesting by pigment antennae

2. e transfer in light-activated reaction centre

3. e transfer to ETC with H pumping

4. Return of e across membrane/ATP synthase

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Photosynthesis studied on 3

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Purple bacteria (1 PS with cyclic e transport)

Cyanobacteria

Algae and higher plants

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light harvesting chlorophylls

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a&b

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Red algae and cyanobacteria: light harvesting antenna

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Phycoerythrobilin and phycocyanobilin

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Light harvesting and photoprotection: carotenoids (2)

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b-carotene and lutein

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another name for lutein

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xanthophyll

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energy transfer rate in chloroplasts

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<< 1ns

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Triplet state?

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Gives free radicals

carotenoids decay them

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pple bact: LH1

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associated with reaction centre, froms core complex

B875

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LH2

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antenna;

9 alpha, 9beta, 9bacteriochlorophyll (B800) , 9bacteriochlorophyll ( B850)

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Requirements for exiton transfer

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<1ps, direct coupling of orbitals, <1.5 nm apart (molecules)

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LH1 (vs LH2)

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alpha and beta concentric rings

reaction centre complex in the middle

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Reaction centre in purple bacteria (photosynth)

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4xbacteriochlorophyll

2xbacteriopheophytin

Qa&Qb

free Fe

4haems

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light harvesting structure in cyanobacteria, structure

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Phycobilisome, 3 proteins, each absorbing light with longer wavelength - unidirectional transfer, to the reaction centre

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Fe deficiency: cianobacteria replaces phycobilisome with

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iron stress induced antenna protein that forms ring around the reaction centre

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light hrvesting complexes in chloroplasts

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LHCI - PSI

LHCII - PSI &PSII

+ loads of pigments

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why manganese?

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lots in oceans

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sources of imbalances in photosystems

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high light levels

fluctuating light levels

PS running at different rates

reducing equivalents being generated faster than cell uses it up

→reactive ox species

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Safety valves for photosynthesis

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PSII &PSI running at diferent rates

LH antenna shifting from PSII if it's running too fast

terminal oxidase (passes electrons from QH2 to o2)

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State transitions (photosynthesis)

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movement of light harvesting antennae away or to PSII to regulate rate of photosynthesis

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Regulation o state transitions

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kinase responds to PQ pool E potential

reduction of PQ pool → activation of kinase

kinase phosphorylates LHC II which leaves PSII

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In cianobacteria proteins that transfer excess e from PS to O2 in controlled way

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flavo-diiron proteins

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Chemiosmotic hypothesis

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1. e transport is coupled to H transport across a membrane

2. Membrane is impermeable to the protons

3. Return of the protons through the membrane is coupled to the ATP synthesis

4. There are other specific transporters in the membrane

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models of coupling e and H flow

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redox loop model

proton pump model

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mechanisms of proton transfer in cpx II and IV

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complex III: Q-cycle (more complex redox loop)

complex IV: redox loop and proton pump

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what kind of molecules are proton uncouplers

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lipophilic weak acids

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what is respiratory control

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stimulation of oxygen consumption by ADP

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the cause of proton leak 2

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•free fatty acids

•specific protein transporters of protons (eg uncoupling proteins - UCPs)

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uncoupling protein in mitochondria of brown adipose tissue (straight forward if think about function), what encodes it

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thermogenin

UCP 1 gene

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brown adipose tissue in cold rodents

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sympathetic nervous system* →noradrenaline (in BAT) ↑ → adrenergic receptors* (surface of bown adipocytes →AC*→cAMP↑→lipolysis* (via lipase) → free fatty acids ↑ (from triacyl glycerol) - substrate for proton leak and mitochondrial respiratory chain. result - heat.

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heat production in mitochondria by plants

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alternative oxidase to oxidise QH2

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inhibitor of intact ATP synthase, what does it block?

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Oligomycin, Fo

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Name of mechanism for ATP synthesis

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binding change mechanism

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binding change mechanism 3

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1. energy (by H flow through F0) releases ATP from catal sites, where it's formed spontaneously

2. ATP binding, ATP formation and release happen at separate interacting sites 1/3 out of phase.

3. binding changes driven by sequential confromational changes in F1, driven by gamma subunit

Front 60

residues of a subunit of ATPase interacting with H

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glu/asp, via histidine

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How abundant adenine nucleotide translocator

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10% of mitochondrial protein

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What does adenine nu trnslocator catalys

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ATP4- for ADP3- exchange across mitochondrial inner membrane

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other mitochondrial carriers 4

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phosphate pyruvate dicarboxylate tricarboxylate

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The two A nu translocator staates, inhibitors which lock them

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C and M states,

carboxyatractyloside and bongkrekic acid

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A nu translocator vs pmf

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exports overall negative charge - favoured by the pmf

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phosphate translocator

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symporter: imports H2PO4- and H+

favoured by pmf

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ATP synthasome

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translocators and ATP synthase together

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closest relative of mitochondria

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Rickettsiaceae

lives intercellularly

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In humans, what's encoded by the mt genome

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complexes I, III, IV and ATP synthase, rRNA and tRNA

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what used for transcription of mt genome

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viral-type RNA polymerase

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how are mt from sperm are got rid of in fertilised egg

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destroyed by ubiquitin-dependent mechanism

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how long ago was mitochondrial Eve

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100,000-200,000 years ago

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How much more quickly mtDNA evolves and mutates, why, consequences?

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10-17x faster than nuclear DNA

reactive ox species from ETC

high polymorphism even between close relatives

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Mitochondrially determined diseases 4

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MELAS - mitochondrial encephalopathy, lactic acidosis and stroke - like episodes

MERRF - myoclonic epilepsy and ragged red fibres

NARP neurophaty, ataxia, retinitis pigmentosa

LHON - leber's hereditary optic neuropathy

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Causes of mitochondrially determined diseases 4

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MELAS - substitution in a mt t-RNA-Leu gene (80%)

MERRF - supstitution in tRNA-Lys gene

NARP - substitution in ATP6

LHON - mutations in NDH genes

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sequence for matrix proteins

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matrix-targeting sequence - cleaved off in the matrix

internal recognition sequences

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nature of mitochondrial signal sequences

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20-30 aa long

on the N-terminus

not strongly homologous

has amphiphilic alpha helix w/ positive charge on one side

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3 membranes in mitochondria

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outer, inner and cristal (ox.phos occurs in cristae)

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cristae connected to inner membrane by /function

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cristal junctions

regulate cell death

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outer and inner membranes connected by

what occurs there

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contact sites

protein transportation

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what do cristal junctions and contact sites depend on

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MICOS mitochondrial contact site and cristae organising system proteins

developed at the time of endosymbiosis

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Mamals: GTPases for

a fussion of outer membranes

b fussion of inner membranes

c fission

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a mfn1 & mfn2

b OPA1

c drp1

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damaged mitochondria vs fusion

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fusion requires membrane potential - damaged mitochondria unable to join the network - get degraded

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process of generating new mitochondria

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mitochondrial biogenesis

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mitochondrial biogenesis involves

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mtDNA replication and incorporation of new proteins and phospholipids.

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mitochondria turnover rate in post-mitotic tissues (eg?)

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heart, brain (no more division)

every 10-25 days

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mt degradation

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by autophagy:

random surrounding of areas of the cytoplasm by the autophagocytic vesicel

which then fuses with lysosomes

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anaerobic metazoa

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shellfish living in sediments

parasitic worms in digestive system

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non-o2 terminal acceptors

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nitrate

nitrite (to NO)

molecules generated during emtabolism

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more degenerate mitochondria

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hydrogenosomes - produce H2

in unicellular eukaryotes living in guts, etc

N. voalis, T. vaginalis

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typical size of chloroplasts genome

mitochondrial

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120-200 kbp chloroplasts

varied eg 6 (plasmodium) - 367 (arabidopsis)

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chloroplasts genome endoces 7

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PSI PSII

b6f complex (pet)

ATP synthase (atp)

protein synthesis

ribulose bis-phosphate carboxylase (rbcL)

rRNAs, tRNAs

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why genes (mt, chl) need to be retained?

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for redox regulation

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stuff chloroplasts

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transcribed by bacterial-type RNA pol

land plants - viral-type RNA pol

uniparentally inherited

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chloroplast structure

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PSI - stromal lamellae, ends of granal

PSII - stacked

cyt b6f - equally distributed

ATP synthase - stromal lamellae

distribution of complexes differs between stacked and non-stacked regions (granal and stromal lamellae)

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non-phontosynthetic chloroplasts?

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developmentally - proplastids, amyloplasts (convertible into chloroplasts)

evolutionarily: non-photosynthetic plants: Epifagus monotropa

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sup with plasmodium

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has photosynthetic ancestry, have remnant chloroplasts. It's essential, target for therapy (as doxycycline, fosmidomycin) target an enzyme in isoprenoid biosynthesis, which is absent in humans, but present in plants, eukaryotic algae and cyanobacteria.