The rate of electron transfer between groups– the distance between donor and acceptor of electron – the free energy change The typical electron transfer rate if groups in contact:

1013/sThe distance between electron-carrier groups: 15 A° 15 A°/1.7A° 9, decrease 10≒ 9 104/sIf no protein mediator: 15 A°/0.8A° 19, decrease 10≒ 19 10-6/s 1 day≒

0.8A° 1.7A°

e- cytochrome c Ⅲ Ⅳ

inverted region

The rate of electron transfer between groups– the distance between donor and acceptor of electron – the free energy change

Ch. 19 ?

Chemiosmotic hypothesis (1961, Mitchell P.) NADH oxidation ADP phosphorylation a covalent high-energy intermediate or an activated protein conformation proton-motive force drives the synthesis of ATP by mitochondrial ATP synthase (F1F0ATPase, complex Ⅴ)

Proton-motive force ﹙ p : the pH gradient ﹚ ﹙ pH﹚

+ the charge gradient [membrane potential ﹙ ]﹚

Testing the chemiosmotic hypothesis

Artificial membrane

Respiratory chain

ATP synthase

Proton gradient

(A purple-membrane protein,pump protons when light)

(from beef heart)

A separate system:

19.2 ATP synthesis Mitchell: chemiosmotic model, proton-motive-force

ADP + Pi + n Hp+ → ATP + H2O + n HN

+

a proton pore associated with ATP synthase

Nelson

ATP synthase mechanism: Mg2+ require

Orthophosphate

HPO42-

The role of proton gradient is to release ATP from ATP synthase but not to form ATP

ADP + Pi + ATP synthase in H218O

isotopic-exchange experiments:

enzyme-bound ATP forms readily in the absence of a proton-motive force

+ ATP synthase

ATP synthase structure

Two functional components: 1. Moving units c-ring, stalk 2. Stationary unit

matrix

(3, 3, , and )

subunits participate directly in ATP synthesis

proton channel complex c-ring (10~14 c subunits)1a, 2b, 1

Boyer PD (2000): binding-change mechanism

subunit make the 3 subunits unequivalent

Loss The conformation of binding ADP and Pi

Tight The conformation of binding ATP

Open The conformation of releasing ATP

A counterclockwise direction in subunit

Binding-change mechanism:

TL

O

Proton drive

The smallest molecular motor

Fluorescence labeled

Polyhistidine tags of N-terminal of subunit

Nicklel ions are coated on glass surface

Only cloned 33 subunits

The subunit was rotated, driven by the hydrolysis of ATP

120° increment/ATP hydrolysis

Near 100% efficiency

Components of the proton-conducting unit of ATP synthase

Two hydrophilic half-channel

Do not span the membrane

Directly about one c subunit, separately

Asp61

a pair of -helices

COO-/COOH

The site of proton entrance

Proton flow/c-ring rotation power rotation, then ATP synthesis

Arg210 in subunit a (02), Ex. 18

[H+]cyto/[H+]matrix 25

hydrophobic interaction

Proton path through the membrane

H+

C ring tightly links to

and subunits

C ring rotate

rotate

360°/3 ATP

10 c subunits/3 ATP

3.33 protons/ATP

NADH: 10 H+, 2.5 ATP

FADH2: 6 H+, 1.5 ATP Ex. 19

116 watts (joule/s) provides energy to sustain a resting person

921 earthquake

§ 18.5 Shuttles: an array of membrane-spanning transporter proteins

Glycerol 3-phosphate shuttle: electrons of cytosolic NADH from glycolysis enter mitochondrial electron

transport chain especially prominent in muscle and some insects lack lactate dehydrogenase

G3P 1,3bisP

against NADH gradient

1.5 ATP formation for

1 NADH from glycolysis

Malate-aspartate shuttle in heart and liver is readily reversible the NADH/NAD+ ratio of the cytosol is higher than that of mitochondria 2.5 ATP formation for 1 NADH from glycolysis

transamination

transamination

ATP-ADP translocase(adenine nucleotide translocase or ANT)

highly abundant in the inner mitochondrial membrane (15%) 30 kd, a single nucleotide-binding site, without Mg2+﹙Ex. 20 ﹚

ADP first entry then coupled to ATP exit, even though the transport rate of ATP is 30-fold higher than that of ADP

high energy consumed, about ¼ of the energy from e- transfer

Bongkrekic acid

Membrane potential

Proton-motive force

Ex. 22

P site

N site

atractyloside

ATP synthasome:

ATP synthase, ATP-ADP translocase, phosphate carrier(electroneutral exchange)

H2PO4-

Mitochondrial transporters

Dicarboxylate carrier

40 genes in human genome are encoded

Consumed a proton during ATP translocationOther metabolites translocated

2e- transfer less than 10H+ formation

or +5

Glucose is completely oxidized Glycolysis + 2

TCA cycle (GTP) + 2

or 32

anaerobic metabolism: 2 ATP

the rate of oxidative phosphorylation is determined by the need for ATP

Respiratory control (or acceptor control) the rate of oxidative phosphorylation is primarily regulated by ADP level

Energy charge regulation

The rate of TCA cycle is controlled by the availability of NAD+ and FAD

ATP synthasome

Uncoupling proteins (UCPs) dissipate proton flow

UCP1 (thermogenin)

Temp.

-adrenergic agonists

triacylglycerol degrade

free fatty acids liberate

Activate UCP-1

generate heat

UCP proteins: generate heat to maintain body temperature in hibernating animals, some newborn animals, and in mammals adapted to cold Brown adipose tissue (also brown fat mitochondria), which is very rich in mitochondria, is specialized for nonshivering thermogenesis (vs. white adipose tissue) regulate the body weight (obesity)[UCP2 and UCP3] increase the evaporation of odoriferous molecules, skunk cabbage

p. 533 7th line: greenish-colored cytochromes

Uncoupler disrupted the coupling of electron transport and phosphorylation – dissipated proton-motive force oxygen consumption, NADH oxidation, no ATP formation heat loss DNP and certain other acidic aromatic compounds used in some herbicides, fungicides, weight- loss drug (?)

Sites of action of inhibitors of electron transport

ferric

ferrous form of heme a3

Oligomycin

Dicyclohexylcarbodiimide

(DCCD)

prevent the influx of protons

through ATP synthase

Alternative mechanisms in plant mitochondriaAraceae: one family of stinking plants

thermogenesis

a cyanide-resistant QH2 oxidase

bypass complex III and Ⅳa rotenone insensitive NADH dehydrogenase, bypass complex Ⅰ a skunk cabbage

Nelson

Mitochondria

¤ semiautonomous organelles

¤ endosymbiotic double membrane, circular DNA, specific transcription and

translation machinery

¤ Maternally inherited

Mitochondria – diseases

a center of energy metabolism Leber hereditary optic neuropathy – NADH-Q oxidoreductase (complex )Ⅰ mutation – resulted in blindness during midlife chance fluctuation percentage the threshold of defect the accumulation of mutations effect the energy transduction, reactive oxygen species (ROS)

generation nervous system and heart are vulnerable– aging, degenerative disorders, and cancer.

Three mitochondrial cell death pathways

Apoptosis inducing factor (AIF)

Apoptotic protease activating factor-1(Apaf-1)

mtPTP

(mitochondrial permeability transition pore)

Programmed cell death

PS

Cysteine protease family

Proton gradient– a central interconvertible currency of free energy

(Mito. inner membrane)

P:O ratio:

the number of molecules of inorganic phosphate incorporated

into organic form per atom of oxygen consumed.

the number of molecules of ATP synthesized per pair of electrons

carried through electron transport.

ATP synthesis: is quantitative as phosphate uptake, conversion

of orthophosphate to organic phosphates.

Electron pairs: are quantitative as oxygen uptake.

matrix NADH: 2.5

matrix FADH2: 1.5

+ 2,4-dinitrophenol: P/O ratio from 2.5 0

Ex. 6

96C

96T 192

97T

97C

98T

98C