Biochem Test Ii West

Front 1

oxidative phosphorylation

Back 1

electron transport and generatioin of transmembrane proton gradient

ATP synthesis from the proton grad

Front 2

2 parts of oxidative phosphorylation

Back 2

1. oxidative-involves oxidation of electron donors like NADH and FADH2

2. phosphorylation- captures energy in the form of ATP synthesis from a proton gradient

Front 3

Where does oxidative phosphorylation occur?

Back 3

in Mitochondria

Front 4

Where do electrons for Oxidative phosphorylation come from?

Back 4

glycolysis
Krebs
Beta oxidation of fatty acids

Front 5

Describe cellular respiration

Back 5

The cell produces CO2 from the Krebs cycle and consume O2 from the ETC. Cellular respiration also deals with the consumption of electrons

Front 6

When NADH and FADH2 from krebs goes on to ETC what happens to the electrons?

Back 6

the e- go from NADH/FADH2 to oxygen and water is released. Since this rxn is so downhill energy is captured as ATP.

2H+ + 1/2 O2 ---> H2O
water is made from the O2

Front 7

membranes of mitochondria

Back 7

inner and outer

Front 8

describe inner mitochondrial membrane

Back 8

large suface area bc many infoldings

impermeable (tightly sealed) unless a transporter is presents (ex is pyruvate transporter)

2 sets of enzyme complex
1. move e- down ETC, they are membrane bound carriers.
2. to make ATP

Front 9

2 sets of enzymes in inner mitochondrial membrane

Back 9

1. move e- down electron transport gradient (membrane bound carriers involved with prton transfer)

2. ATP synthesis

Front 10

Describe outer membrane

Back 10

True membrane but leaks so it allow metabolites that are small and ions through holes called porins. Therefore, things like pyruvate can get in mitochondrial matrix

Front 11

are matrix and cytoplasm mixed?

Back 11

no mit. matrix is isolated from cytoplasm

Front 12

what is between inner and out mit membrane?

Back 12

intermembrane space is between the inner and outer mit. membrane. It is similiar to cytoplasm but it holds unique proteins and has a special role in oxidative phosphorylation

Front 13

overview of Oxidative phosphorylation

Back 13

electrons flow through the chain of membrane bound carriers in the inner mit. membrane

Free energy released is used to transport protons uphill across the inner membrane (oxidative)

Transmembrane flow of protons down their conc. graient provides free energy for ATP synthesis via ATP synthase (Phosphorylation)

Front 14

properties of NADH

Back 14

good at carrying e- in aqueous solution but not in membrane

v. mobile to carry around e-

Carries 2 electrons

always in NAD form to capture e- from catabolic pathways then the NADH form goes to ETC

Front 15

function of nicotinamide nucleotide linked dehydrogenasees

Back 15

collec e- from catabolic rxns and transfer them to water (NADH)

Front 16

NADPH

Back 16

seperate that NADH

always in NADPH form to give e- for anabolic rxns

Front 17

orgin of e- from catabolism

Back 17

NADH
FAD
FMN

Front 18

FMN and FAD properties

Back 18

stuck to protein so very immobile

can carry one electron as (semiquninone) or two electrons (FADH2 or FMNH2) so very versatile

Front 19

What does FAD and FMN reduction potential depend on?

Back 19

protien microenvironment

Front 20

Since NADH can only carry e- in aqueous solution, what is need to transfer e- to Q?

Back 20

need protein prosthetic groups

Front 21

Describe Ubiquinone (coenzyme Z)

Back 21

it is a downstream electron reducing equivalent

it has long hydrophobic tail with 50 Carbon units it the lipid bilayer.

Quninone is the active group and it has 2 ketone carbonyls that can be reduced to hydroxyls

Q carries E- is the membrane

Front 22

How many e- can Q carry?

Back 22

one or two bc has two carbonyl ketone groups

carry one e- (semiquinone)

Carry two e-(fully reduced Q)

Front 23

cytocromes

Back 23

have Fe containg heme prosthetic group in center to carry the e-

Type of protien prosthetic group to get e- from NADH to Q.

Front 24

3 classes of cytochromes

Back 24

a,b,c

cytochrome protein with heme group is colored bc of the resonance in the double bond. so a,b,c differentiated by light absorbtion

Front 25

difference is cytochrome a, b, and c

Back 25

a has long carbon chain that is anchored to a lipid bilayer. it is an integral membrane protein

b is a porphin ring also a integral membrane protien

c has a group with a convalently linked cysteine. c is water soluable but ionically associated with outer suface of inner mitochondrial membrane

Front 26

purpose of difference in cytochrome a, b, c

Back 26

the cytochromes are protein prosthetic group. Each type of cytochrome is found at a different level in the ETC bc each one hold e- at a different affinity and have different reducing potentials

Front 27

what does absorbtion spectrum of cytochrome c tell us

Back 27

reduced cytochrome c (fe 2+) has major absorbtion at 400 nm and and minor absorbance at 540 nm. The oxidized (fe 3+) absorbance goes down at 400 nm and 540 nm bc color changes. So you can tell how oxidized or reduced the cytochrome is and determine at what place it is in ETC

Front 28

Iron sulfer proteins

Back 28

used to hold e-

Dont use heme group but do have iron groups that bind to sulfer atoms of side chains of cystiene residues.

2 arms fold to make horse shoe so adjacent sulfidyl groups coordinate iron and inorganinc sulfate is in v. center.

the Fe groups hold the e-

Front 29

What are Rieski protiens?

Back 29

iron sulfur protiens

Front 30

5 major classes of e- transporters

Back 30

NADH
FADH2
Q
cytochromes
Iron sulfer proteins

Front 31

describe standard reduction potential of ETC

Back 31

net is E=very + therefore G is very negative so alot of nrg is there so it takes several steps bc there is so much energy it is too much energy for just one enzyme/protein to handle. So bits of nrg are captured at each step. ETC has many stair steps to give little energy at each step

Front 32

How is energy saved in ETC?

Back 32

energy is basically saved as acid most stair step of the way. Acid is pumped from matrix across intermitochondiral membrane and stored in the cytoplasm to make a huge proton gradient. The cytoplasm is approx 1 pH unit lower than the matrix

Front 33

4 ways to interfere with oxidative phosphorylation

Back 33

1. ihibition of electron transfer

2. inhibition of ATP synthase

3. uncoupling of phosphorylation from eleltron transfe

4. inhibiton of ATP-ADP exchange

Front 34

how does cyanide intere with ox phos?

Back 34

by inhibition of ETC bc it binds to chytocrome oxidase instead of oxygen

Front 35

How does carbon monoxide interfere with oxphos?

Back 35

by inhibition of ETC bc it binds to chytocrome oxidase instead of oxygen

Front 36

How does Rotenone interfere with ox phos?

Back 36

it inhibits ETC by preventing electron transfer from Fe-S center to ubiquinone (inhibits e- transfer from NADH to Q)

Front 37

how does oligomycin interefere with oxphos?

Back 37

it inhibits ATP syntase by blocking proton flow

Front 38

3 compounds that inferefere with oxphos by uncoupling phsophoylation from electron transfer

Back 38

DNP
Valinomycin
Thermogenin

Front 39

How does Antimycin A interfere with oxphos?

Back 39

inhibits ETC bc it blocks e- transfer from cytochrome b to cytochrome C1

Front 40

ETC

Back 40

(reduced end)
NADH
Q
Cyt b
Cytc1
Cytc
Cyta
O2
(oxidized end)

Front 41

What happens with antimycin A?

Back 41

block e- from going from cyt b to cyt c1 so cytb gets way reduced and cyt c gets really oxidized and you can see this with a spectrophotometer looking at absorbance

Front 42

what does cyanide and carbon monoxide block?

Back 42

transfer of e- from cyt a to oxygen.

Front 43

how can the 4 multienzyme complexes wi the inner mit membrane be isolated/

Back 43

fist use digitonin, a mild detergent, to strip away the outer mit membrane

then put the mit. in water and it will lyse breaking apart the inner mit mem. into fragments (osmotic rupture)

Take a fragment and use a stron nonionic detergent to seperate the four multienzyme complexes then seperate with chromatography to see how they work individually

Front 44

4 enzyme protin complexes of the mitochondrial electron transfer chain

Back 44

I NADH dehydrogenase

II Succinate dehydrogenase

III ubiquinone cytochrome c
oxidoreductase and cyt c

IV cytochrom Oxidase

all are found in the inner mit. membrane

Front 45

6 electron carriers

Back 45

NADH
FADH2
Heme
FeS
Cu
Q


*remember that metals aid in carrying electrons!

Front 46

4 ways to get electrons to Q which is in the center of the inner mit. membrane

Back 46

I. NADH from the matrix gives its e- to FMN and FeS which then give the e- to Q in the inner mit membrane space. Q can then diffuse into intermembrane space in mit. and go anywhere

II. membrane bound succinate dehydrogenase from matrix side makes FADH2. e- then go to FeS then to Q in inner mit membrane

III membrane bound acyl Co A dehydrogenase from beta oxidatatioin on inner mit membrane (matrix side) passes e- and goes through 3 subunits then to Q

IV. weird only one from cytoplasmic side. glycerol 3 phosphat dehydrogenase gives electrons to FAD then goes to Q

Front 47

Describe how complex I transports e- and protons across membrane

Back 47

NADH from matrix gives 2 e- to FMN then goes to FeS then to Q.

Also protons from the matrix leave and diffuse across inner mit membrane and into intermembrane space (cytoplasm)

For every 2e- that go through complex I, 4 protons are transported to intermembrane space to conserve energy

NADH + 5H+n + Q
-> NAD+ + QH2 + 4 H+p

so the H from NADH is added to Q and a H from the matrix (n) is added to Q to make QH2 and the other 4 H+ enter intermembrane space

Front 48

Describe complex II

Back 48

Succinate binds to substate binding site on complex II and want to go to QH2 (made from complex I) but cant bc it is so far away so it uses 5 steps each with a different Fe carrier at each step. Each step is less than 10 A apart from the other so the e- moves easily.

A heme b is also in complex II to grap any electrons that accidently leave the path.

Front 49

Describe complex III

Back 49

Transfers one e- from QH2 to Cyt C and the other e- to semiquinone (Q-) to make another QH2

complex III is where all tributaries converge to give up 2 e-

QH2 + cyt c(oxidized) + 2 H+n
-> Q + 2 cytc (reduced) + 4Hp

the 2 H+n comes from QH2

so 4H+ enter the cytplasm

Front 50

How many electrons can each eytochrome carry?

Back 50

ONLY ONE e-

Front 51

Describe complex IV

Back 51

4 cyt C with 4 e- (1 e- per cyt c) comes in via Cu centers. An oxygens is captured by a heme group and e- is put on oxygen. protons collected from matrix add to oxygen to make water. 4 H+ are pumped into the cytoplasm per 2NADH or 1 Oxygen

4 Cytc (red)+ 8H+n + O2
->4 cytc(ox) + 4H+(p) + 2H20

4 of the H+ from matrix go to cytoplasm and the other four are used to make 2 waters

Front 52

How many e- does each NADH give?

Back 52

enough to make only 1/2 of an oxygen and to pump 2 H+ to cytplasm. So you need tow NADH to make 1 Oxgen and 4 H+ to be pumped

Front 53

summary of e- and proton transport

Back 53

NADH give e- to I and 4 H+ transferred to cytoplasm. succinate gives e- to to all the e- goes to Q which is a lipid carrier lipid soluable complex that picks up the e- and gives them ti complex III and here 4 H+ are transferred to cytoplam. Complex III gives e- to Cyt C and in complex IV Cyt C gives e- to water and 2 H+ go to cytplasm

Front 54

final product of ETC

Back 54

water

Front 55

Purpose of proton motive force

Back 55

generate ATP

Front 56

2 parts of proton motive force

Back 56

1. chemical potential energy H+ gradient

2. electrical potential enrgy (charge seperation)

basically the G is a ratio of concentraion of solute across membrane and electric field in Volts

Front 57

what is the gradient an example of ?

Back 57

potential energy

Front 58

how is energy from oxidation recovered?

Back 58

90% of energy recovered in proton gradient so it is very efficient

Front 59

How is proton gradient used?

Back 59

to do work to put ADP + P to make ATP. It involves a mechanical method. Protons come into F0 which is transmembrane and causes F1 to turn making mechanical motion

Front 60

Describe chemiosmotic model

Back 60

2 parts
1. electron transport chain
2. ATP synthase brings back the protons

cannot make ATP without pumping protons this is known as coupling

Front 61

when using drugs to look at coupling of proton pumping and ATP synthesis, what happens when succinate, pyruvate, or CoA is addded?

Back 61

they are e- donors so increase oxygen consumption, proton pumping and ATP synthisis bc they are all coupled

Front 62

when using drugs to look at coupling of proton pumping and ATP synthesis, what happens when CN- is addded?

Back 62

inhibits the ETC so there is no proton gradient and therefore no ATP is made

Front 63

when using drugs to look at coupling of proton pumping and ATP synthesis, what happens when oligomyacin is addded?

Back 63

oligomycin inhibits ATP synthase so stops the ETC too but takes a second.

Front 64

when using drugs to look at coupling of proton pumping and ATP synthesis, what happens when the ionophore DNP is addded?

Back 64

DNP is soluble in the membrane. It carriers protons and breaks down the proton gradient. When DNP is added it blocks ATP synthesis but keeps consuming oxygen and carrying e- so it basically uncouples the two processes

Front 65

Rxn coordinat for ATP synthase. How is it unique?

Back 65

the ATP synthase allows ATP synthsis to have lower enegy that ADP + P. when the enzyme is bound to ATP it has lower energy that regular ADP + P but energy is needed to uncouple the ATP synthase and ATP bc they are v. tightly bound. The ATP wo the enzyme is hi energy

Front 66

2 parts of ATP synthase

Back 66

1. embeded in membrane (F0)
2. stalk that sticks out of membrane (F1)

Front 67

describe F1 of ATP synthase

Back 67

3 identical alpha/beta subunits. 1 binds ATP, 1 binds ADP, and 1 is empty

basically 3 dimers all in a different state

also has gamma chain which is off center and rotates and bumps the dimers

Front 68

describe F0 and how F1 and F0 fit together.

Back 68

F0 is inside the membrane. It is composed of alpha helices lined up longitudinally. the F1 gamma subunit sits on the F0

Front 69

Why is it called F0

Back 69

for F oligomycin

Front 70

How does F0 and F1 work together as ATP synthase?

Back 70

downhill movement of H+ throgh the F0 pore drives the rotation of F0 and the attached gamma from F1. the rest of F1 (alpha and beta subunits) are stationary bc anchored to b2 stator. The off center gamma subunit touches the alpha/beta subunits from the inside and induces the release of the bound ATP

Front 71

how many ATP are made for each turn of ATP synthase?

Back 71

3 ATP are made per turn

Front 72

3 purposes of proton motive force

Back 72

1. make ATP
2. get ATP into mit matrix
3. get phosphate into matrix

Front 73

how does ATP get from the matrix to the intermembrane space?

Back 73

by carrier called adenine nucleotide translocase. It is an antiporter bc it exchanges one ADP into matrix for one ATP out of matrix and into intermembrane space. It is driven by transmembrane electrochemical gradient

Front 74

How does phosphat get into the matrix?

Back 74

uses phosphate symporter as protons come down coc gradient phosphate comes with them going up their conc gradient.

driven by proton gradeint.

Front 75

How does NADH get across iner mit membrane and into the matrix?

Back 75

in intermembrane space NADH gives H to oxaloacetate to make malate, the malate has transporter to get to matrix. In matrix the malate is converted to oxaloacetate by giving proton to NAD to make NADH. This NADH can go to ETC. problem is the oxaloacetate could run out so need to convert it to the aa aspartate by aminiation. the aspartate then goes back through transporter to intermembrane space. the aspartate give it amino group to aKg to make glutamate and asp becomes oxaloacetate. the glutamate goes back to matrix.

Front 76

describe the glycerol 3 phospate shuttle

Back 76

used in skeleton and brain
makes only 1.5 ATP not 2.5 bc uses FADH2 instead of NADH

glycerol derivitive dihydroxyacetone phosphate takes e- from NADH and to make glycerol 3 phosphate, the glycerol 3 phosphate gives e- to FAD to make FADH2 which gives e- to Q.

Front 77

how many ATP are made from one NADH? one FADH2?

Back 77

2.5 ATP made per NADH

1.5 ATP made per FADH2

Front 78

Amount of ATP made from complete oxidation of glucose

Back 78

30-32

most ATP made from ox phos
only 4 come from substrate level phosphorylation

Front 79

main ways to regulate oxidative phosphorylation

Back 79

acceptor control ration. need ADP in matrix

Mass action raton which is ATP/ADP is v. hi so always have ATP

Front 80

Protien 84aa inhibitor IF1

Back 80

since ATP synthase can go forward or backward it inhibits ATP synthase in ischemia w/ lo pH from pyruvalte or lactate build up

Front 81

Thermogenin

Back 81

like DNP. It uncouples ATP synthesis from oxidation so allows ETC to run without ATP syntheiss. THe energy of oxidation is lost as heat. This is really good for brown adipose tissue bc has lots of mitochondira with cytochromes so baby can get warm

Front 82

with product inhibiton what happens when there is alot of acetyl CoA?

Back 82

inhibit pyruvate dehydrogenase

Front 83

with allosteric regulation, what happens to PFK1 with hi Citrate?

Back 83

Citrate will diffuse back into the cytoplasm and inhibit PFK1 with is the regulatory step of glycolysis.