BMB- bioenergetics

why cells need energy

to concentrate substrate molecules at high enough concs
convert substrate to complex ordered macromolecules e.g. DNA

2nd law of thermodynamics

processes can only go in direction of net disorder, increasing entropy and free energy decrease

energy coupling

couple unfavourable processes with spontaneous disordering reaction
use chains when driving reacting runs down

intermediates of energy

electrons

protons

phosphoryl group

del G

= -TdelS

redox potential - Eh

measure of affinity of electrons of something in its oxidised form

large positive Eh

high affinity for electrons

takes electrons from molecules with lower Eh

large negative Eh

low affinity for electrons

donates electrons to molecules with higher Eh

proton motive force

difference in electrical potential between P and N phase - pH potential

photosynthesis photon conversions

2 moles photons enough to convert NADP to NADP

respiration conversions

1 mole electrons from NADH to oxygen is enough to move 5 protons per electron

3 protons needed per ATP

basic pattern of ETCs

electron donor system - Q - bc complex - soluble c - electron acceptor system

evolution of ETCs

primitive photosystem had homodimeric reaction centre

diverges into type I and II reaction centres

type I and II reaction centre proteins duplicate and diverge to become heterodimeric

type I and II reaction centres joined

types of electron carriers

small organic molecules - H carriers

metals - single electron carriers

examples of electron carriers

quinones, NAD, FAD, tyrosine, haems, chloropyll, Mn centres, Fe-S

quinones

Em = +60mV

1H or 2H

plastoquinone in chloroplasts

phylloquinone in PSI

Ubiquinone in mitochondria

NAD

2 electron and H

nicotinamide ring accepts electron

FAD

1H or 2H

1H = semiquinone

couples 2 electron to 1 electron processes

tyrosine

1H

Em = 1V so needs strong oxidising agent to remove H

oxidised form strong

haems

cytochromes distinguished by absorption spectra

a = 600nm

b = 560nm

c = 550nm

Fe in centre of 4 pyrrole rings

chlorophyll

Mg II

a, b, d, f have different absorbance spectra due to different groups

Mn centres

couples 4 electron reaction (H2O to O2) to single electron turnovers in PSII

Fe-S

various stochiometries

rieske

rules for movement of electrons through proteins

distance less than 14A

free energy difference shouldn’t be too large

response of donor and acceptor to change charge

dielectric constant of intervening protein

overview of mitochondrial electron transfer - major complexes

NADH - CI/CII - Q - CIII - cytc - CIV - H2O

ratio of complexes in mitochondrial ETC

I:II:III:IV:cytc:Q = 1:2:3:6:6:60

several electron sources funnel into Q - CI, CII, ETF, FAD

structure of complex I

14 su in bacteria, 31 su in mammals

peripheral arm protrudes into matrix - FMN and 8Fe-S clusters

peripheral arm can change to open or closed conformation linked to proton pumping

electron transfer through complex I

NADH - FMN - 8 Fe-S - Q site

complex I domino model

string of charged residues along middle of complex and peripheral arm movement drives transfer of charges

complex II electron transfer

FAD - 2Fe-S - 4Fe-4S - 3Fe-4S - Q - b haem

oxidises succinate to fumarate extracting 2 electrons

complex II - backwards

upon reperfusion after ischaemia - ROS generation

fumarate accumulation so forms succinate

increased QH2/Q ratio

RET in complex I

complex III structure

cytochrome bc1

dimer - 11 su per monomer

TM domain, matrix domain, IMS domain

overall oxidises QH2 and reduces cyt c

ISP head

Q cycle

1. QH2 binds Qo site - 1 electron to Fe-S, now QH in Qo

2. ISP head rotates - Fe-S electron to cyt c, QH electron transfers through b haems to another Q in Qi site (now QH)

3. ISP head rotates back - another QH2 joins, QH in Qo site

4. repeat step 2 - QH reduced to QH2 in Qi site, returns to Q pool

overall changes in Q cycle

net 1 QH2 oxidised to Q

2 cyt c reduced

4 protons transferred to IMS

2 protons taken up

evidence of Q cycle

ERR studies tracks unpaired electrons

sequences and structures of Q sites and b haems

stoichiometry of proton transport

single turnover experiments in photosynthetic bacteria

removal of electrons from bc complex causes cyt c reduction

mutations in ISP

complex IV electron transfer

reduced cyt c - Cua - haem a - cyt a3 - Cub

when cyt a3 and Cub are reduced, oxygen binds haem a to make H2O - 4 protons from matrix as substrate, 4 protons pumped across

supercomplexes

respirasome - I, III and IV seen in cryo EM

reasons to form supercomplexes

reduced diffusion distance

more efficient

less ROS

evidence for order of complexes in ETC

reoxidation of reduced chain - follow order of reoxidation spectroscopically

inhibitors - antimycin A complex II, cyanide complex IV

thermodynamic data - Em values for components, ascending order = ETC order

S.cerivisae respiratory ETC

NDH-2 instead of complex I

no proton pumping

plant respiratory ETC

NDH-2

NADPH- Q oxidoreductases

alternative oxidases

generates heat if no proton transport

non photosynthetic bacteria respiratory ETC

use Fe II or H2 as donors and nitrate or fumarate as acceptors

basic steps in electron transfer in photosynthesis

1. light harvesting by pigment antennae system

2. primary electron transfer in light activated reaction centre

3. electron transfer down ETC with proton transfer

4. return of protons through ATP synthase coupled with ATP formation

light harvesting pigments

chlorophylls, phycoerythrobilin, beta-carotene

chlorophylls

a and b = light harvesting in plants and algae

a = reaction centre

phycoerythrobilin

antennae in cyanobacteria and red algae

beta-carotene

light harvesting and photoreception in PSI and II

LH2 structure in purple bacteria

9 alpha subunits surrounded by beta su in ring

B800nm between each beta su

ring of B850nm between alpha and beta rings

LH2 light harvesting

1. absorbed at 800nm

2. transferred by RET from B800 to B850

3. excitation between B850 molecules or to LHC1 b875

4. reaction centre

structure of LH1

alpha and beta rings

B875 between alpha and beta rings

reaction centre in middle

gap in ring for quinone entry

structure of LH1 reaction centre

4 su - H, M, L, cyt c

4BChl, 2 bacteriopheophytin, Q a and b, Fe, 4 haems

electron transfer in reaction centre

special pair through BChl - Qa - Qb

photosynthetic electron transfer - cyclic phosphorylation in purple bacteria

P870 - B870* - pheo - Q - cyt bc1 - cytc2 - P870

doesn’t generate reducing equivalents

cytochrome bc1 intersects with respiratory ETC

cyanobacteria light harvesting

phycobilisome - allophycocyanin, phycocyanin, phycoerythrin

unidirectional electron transfer - 565-575nm, 615-640nm, 650-695nm

light harvesting in chloroplasts

LHC1 binds PSI

LHC2 binds PSII

PSII monomer binds 3 LHC2 then forms dimer = supercomplex

electron transport - Z scheme

P680 - P680* - Pheo - PQ pool - cyt b6f - plastocyanin - P700 - P700* - NADP

electron transfer through PSII

Chl special pair - CHl b1 - pheophytin - Qa - Qb

P680 takes electron from tyrosine (from OEC Mn4) to replace

OEC

Mn4 takes 4 electrons from 2H2O to produce oxygen

allows oxidising units to accumulate before oxygen production

avoids ROS production

cytochrome b6f electron transfer

has Q cycle similar to complex III

oxidises quinone - c type cytochrome or plastocyanin

PSI electron transfer - linear

Pc - Chl a special pair - Q - 3 Fe4-S4 - Fd - FNR - NADPH

PSI electron transfer - cyclic

Pc - Chl a special pair - Q - 3 Fe4-S4 - Fd - PQ - cytb6f - Pc - PSI

safety valves in photosynthesis

xanthophyll cycle, PSBS, PSII and PSI, orange carotenoid protein, terminal oxidase, intersection of pathways

xanthophyll cycle

increased light = decreased lumen pH = conversion of violaxanthin in LHC to zeaxanthin by de-epoxidation

enhances ROS quenching

PsBs

pH sensitive antennae protein

enhances non photochemical quenching

synergistic with violaxanthin

PSII and PSI

680nm vs 700nm - different rates of transfer

if PSII too fast, LH antennae shift away - state transitions due to kinase which responds to redox state of PQ pool

orange carotenoid protein

may modulate phycobilisome

dark-stable form activated by blue-green light

terminal oxidase

pass electrons from reduced PQ to Q2

intersection of pathways

cyanobacteria respiratory and photosynthetic

redox loop proton transport

protons carried with electrons as hydrogen

alternating carriers of electrons and H = net proton translocation

e.g. Z scheme

proton pumping

electron and proton flow is separate but coupled by conformational change

e.g. complex I and IV

evidence of chemiosmosis

pH change, uncouplers, RQR, pmf measurement

pH change experiment

give mitochondria reduces species but no oxygen

can see pH change when oxygen added

uncouplers experiment

proton ionophores dissipate proton gradient

allows electron transport without ATP synthesis

lipophilic weak acids

respiratory control ratio

measuring oxygen consumption over time

state 3 = increased ADP, oxygen consumed rapidly, pmf decreases

state 4 = increased pmf due to decreased ADP stopping proton transport

more proton leak in state 4

RQ = state3/state4 = measure of leakiness

pmf measurement

electrostatic difference calculated from distribution of membrane permanent cations at eqm

pH difference calculated from distribution of weak acid or base that can cross membrane when protonated - nernst equation

structure of ATP synthase - Fo

a, 2 b and 8-15 c subunits

links H+ transport to ATP synthesis

can be blocked by DCDD on asp/glu in c su

acts as proton channel in absence of F1

ATP synthase structure - F1

3 alpha, 2 beta, gamma, delta, epsilon

alpha and beta = head

beta = ATPase activity

principles of mechanism of ATP synthase

primary use of energy from pmf is to make ATP and promote release

energy linked to substrate binding - formation of tightly bound ATP and product release, occurs at 3 sites 120 degrees out of phase

binding changes required are driven by sequential conformational changes in F1 driven by gamma rotation

binding change mechanism

1. L state - ADP and Pi bind

2. T state - ATP forms

3. O state - conformational change allows release of ATP

gamma rotation

gamma is bent and alpha helical

helped by hydrophobic ring

proton movement in ATP synthase

1. proton enters and binds c1

2. proton displaces arg210 toward c2

3. arg120 binds c2 and displaces proton that was bound to it

4. proton leaves from other half channel

5. c ring rotates, arg210 now bound c2

6. c2 now adjacent to P site half channel

gearing of c ring

each 360 degree rotation generates 3 ATP

H/ATP ratio depends on number of c subunits

control of ATPase

IF1 in mammals inhibits ATPase activity

translocators out of mitochondria/chloroplasts

adenine nt transporter - exchanges ATP for ADP, overall negative charge transported out

phosphate translocator - imports phosphate and protons

mitochondrial genome

encodes components of complexes

variation in genome size and genetic code

transcribed by viral type RNAP

increased mutation rate - ROS, poor repair systems, lack histones

mitochondrial diseases

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

MERRF = myoclonic epilepsy and ragged red fibres - substitution in tRNA of lysine

NARP - substitution in ATP6

mitochondrial proton transport - into mitochondria

TOM

mitochondrial transport - to matrix

TIM23 + PAM recognition and cleavage by MPP

mitochondrial transport - to IMS

bypass TIM23, use MIA

mitochondrial transport - to outer membrane

SAM

mitochondrial transport - direct to outer membrane

MIM1 then SAM without TOM

mitochondrial transport - inner membrane

TIM22

mitochondrial transport - inner membrane from synthesis in matrix

OXA

fission-fusion of mitochondria

controlled by GTPases

mfn 1 and 2 regulate outer membrane fusion

OPA1 regulates inner membrane and cristae remodelling

fission requires Drp1

anaerobic mitochondria - hydrogenosomes

produce hydrogen

pyruvate and CoA reduces Fd

reduced Fd reduces protons to H2