BCHE 5190 Dr. Goodwin Exam 3 Review

what dilemma is dealt with in respiratory electron transport (RET) and oxidative phosphorylation?

there is a cytosolic NADH dilemma

what are the issues of the cytosolic NADH dilemma?

• NADH cannot cross the mitochondrial inner membrane

• NADH can only access complex I from the matrix-side of the inner membrane

what can eukaryotes do to deal with this cytosolic NADH dilemma?

they have two mechanisms

1. FAD-dependent glycerol-3-phosphate shuttle

2. FAD-dependent malate-aspartate shuttle

glycerol-3-phosphate

glycerol-3-phosphate dehydrogenase

G3P

G3P DH

dihydroxyacetone phosphate

DHAP

glycerol-3-phosphate shuttle

the use of cytosolic G3P DH to convert DHAP to G3P, using NADH and H+ and turning it into NAD+

G3P is converted back to DHAP by inner-membrane bound G3P DH, using FAD and turning it into FADH2. also using UQ to turn FADH2 to FAD and converting UQ to UQH2

are there any H+ translocated at the first step of DHAP conversion to G3P using membrane-bound G3P DH?

there are NO H+ being translocated, BUT 2e- are introduced to the RET

after the conversion of UQ to UQH2, what complexes are used and how many H+ and/or ATP are used?

Complex III 6 H+

Complex IV 1.5 ATP

ubiquinone

ubiquinol

UQ

UQH2

what gives NADH in the cytosol?

glycolysis

what must be available for the two FAD-dependent shuttles of the cytosolic NADH dilemma?

oxygen

draw the G3P shuttle

malate-aspartate shuttle

ims/cytosol: aspartate is converted into oxaloacetate using alpha-ketoglutarate and turning it into Glutamate. NADH is then turned into NAD+, converting oxaloacetate into malate

there are shuttles in the inner membrane which allow the aspartate, alpha-ketoglutarate, and glutamate to travel from the ims/cytosol to the matrix.

matrix: malate from ims/cytosol is converted back to oxaloacetate by NAD+ to NADH and H+. then oxaloacetate is converted to asparatate by Glutamate to alpha-ketoglutamarate

purpose of the malate-aspartate shuttle?

to transport the e- from the cytosol to the matrix so they can be used for oxidative phosphorylation. NADH is impermeable to the inner membrane

what can be expected for the amount of NADH from the cytosol and in the matrix, from the malate-aspartate shuttle?

there is an equal amount in each

what can be expected for the ATP yield from the malate-aspartate shuttle?

no loss of ATP, yielding 2.5 ATP!

the malate-aspartate shuttle more susceptible to what?

equilibrium pressure

what is the P/O ratio of NADH? (P/O = ATP per NADH oxidized)

2.5

which complex is NOT involved in succinate oxidation?

Complex I

The P/O ratio for NADH generated in the cytosol is …

1.5 or 2.5, depending on how NADH into the cytosol processing happens

what element would be a problem for getting NADH in the cytosol via glycolysis?

cyanide

draw the malate-aspartate shuttle

draw the overall reaction of pyruvate carboxylase

biotin

a cofactor required by pyruvate carboxylase and enzymes that catalyzes other similar reactions. it is covalently bound to the enzymes that use it by way of a lysine residue

how many parts does pyruvate carboxylase occur in?

two parts

draw the pyruvate carboxylase mechanism

pyruvate carboxylase

a homotetramer, each subunit composed of four domains

• Biotin Carboxylase (BC) Domain (1)

• Biotin Carboxy Carrier Domain (1)

• Carboxytransferase (CT) Domain (1)

• PC Tetramerization Domain (1)

• Biotin Carboxyl Carrier Domain (2)

• Biotin Carboxylase (BC) Domain (2)

• Carboxytransferase (CT) Domain (2)

• PC Tetramization Domain (2)

biotin carboxyl carrier domain

transfers between the active site of the BC domain of the subunit 1 and the CT domain of subunit 2

fishing with biotin

transferring intermediates from one active site to another, the biotin carboxy carrier domain parks here and transfers over to biotin carboxyl group

what are the cofactors of RET?

NADH

flavins, FAD/FADH2, FMN/FMNH2

iron-sulfur clusters

ubiquinone (Coenzyme Q)

hemes

copper

NADH cofactor of RET

• only appears at the first step of RET, i.e., when the e- transfer to Complex I

• HYDRIDE-ONLY DONOR (2e- transfer only H:-)

flavins, FAD/FADHS, FMN/FMNH2 cofactor of RET

• serve as adaptors between H:- ONLY DONORS and 1 e- ONLY ACCEPTORS

• ACCEPTS H:- for direct and complete reduction

• DONATES e- one-at-a-time to one e- only acceptors

FAD/FADHS, FMN/FMNH2 direct and complete reduction

H:- + H+ + FMN → FMNH2

H:- + H+ + FAD → FADH2

where does the direct and complete reduction of FMN and FAD occur?

• NADH (complex I)

• succinate (succinate DH/complex II)

• G3P, membrane-bound G3P DH

• fatty acyl-CoAs

draw the oxidation of FADH2, FMNH2

• a relatively stable semiquinone radical allows for one-at-a-time e- transfer

• flavins frequently donate to Fe/S cluster

iron-sulfur clusters (Fe/S)

• one e- only acceptors/donors

• DO NOT typically accept or donate e-

• although have multiple irons per cluster, only one e- donated at a time

• typically bound to proteins via Cys residues, with a special exception to Rieske-type Fe/S cluster

example of multiple irons per cluster

2Fe-2S, 4Fe-4S cluster

4Fe/4S cluster

3 Fe^III / 1 Fe^II → oxidized

2 Fe^II / 2 Fe^II → reduced

ubiquinone cofactor of RET

• highly hydrophobic, not associated with any one complex or protein

• freely mobile within the mitochondrial inner membrane

• acts as electron carrier between complexes

• capable of one e- and two e- transfers, BUT in RET always one e- transfer

• shuttles between one e- only donors and one e- acceptor

  • Fe/S clusters donate; Fe/S clusters or hemes accept

• operates in a “Q cycle”

Q Cycle

• as UQ is reduced by Fe/S clusters (e.g. Complex I), H+ must be delivered from somewhere, the matrix

• as UQH2 is oxidized, H+ must be off-loaded somewhere, the ims

• as UQ/UQH2 redox cycles, there is a net transfer of H+ across the inner membrane (matrix→ims)

draw the Q cycle conceptually

hemes cofactor of RET

• prosthetic group for “cytochromes”

• Fe-containing prosthetic group → cycles between ferric (Fe^III) and ferrous (Fe^II) oxidation states

• ONE e- DONORS/ACCEPTORS

• DO NOT DONATE/ACCEPT H+

• structure consistent w/ e- transfer properties

• Cytochrome C → Heme C

• Cyt a₃

cytochrome C

• heme C

• “hexacoordinate”

• precludes all binding of other “exogenous” ligands (e.g., O2)

• typical arrangement for most hemes in RET

hexacoordinate

all six sites for ligand association are occupied will accomodate e- transfers Fe³⁺/Fe²⁺

Cyt a3

• binds to its heme differently

• “pentacoordinate”

• open sites allows for direct binding of O₂

• open coordination site means that other ligands can also bind (e.g. CN^-, N3^-, CO)

“heme poisons”

molecules that can lock onto open coordination sites and refuse to let go on the cyt a3 heme

copper cofactor of RET

• found only in complex IV

• Cu_A and Cu_B

Cu_A

one e- transfer only

Cu_B

assists cytochrome a3 in O2 binding and reduction

molecule? circled?

nicotinamide adenine dinucleotide (NADH) (reduced)

where the hydride transfer occurs

molecule? circled?

flavin adenine dinucleotide (FAD)

the business end

molecule? circled?

flavin mononucleotide (FMN)

business end

molecule? circled?

ubiquinone (oxidized form)

business end

molecule? circled?

CuA center

business end

molecule? circled?

CuB center

business end

molecule? circled?

Rieske-type [2Fe-2S] cluster

molecule? circled?

[2Fe-2S] cluster

business end

molecule? circled?

[4Fe-4S] cluster

business end

what component does each flavoprotein have in common? ex: Complex I (NADH, coenzyme Q oxidoreductase), Complex II (succinate, coenzyme Q oxidoreductase), fatty acyl-CoA DH, G3P,

Flavins (FMN, FAD) and Fe/S clusters

what cofactor/s are ONLY hydride donors?

NADH

flavins, FAD/FADH2, FMN/FMNH2 (adaptors)

what cofactor/s are ONLY hydride acceptors?

NAD+

FAD/FMN

accepts H:- from H:- only donors to make FADH2/FMNH2

FADH2/FMNH2

transfers one e- (and H+) to a Fe/S cluster to make FADH^./FMNH^.

FADH^. / FMNH^.

transfers one e- (and H+) to Fe/S cluster to convert into FAD/FMN

OR

transfers one e- is donated by Fe/S cluster and pairs with FADH./FMNH. and it would pick up a free electron to become FADH2/FMNH2

UQ (CoQ) to UQH. (semiquinone radical/CoQH.)

one e- from a Fe/S cluster and one H+ from the matrix

UQ is fully oxidized

UQH. (CoQH.) to UQH2

one e- from Fe/S cluster and one H+ from the matrix

UQH2 fully reduced

UQH2 to UQH.

one e- accepted by Rieske Fe/S cluster and one H+ to IMS

UQH. to UQ

one e- accepted by heme and one H+ to the IMS

heme a

• found in a-type cytochromes

• has an aldehyde and a hydrophobic tail

heme b

• aka Iron protoporphyrin IX

• found in b-type cytochromes

• no tail or covalently linked protein like hemes a and b

heme c

• found in c-type cytochromes

• has two covalently linked proteins by the Cys side chains

which heme is this?

heme a

which heme is this?

heme c

which heme is this?

heme b

why are proteins necessary for RET?

1. to translocate H+ across the membrane

2. so cofactors can be held in a close proximity to one another to faciliate e- transfer at sufficient rates

3. to reduce protential tuning

4. to prevent premature, incomplete, or uncontrolled reduction of O2

H+ translocation requires which kind of proteins?

transmembrane proteins

why must cofactors be held close together?

to faciliate sufficient transferance of e- at a sufficient rate

what is the typical Å (angstrom) separation between a donor and acceptor?

usually 14Å to produce a e- transfer rate of about ~2,000 per second

for every 1.7Å separation between donor and acceptor, how is the rate affected? what if the distance was 28Å?

the rate DECREASES by a factor of 10

at a distance of 28Å, the rate would be 1/day

how many e- transfers are needed from one end of the RET to the other?

about 30

reduction potential tuning

• creates a thermodynamically favorable process step-to-step

• “refers to the experimental or structural methods used to deliberately shift the standard reduction potential (E°) of a redox-active center to make it easier or harder to reduce or oxidize”

what tunes the reduction potential of cofactors?

the protein environment

what is an extreme example of a reduction potential tuning?

2Fe-2S (Cys) v.s. Rieske 2Fe-2S (His)

there is a 450 mV difference in potential

the regular clusteres come at the top while the Rieske come “much later”

what is the goal of the reduction of O₂?

to make it all the way to 2H2O without the issue of uncontrolled, premature, or incomplete reduction of O₂. complex IV makes sure this is possible.

draw the reduction of O₂ to H₂O₂

what is the enzyme that is considered the second line of defense to prevent the incomplete/uncrontrolled reduction of O₂?

the protein superoxide dismutase

2O₂^.- → H₂O₂ + O₂

what is considered the third line of defense to prevent the incomplete/uncrontrolled reduction of O₂?

“sacrificisl e- donors” Vitamin C and E

catalase

enzyme that converts H₂O₂ to H₂O and O₂

Complex II

• aka succinate oxidoreductase, formal biochemical name: succinate:ubiquinone oxidoreductase

• ~140 kD; 4 subunits

• contains: FAD, 3 Fe/S clusters, and 1 heme b

• there is no H+ translocation at this complex; however, e- are introduced at RET here

• inhibited by thenoyltrifluoroacetone carboxin

how does FAD contribute to Complex II

covalently bound to the enzyme, it is a prosthetic group

is heme b playing a part in e- transfer in Complex II?

no, it is not playing a part in the e- transfer sequence. but it could be a possible defense against O2^.- production

draw the catalytic cycle of succinate dehydrogenase/Complex II concerning RET?

what does thenoyltrifluoroacetone carboxin do?

it blocks UQ from binding and reducing

Complex III

• biochemical name: ubiquinol:cytochrome C oxidoreductase

• 500 kD; 2 sets of 11 subunit multimers

  • one set extends into the mitochondrial matrix

  • another set extends into the IMS

• each 11 multimer set contains a Riekse Fe/S cluster

• cytochrome C

• there are 4 H+ translocated across the inner membrane for each e- pair transferred

• inhibitted by: myxothiazol, antimycin A, stigmatellin

how many Å does Complex III extend into the mitochondrial matrix?

75Å, so it extends more into the matrix than it does the IMS

how many Å of Complex III extends into the IMS?

40Å

what cofactor binds to Complex III in the IMS portion?

cytochrome C

membrane-spanning region of Complex III

• each has 11-subunit multimer which contain two UQ/UQH2 binding sites

• Q_P and Q_N sites

Q_P

Q “positive,” on the IMS side of the membrane

PIMS