Ch 19 Oxidative Phosphorylation

Energy from what is used to synthesize ATP in animals?

Reduced fuels (Carbohydrates, lipids, and amino acids)

Where are electrons from reduced fuels transferred to?

Reduced coenzymes (NADH or FADH2)

In oxidative phosphorylation energy from what is used to synthesize ATP?

Energy from NADH and FADH2

Oxidation of Acetyl-CoA produces what products?

NADH and FADH2

In eukaryotes what is the ultimate electron acceptor?

Oxygen

What type of energy is used to phosphorylate ADP?

Oxidation energy

What does chemiosmotic theory explain?

The mechanism by which electron transport enables ATP production through creation of a transmembrane difference in proton concentration.

What four distinct compartments make up the mitochondrion?

1. Outer membrane

2. Intermembrane Space (IMS)

3. Inner Membrane

4. Matrix

Is the outer membrane of the mitochondria porous?

Yes, allows passage of metabolites

What environment is the IMS of the mitochondria most like?

Similar environment to cytosol due to porous outer membrane. Slightly lower pH due to higher proton concentration.

Is the inner membrane of the mitochondria porous?

No, relatively impermeable, with a proton gradient across it

Where are the electron transport chain complexes found?

In the inner membrane of the mitochondria

What is the Cristae and what purpose does it serve?

Cristae are folds of the inner membrane of the mitochondria which serve to increase the surface area

Where does the CAC occur and parts of lipids and amino acid metabolism occur?

The matrix of the mitochondria

Does the matrix of the mitochondria have a higher or lower proton concentration than the IMS?

Lower proton concentration (Higher pH)

Is the reaction ADP + Pi → ATP thermodynamically favorable?

No, highly thermodynamically unfavorable

How is energy provided to phosphorylate ADP?

Flow of protons down the electrochemical gradient

How is energy released by electron transport used?

Used to transport protons against the electrochemical gradient

Chemiosmotic energy couple requires what?

Membranes

What type of membrane is needed for ATP synthesis? What type in bacteria? What type in eukaryotes?

ATP synthesis requires a membrane that is impermeable to ions.

Plasma membrane in bacteria

Inner membrane of mitochondria in eukaryotes

Membrane must contain proteins that couple the (uphill/downhill) flow of electrons in the electron-transfer chain with the (uphill/downhill) flow of protons across the membrane?

Downhill flow of electrons coupled to the uphill flow of protons.

Membrane must also contain a protein that couple the downhill flow of protons to what reaction?

Phosphorylation of ATP

What are the three major steps required for cellular respiration occurring in the oxidative phosphorylation step?

1. Generation of high transfer potential e-

2. Flow of these e- through R-chain

3. Synthesis of ATP

What terms define the two sets of redox pairs in a redox reaction?

Reductant / Oxidized Reductant

Oxidant / Reduced Oxidant

What type of energy is utilized in the formation of H2O?

Oxidation energy

What determines the direction of the flow of electrons

The atoms that are the stronger oxidants because they have a higher affinity for the electrons

Is Fe+3 or Cu+1 the stronger oxidant? Which has a stronger affinity for its electrons?

Fe+3

Does a stronger or weaker oxidant have a higher reduction potential? (Greater tendency to gain electrons)

Stronger oxidant

Does a stronger or weaker oxidant have a lower reduction potential? (Greater tendency to lose electrons)

Weaker oxidant

What is reduction potential? (E degree)

Affinity between an electron-donor and its electrons

What force is used to describe the tendency of electrons to flow towards the stronger oxidants?

Electromotive Force

The reduction potential of any redox pair is relative and is measured against what?

A “reference redox pair” (H2 → 2H+ + 2e-)

What unit is reduction potential measured in?

Volts

In order for a reaction to be spontaneous the free energy change is negative. In this type of reaction is the reduction potential positive or negative?

Positive E

What formula is used to express how much energy is generated during a redox reaction?

Standard delta G = -nF(standard reduction potential change - Delta E)

n = number of electrons being transferred

F = faraday’s constant (96.5kJ/molV)

In biological electron transport what is the reductant?

NADH

How is reduction potential calculated?

Delta E = E(oxidant-e acceptor) - E(reductant-e donor)

When NADH is oxidized how much free energy is made available for ATP synthesis?

-220 kJ/mol

How are electrons transferred?

Electron pass through a series of membrane-bound carriers.

How do the protein complexes function in an addition to their purpose for transporting electrons? What purpose does this serve?

These complexes act as proton pumps which generate the proton gradient

ETC complexes contain a series of e- carriers and multiple redox centers consisting of what three things?

1. Flavin Mononucleotide (FMN) or Flavin adenine dinucleotide (FAD)

2. Cytochromes a, b, or c

3. Iron-sulfur cluster

What molecules are the initial electron acceptors for complex 1 and 2? How can they carry two electrons?

FMN or FAD can carry two e- by transferring one at a time

What are the three ways in which e- transfer occurs in oxidative phosphorylation?

1. Direct transfer (Fe3+ → Fe2+)

2. Transfer of H atom (H+ + e-)

3. Hydride ions (:H-)

What is the key difference between NAD and FAD ability to carry e-?

NAD can only carry two e- and pick up/drop off two e- never one

FAD can pick up one or two e- and drop off one or two e-

NAD linked dehydrogenases remove how many H atoms? What happens to them?

2 H atoms. One is transferred as hydride ion (:H-) and a proton which is released in the media and is water soluble

Can any part of NADH cross the inner mitochondrial membrane?

Not the whole molecule but the electron they carry can

Do flavin containing carrier bind tightly with protein sometimes even covalently?

Yes

Do e- carriers have the same reduction potentials when bound to a protein versus free in solution?

No, different reduction potentials

Is the standard of reduction potential for FAD based on a flavo-protein or an isolated FAD/FMN?

Flavoprotein

How do flavin-containing carriers act as intermediates for e- transfer?

They act as intermediates because they can accept one or two electrons

What are cytochromes? How many are there? How do they differ generally speaking?

Cytochromes are one electron carrier consisting of a cyclic porphyrin structure with four nitrogen atoms coordinated about a central Fe ion (Fe2+ or Fe3+). There are 3 kinds a, b, and c. They differ by additions to their ring structures.

What is unique about the heme in cytochrome a? What is the name of this heme?

Heme A is found in cytochrome a. It has a long isoprenoid tail attached to one of the five-membered rings

What type of porphyrin ring is found in cytochrome b? Where is it also found?

Iron protoporphyrin IX which is also found in hemoglobin and myoglobin

What type of heme is found on cytochrome c? How is it bound?

Heme C is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues

What is the general purpose of cytochromes in the ETC?

Help from the e- wire (electron carriers)

What is another term for Coenzyme Q (Q)?

Ubiquinone

Why is ubiquinone helpful for transport of electrons?

It’s readily accepts electrons and it is a lipid soluble compound so it can freely diffuse through the mitochondrial inner membrane.

When ubiquinone accepts electrons what else does it pick up? And how many of each does it pick up? What is the fully reduced form called (QH2)? Where does it take electrons from and where does it drop them off?

CoQ also picks up H+. Takes 1 or 2 e- and equal amounts of H+. Ubiquinol is fully reduced QH2 form. Takes e- from complexes I and II to complex III.

What are iron-sulfur centers? How are they coordianated? What is the Fe to S ratio? Where are they most abundant?

Iron sulfur center are one e- carriers coordinated by 4 Cys residues in the protein complex. There a 1:1 ration of Fe to free S, not from Cys. They are abundant in complexes I and II.

What does the standard reduction potential of the iron in iron sulfur centers depend on?

Type of center (2Fe-2S vs 4Fe-4S) and its interaction with the associated protein

What is the name of complex I in the ETC? What prosthetic groups does it have?

NADH dehydrogenase or Ubiquinone oxioreductase. FMN and Fe-S.

What is the name of complex II in the ETC? What prosthetic groups does it have?

Succinate dehydrogenase. FAD and Fe-S.

What is the name of complex III in the ETC? What prosthetic groups does it have?

Ubiquinone-cytochrome c oxioreductase. Hemes and Fe-S.

What is the name of complex IV of the ETC? What prosthetic groups does it have?

Cytochrome oxidase. Hemes; CuA and CuB.

What are the three methods to determine the sequence of e- transfer?

1. Determine the reduction potential of the individual e- carriers (low→high)

2. By providing electron carriers and O2 then analyzing which carrier is oxidized first. Carrier closest to O2 will be oxidized first because O2 is the strongest oxidizer in the chain.

3. Use specific inhibitors that prevent transfer of electrons between specific carriers

At e- moves down the ETC how does the components affinity for e- change?

It increases

How do electrons flow relative to reduction potential?

Low to high

In vitro, does isolated ATP synthase still have full function?

No only ATP-hydrolyszing (ATPase), not ATP-synthesizing activity.

What type of compound is Digitonin?

Detergent

Which complexes in the ETC pump protons? How many do they each pump?

Complexes I, III, and IV pump protons. Complex I pumps 4 H+, Complex III pumps 4 H+, and Complex IV pumps 2 H+.

What is the intermembrane space also referred to when considering the ETC?

The P side. Positive side.

What is the matrix also referred to when considering the ETC?

The N side. Negative side.

What occurs in complex I of the ETC?

NADH bind to the NADH binding site in the matrix. A non covalently bound FMN accepts 2 e- from NADH. Several Fe-S centers pass one electron at a time toward the ubiquinone (Q) binding site. A series of amino acids undergo protonation and deprotonation to pump four protons per NADH from N side to P side. 2 H+ are picked up by Q.

NADH + Q + 5H+n → NAD+ + QH2 + 4H+p

What happens in complex II in the ETC?

FAD accepts 2 e- from succinate and e- are passed one at a time via three Fe-S centers which becomes reduced QH2.

How many Fe-S clusters are in complex I?

6

How many Fe-S clusters are in complex 2?

3

How many subunits make up complex 2 of the ETC? How many of the sub units are transmembrane and how many are cytoplasmic extensions? Are there any prosthetic groups? If so, what are they called? Where are they found and what is their purpose?

4 subunits (A, B, C, and D). 2 transmembrane and 2 cytoplasmic extensions. Heme b is sandwiched between subunits C and D. It serves to prevent formation of reactive oxygen species by e- that go astray.

What enzyme is involved both in ETC and CAC?

Succinate dehydrogenase

Which complex in the ETC is a dimer?

Ubiquinone-cytochrome c oxioreductase

What is the functional core of the monomers composing complex III?

Cytochrome b, 2 hemes, Rieske iron-sulfur protein.