Lecture 17 Review

Why does the NADH produced by aerobic glycolysis require a shuttle system in order to donate electrons to the electron transport system? Which two shuttle systems accomplish the transport of NADH/electrons into the mitochondria? Why is less ATP produced when the glycerol 3-phosphate shuttle is used?

A shuttle system is needed to accomplish the translocation of NADH from the cytoplasm to the mitochondria. the glycerol-3-P shuttle is predominant in the muscle cells. The reducing equivalents from cytoplasmic NADH are carried into the matrix by the glycerol-3-P shuttle. The end product of this process is FADH2 rather than NADH. the net cost of htis transport is that 1 ATP equivalent is lost during the product because FADH2 only yields 1.5 ATP in the oxidative phosphorylation system. Electrons from NADH are used to reduce dihydroxyacetone phosphoate to glycerol-3-P. the glycerol-3-P is reoxidized by electron transfer to an FAD prosthetic group of a dehydrogenase that's located in the inner mitochondrial membrane. Subsequently, the electrons are transferred to CoQ forming QH2 which enters the electron transport chain. The Malate-Aspartate shuttle is predominant in the heart and liver. It is mediated by two membrane transport systems and four enzymes. the major benefit of this elaborate process is that the electrons from cytoplasmic NADH end up in mitochondrial NADH, so there is no reducing power lost in this process. The process begins with the reduction of oxaloacetate to malate. The shuttle process involves two antiporters; one antiporter exchanges malate for alpha-ketoglutarate, and the other exchanges aspartate for glutamate. The overall reaction scheme involves the sequential formation of malate, oxaloacetate, a transamination reaction to form alpha-ketoglutarate and aspartate, and a second transamination reaction to form glutamate and oxaloacetate

What are the two components of the ATP synthase, and where are each of these components located?

ATP synthase is made up of an F0 component which is embedded in the inner mitochondrial membrane and an F1 component which is a peripheral protein assemby. The F0 component consists of two different subunits, and the F1 component contains five different subunits

Why does the ATP yield in oxidative phosphorylation differ depending on whether the initial electron donor is NADH or FADH2?

Because electrons that enter the electron transport system from NADH go through complex 1, which pumps protons and then goes through complexes 3 and 4 which also pump protons. Whereas, electrons from FADH2 pass through complex 2, which does not pump protons, and then goes on through complexes 3 and 4, which do pump protons. As a result there is less of a proton gradient produced from FADH2 and thus less ATP is produced

What are the four complexes involved in the electron transport system? Which three complexes function when NADH is the electron donor and which three function when FADH2 is the electron donor? What is the order of electron flow through these complexes?

Complex 1: NADH-Q-oxidoreductase Complex 2: Succinate-Q-reductase Complex 3: Q-cytochrome c-oxidoreductase Complex 4: cytochrome c-oxidase Complex 1, 3, and 4 function when NADH is the electron donor Complex 2, 3, and 4 function when FADH2 is the electron donor

How are sequences of cytochrome c used by evolutionary biologists?

Cytochrome c is a comparatively small protein with only 104 amino acids. That makes it relatively easy to purify. Of the 104 amino acids about 27 ar invariant in 60 widely divergent pieces of organisms. These amino acids are probably critical to cytochrome c function. Mutations causing changes in these amino acids would inactivate cytochrome c and so such mutations are lethal and not maintained in the gene pool. Another 14 are highly conserved, meaning that amino acid substitutions at those positions generally replace one amino acid with a similar amino acid, for example replacing Leucine with Isoleucine. The rest of the amino acids appear to be less critical in cytochrome c function, so a variety of changes have occurred in these amino acids. It's possible to construct an evolutionary tree based on the number of amino acid changes from one species to another

What are cytochromes? what is the nature and function of heme cofactors? What roles do cytochromes play in the electron transport system?

Cytochromes are proteins with a heme prosthetic group covalently bound via thioether bonds. Cytochromes (b, c, c1, a, a3) differ in: - protein structure - heme structure - absorption spectra - reduction potential - role in electron transport The soluble protein, cytochrome c shuttles electrons between the coenzyme Q cytochrome c reductase and cytochrome c oxidase complexes. Cytochromes b and c1 are components of cytochrome c reductase Cytochromes a and a3 are components of cytochrome c oxidase All contain heme prosthetic groups with a tetrapyrrole organic structure and a central iron ion

What amino acid is attached to the iron ion in some iron sulfur proteins? A) methionine B) serine C) alanine D) cysteine E) threonine

D) cysteine

The electron transport process has two major products, which are a proton gradient and: A) ATP B) NADH C) FADH2 D) Oxygen E) Water

E) Water

How does the electron transport create a proton/hydrogen ion gradient? Which mitochondrial compartment is depleted of protons and which is enriched?

Electron transport creates a proton gradient by transporting protons out of the mitochondrial matrix by the flow of electrons through various protein complexes (complex 1, 2, 3, and 4) within the mitochondrial membrane. The inner membrane is enriched with protons. The mitochondrial matrix is depleted of protons

What molecule is the ultimate electron acceptor for electron transport? What are the two products of the electron transport system?

Oxygen is the ultimate electron acceptor for electron transport The two products of the electron transport system are water and a proton gradient

What role does each of the following proteins, electron carriers, or metabolites play in oxidative phosphorylation? - NADH - FADH2 - FMN - Non-heme iron-sulfur complexes - Ubiquinone - Cytochromes b, c1, c, a, a3 - Oxygen - water - hydrogen ions

- NADH - electron donor - FADH2 - electron donor - FMN - first acceptor of electrons from NADH in complex 1 - Non-heme iron-sulfur complexes - iron atoms participate in oxidation reduction reactions - Ubiquinone - carries electrons between complex 1 and complex 3, complex 2 and complex 3, and within the cytochrome c reductase complex - Cytochrome c shuttles electrons between the coenzyme-Q-cytochrome c-reductase and cytochrome c- oxidase complexes - cytochrome b and c1 are components of cytochrome c reductase - cytochrome a and a3 are components of cytochrome c oxidase - oxygen - electron acceptor - water - product - hydrogen ions - their gradient drives ATP synthase to form ATP

What is ubiquinone/coenzyme Q and what are its various functions?

- lipid soluble cofactor - can accept 2 electrons and 2 protons - It's reduction occurs in a sequential manner. The addition of one electron and one proton produces semiquinone, a free radical with an unpaired electron. Semiquinone is a very reactive and unstable intermediate which has to be closely sequestered within the active sites of enzymes. The addition of a second electron and proton produces the stable reduced intermediate ubiquinol. - The coenzyme Q molecule carries electrons within the inner mitochondrial membrane between complex 1 and complex 3. It also functions to carry electrons between complex 2 and complex 3 and also within the cytochrome c reductase complex

Complex IV/cytochrome c oxidase accepts one electron from each of four molecules of cytochrome c and donates those electrons to one molecule of O2 to form two water molecules. What types of intermediates are involved in this process? What metal ions are involved, and how do they change their oxidation state?

All four electrons enter this reaction scheme provided by reduced cytochrome c. The end result of this series of rxns is the reduction of O2 to form two molecules of water. It takes two electrons to rescue each O atom to water, so four electrons are required. To begin the complex contains Fe3+ and Cu2+. The addition of one electron from cytochrome c reduced Cu2+ to Cu+. The addition of a second electron reduced Fe3+ to Fe2+. At this point, O2 binds to one of the ligand binding sites on the iron ion. Then an internal oxidation-reduction rxn takes place. Both Fe2+ and Cu+ lose an electron and the molecular oxygen is converted to a double anion. In the next step, one electron and two protons are added and molecular oxygen is split into two unconnected oxygen toms. One of the O atoms attached to Cu2+ gains two protons and two electrons to form water and the other O attached to Fe4+ ends up carrying a -2 charge. The addition of one electron and two protons produced a second water molecule and return the Fe to the +3 state. At this point the cycle is complete.

How does uncoupling electron transport from ATP production by the UCP-1 Protein allow for the production of extra heat? How does this protein dissipate a proton gradient? what is the different between an uncoupler and an inhibitor of electron transport?

An uncoupling protein (UCP-1) acts as a proton channel. That allows protons to enter the matrix of the mitochondria without passing throught eh ATP synthase. this mechanism dissipates the proton gradient and generates heat, but eliminates the synthesis of ATP. In addition to natural systems for uncoupling oxidative phosphorylation by dissipating the proton gradient, there are also natural and chemical substances that work directly to inhibit the flow of electrons through the electron transport system. there are a variety of such inhibitors that exhibit specific inhibition at each of the electron transport complexes. In the presence of these inhibitors, no NADH is oxidized.

When FADH2 is the electron donor, what is the best current estimate of the ATP yield in oxidative phosphorylation? A) 1 B) 1.5 C) 2 D) 2.5 E) 3

B) 1.5

Oxidative phosphorylation includes two component parts, the electron transport system and the ATP synthase. What are the functions of each of these systems, and where are these systems located within the mitochondria? What ion links the two systems?

Electron transport is a process in which the transport of protons out of the mitochondrial matrix is energized by the flow of electrons through various protein complexes within the mitochondrial inner membrane. Electron transport leads to the formation of a proton gradient with the high proton concentration in the inner membrane space and a low proton concentration in the mitochondrial matrix. The ATP synthase enzyme uses the proton gradient formed by the electron transport system to drive the synthesis of ATP. Protons flow downhill from the inner membrane space through the ATP synthase protein. The downhill flow of protons from an area of high concentration in the inner membrane space to an area of low concentration in the mitochondrial matrix through the ATP synthase energizes the synthesis of ATP from ADP and inorganic phosphate. The hydrogen ion links the two systems

What is the sad story of scientific equivocation about the ATP yield from oxidative phosphorylation? What is the current dogma concerning ATP yield from NADH and from FADH2? Why was this yield so ambiguous in the first place?

Electrons that enter the electron transport system from NADH yield 2.5 ATP. Electrons that enter the electron transport system from FADH2 yield 1.5 ATP. this is because complex 1 pumps protons, but complex 2 does not, so NADH yields a larger proton gradient than FADH2, promoting the production of ATP. Taking into account these estimates of ATP yield per NADH and FADH2, and the energy cost of moving reducing power from cytoplasm into the mitochondria, the complete oxidation of one glucose molecule should yield about 30 ATPs

What is FMN? How and where does FMN function in electron transport?

FMN (aka Flavin mononucleotide) is the first acceptor of electrons from NADH in complex 1. The reduction of FMN to FMNH2 occurs on the same isoalloxazine ring and has essentially the same chemistry as the conversion of FAD to FADH2. When the FMN cofactor is reduced to FMNH2, it accepts 2 electrons and 2 protons on the isoalloxazine ring. The protons occupy sites where there were paired electrons on two of the ring N atoms. There is a shift in the double bond pattern on two of the isoalloxazine rings when the additional electrons are added to the system

What is the function of each of the four complexes in the electron transport system? What donates electrons to each complex and what carries the electrons away? What electron carrier is water soluble? What electron carrier is a lipid soluble cofactor that shuttles electrons from one complex to another?

High potential electrons from NADH enter the system at complex 1. Electrons flow from NADH to coenzyme Q through complex 1. The flow of electrons through complex 1 is coupled to the pumping of 4 protons out of the matrix into the space between the inner and outer mitochondrial membrane. Electrons from FADH2, which have a lower potential than those from NADH flow to coenzyme Q through complex 2. This complex does not pump any protons. 2 electrons are carried through the mitochondrial membrane from complex 1 or complex 2 to complex 3 by reduced cocenzyme Q. This coenzyme is lipid soluble and always stays in the membrane. Two electrons flow form QH2 through complex 3 to the water soluble protein cytochrome c. The flow of electrons through complex 3 is coupled to the net transport of 4 protons into the space between the inner and outer mitochondrial membrane and the uptake of 2 protons from the mitochondrial matrix. Each molecule of reduced cytochrome c carries one electron from complex 3 to complex 4. 4 electrons from 4 molecules of cytochrome c flow through complex 4 to react with oxygen and form water as the final product of the respiratory chain. This process is coupled ot the uptake of four protons from the matrix, which react with molecular oxygen to form 2 water molecules and the transport of four additional protons from the matrix to the space between the inner and outer membrane

What is the Nernst Equation, and how is this equation used to chart the changes in reduction protential that occur as electrons pass through the electron transport system? How is the voltage change in a reaction related to the free energy change?

The Nernst equation is similar to the free energy equation in that it allows us to calculate the potential energy of chemical reaction. The Nernst equation is used for oxidation-reduction reactions, and allows us to calculate the potential energy of chemical reactions, and the energy values are expressed in volts, not Joules or calories. The reduction potential for a reaction is equal to the standard state reduction potential at pH 7 plus (RT/nF)ln(electron acceptor/electron donor) The standard free energy change is equal to -nF(deltaEo'), where (deltaEo') is the change in standard state reduction potential

Electron complex III accepts two electrons from one molecule of coenzyme Q and donates two electrons one at a time to two different molecules of cytochrome c. What mechanism is involved in the two step process of electron transfer by this system?

The QH2 produced in complex 1 diffuses through the hydrophobic membrane to complex 3. QH2 feeds 2 electrons into the complex, but cytochrome c only accepts one electron, as a result it takes 2 cycles of reduction involving 2 cytochrome c molecules to effectively convert coenzyme Q from the reduced to the oxidized form. In the first cycle, the reduced CoQ donated 2 electrons, 1 electron goes through the iron sulfur protein and a protein bound cytochrome c1 to cytochrome c. The other electrons goes to a protein bound coenzyme Q molecule to form a free radical, then, through two variants of cytochrome b and finally ends up in a second coenzyme q as a free radical with an unpaired electron. This free radical intermediate is extremely reactive and could damage the cell if it were set free from the surface of the enzyme. Fortunately, the enzyme complex holds the intermediate closely until further rxn occurs. In the next cycle, a second molecule of reduced CoQ donates two electrons. One electron follows the same pathway as in the first cycle, through a Fe-S protein and a protein bound cytochrome c1 to cytochrome c. the second electron follows the pathway used yb the second electron in the first cycle and it ends up converting the free radical back to reduced cytochrome Q. The result of this is that two molecules of reduced coQ donate four electrons. Two of those electrons produce two molecules of reduced cytochrome c and the other two electrons end up regenerating one molecule of reduced coenzyme Q, QH2

What roles do the a and c subunits play in the lipid soluble F0 complex? How does the protonation, deprotonation and rotation of the c ring allow protons to pass through the two half channels of the a subunit?

The c subunit consists of two alpha-helix structures with a negatively charged aspartate in the center. the a subunit consists of a cytoplasmic half channel and a matrix half channel. Each of the c subunits consists of two alpha-helices, between 10 and 14 of the c subunits form a membrane spanning ring and aspartic acid residue, in one of the helices, lies in the center of the membrane. These aspartate residues are protonated and deprotonated during the passage of protons around the ring. The a subunit appears to contain two half channels that allow protons to enter and pass part way but not completely through the membrane. Proton movement across the membrane drives rotation of the ring. A proton enters from the inner membrane space into into the cytoplasmic half channel of the a subunit to neutralize the charge on an aspartate residue in a c subunit. Once the charge is neutralized, the c ring can rotate clockwise by one c subunit. This moves another protonated aspartic acid residue out of the membrane and into contact with the matrix half channel. This proton can diffuse into the matrix resetting the system to its initial state. In summary, each proton enters the cytoplasmic half channel, follows a complete rotation of the c ring and exits through the matrix half channel

How is the rotation of the gamma subunit driven by the flow of protons through the system, and how does this rotation drive the synthesis of ATP?

The flux of protons through the F0 component drives the rotation of the c ring, which in turn drives the rotation of the gamma subunit of the F1 component, which in turn powers the synthesis of ATP from ADP and inorganic phosphate. This constitutes the phosphorylation part of oxidative phosphorylation.

What roles do the 3 α and 3 β subunits play in the water soluble F1 complex, and what is the function of the central γ subunit? Which subunit rotates?

The gamma subunit passes through the middle of the a3b3 hexamer which consists of alternative alpha and beta subunits. At any given time, each of the 3 beta subunits exists in a different nucleotide binding form designated O, L, and T. The alpha subunits bind ATP but don't participate in any catalytic or transport reaction. Proton driven ATP synthesis involves a binding change mechanism in which three sequential 120 degree rotations of the gamma subunit drives the beta subunits through the three different forms, T (tight), O, (open), and L (loose) The subunit in the T form converts ADP and inorganic phosphate to ATP but does not let ATP be released. When the gamma subunit is rotated by 120 degrees in a counter clockwise direction, the T form is converted into the O form allowing ATP release. Then ADP and inorganic phosphate can bind to the O form. An additional 120 degree rotation traps ADP and inorganic phosphate in the L form

What are iron sulfur proteins? What types of iron sulfur proteins function in electron transport, and how do these proteins differ from each other? What is the significance of differing reducing potentials?

There are three types of iron-sulfur clusters involved in the electron transport scheme, actually non-heme iron proteins containing these types of clusters are found in complexes 1, 2, and 3. The least complicated contains 1 iron and 4 sulfur atoms of 4 cysteine residues. The next most complicated contains 2 iron and 4 cysteine sulfur atoms and 4 inorganic sulfur atoms. The most complex is a cubic structure which contains 4 iron, 4 cysteine sulfur atoms, and 4 inorganic sulfurs The iron atom participates in oxidation-reduction reactions by accepting or donating electrons. The proximity of the S atoms influence the oxidation state of the iron ions and helps determine their energy levels

What are absorption spectra, and how can these spectra be used to study the function of various electron carriers?

There is a significant difference in the absorbance spectrum of NAD+ and NADH. Such alterations in the absorption spectra occur in various cofactors depending on their oxidation states. These changes are useful in following the progress of a biochemical reaction

Overall the drop in reduction potential from NADH to water is equivalent to the energy needed to synthesize six or seven molecules of ATP, yet only about 2.5 molecules of ATP are actually synthesized? How does this apparent thermodynamic inefficiency allow ATP synthesis to be achieved? What would happen if the system were 100% efficient?

To apparent thermodynamic inefficiency in the synthesis of ATP allows ATP synthesis to be achieved because there is more than enough drop in voltage in each complex (I, III, IV) to account for synthesis of one ATP. If the process were 100% efficient, there would be enough energy released by each complex to synthesize at least two molecules of ATP