15. Biochemistry ETC - Lecture notes 15 PDF

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Electron Transport Chain ETC overview ● Oxidative phosphorylation captures the energy of high-energy electrons to synthesize ATP o Electrons flow from NADH and FADH2 to O2 occurs in the electron-transport chain or respiratory chain (Aerobic) ● ETC occurs in the MITOCHONDRIA ● Both the citric acid cycle and oxidative phosphorylation are called cellular respiration or simply respiration The mitochondria

The outer membrane has pores that make it permeable to small molecules and ions, but NOT to proteins. The convolutions of the inner membrane, called cristae, provide a very large surface area. The inner membrane of a single liver mitochondrion may have more than 10,000 sets of electron-transfer systems and ATP synthase molecules, distributed over the membrane surface. A typical animal cell has hundreds or thousands of mitochondria.

Mitochondria is composed of roughly 70% protein and 30% lipid Inner membrane is impermeable to most molecules Inner membrane is the site of electron transport and ATP synthesis. The citric acid cycle and fatty acid oxidation occur in the matrix

Chemiosmotic Theory ● Early Scientist thought ETC was a series of “high energy” phosphorylation reactions ● The chemiosmotic theory differs from this initial thought

o Transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions ● The transfer of electrons down an electron transport system through a series of redox reactions releases energy o This energy release allows the transport of H+ across a membrane o Accumulation of H+ forms an electrochemical gradient ▪ Creates a Positive (P) side in the intermembrane space, and a negative (N) side in the matrix ● Proton motive force (PMF) provides energy for ATP synthases to catalyze synthesis of ATP by H+ reentering down concentration gradient REDUCTION POTENTIAL E’0 (YOU KNOW THIS) ● Just as a chemical gradient forms due to proton removal there is a potential between the intermembrane space and the matrix. o Electrochemical gradient! ● Reduction potential is a measure of a molecule’s tendency to acquire electrons and become reduced. o A strong reducing agent readily donates electrons and has a negative E0’ o A strong oxidizing agent readily accepts electrons and has a positive E0’ ▪ Standard free-energy change is related to the change in reduction potential 0'

'0

'0

'0

∆ G =−nF ∆ E =− nF( Eacceptor−E Donor )

Where n is the number of electrons transferred and F is the Faraday constant 96485 J mol-1V

● Electrons can be transferred in 3 different types of reactions that occur during oxidative phosphorylation: 1) Direct transfer of electrons (reduction of Fe3+ to Fe2+) 2) Transfer as an H atom (H+ + e-) 3) Transfer as a hydride ion (H:-) which bears 2 e- (NADH) ● In biological electron transport, each redox reaction in the sequence is exergonic under standard conditions o The free energy released is used to pump protons, storing this energy as the electrochemical gradient +¿+H 2 O ∆ G o ' =−220 kJ /mol ¿ +¿ → NAD 1 NADH + O 2+ H ¿ 2 ELECTRON TRANSPORT

To summarize what we will soon go through, complex I and complex II will transfer electrons from NADH and FADH2 respectfully to coenzyme Q in a process known as the Q cycle these electrons will then be passed onto complex III (cytochrome b). Which will then be transferred to cytochrome c, and then to cytochrome a and a3 of complex IV and finally to oxygen. Electrons flow down the gradient from areas of lower (negative) reduction potentials to higher (positive) reduction potentials. Here are some other facts to know before looking at the process piece by piece FADH2 has a lower reduction potential compared to NADH, FADH 2 pumps fewer electrons which leads to less ATP produced. NADH feeds into system at complex I and FADH 2 ad complex (II) Cofactors in Electron transport ● Flavoproteins contain a very tightly, sometimes covalently bound flavin nucleotide, either FMN, or FAD. o Links 2e- and 1e- processes by accepting 1 electron to form the semiquinone or accepting 2 electrons to form FADH2 or FMNH2 and then transferring those 2 eo FMN and FAD are redox cofactors and their redox potentials, unlike NAD or NADP, are associated WITH their associated protein and not as free FMN or FAD. o FMN is utilized in complex I, while FAD is utilized in complex II. ● Iron-Sulfur Proteins (FeS, Fe2S2 Fe4S4)

o Found only in complexes I, II, and III o The Fe-S complexes are the simplest with a single Fe atom complexes to four Cys-SH groups to o More complex centers such as (Fe4S4) contains four Fe atoms complexed to four Cys-SH and four inorganic sulfates o Rieske iron-sulfur complex are different in that they contain His residues instead of Cys residues! o All iron-sulfur cluster participate in single electron transfers where the loss or gain of an e- is possible converting between Fe2+ and Fe3+

● Coenzyme Q also known as ubiquinone

o Lipid-soluble (hydrophobic) benzoquinone with a long isoprenoid side chain. o Rapidly diffuses within the inner mitochondrial membrane to shuttle protons and electrons (Q pool) o Ubiquinone can accept one electron to become the semiquinone radical (*QH) or two electrons to form ubiquinol (QH2) ▪ Just like flavoproteins can act at the junction between a two-electron donor and a one-electron acceptor. o Can carry both electrons and protons, creates central role in coupling electron flow to proton movement. ● Cytochromes are proteins with characteristic absorption of visible light, due to their iron containing heme prosthetic group

The hemes of a and b cytochromes are tightly, but not covalently, bound to their associated proteins; the hemes of c-type cytochromes are covalently attached through Cys residues. As with flavoproteins the reduction potentials depend on the interaction with its protein side chains. o Different cytochromes have different absorptions, and all are one electron carries with reduced form being Fe2+ and oxidized form being Fe3+ o Complex II/III contain Cytochrome b

o Complex III also contains Cytochrome c1 o Complex IV contains cytochromes a and a3 which we will see interact with copper. ● More cool facts about the cytochromes (COLORS!) o Cytochromes b, c, and c1 all contain the same heme found in hemoglobin and myoglobin ▪ Cytochrome b iron is complexed with protoporphyrin IX emits blue ▪ Cytochrome c and c1 iron is complexed with Heme c emits red ▪ Cytochrome a and a3 iron is complexed with Heme a emits green Complex I: NADH-Coenzyme Q reductase (AKA NADH dehydrogenase)

Here shows the process of the electron transfer within NADH dehydrogenase, where complex I catalyzes two simultaneous and obligately coupled processes: (1) the exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, and (2) the endergonic transfer of four protons from the matrix to the intermembrane space. +¿ + ¿ + QH 2+4 H¿P ¿ +¿+Q→ NAD ¿ NADH+5 H N The above overall reaction of the process shows that a total of four protons will be pumped to the intermembrane space

Complex II: Succinate Coenzyme Q Reductase (AKA Succinate dehydrogenase)

The reaction for complex II is utilized for substrates with a more positive redox potential than NADH such as succinate. The electrons will be passed on to Q through complex II instead of complex I. Electrons will be passed to FAD and one at a time through iron-sulfur clusters and eventually reduce coenzyme Q. Important to know that electrons are passed one at a time through the iron-sulfur centers and there is NO PROTON TRANSPORT Complex III: Cytochrome c Oxidoreductase (bc1)

Looks confusing! But actually quite simple, the purpose of complex III is to mediate electron transport from coenzyme Q to reduce two molecules of Cytochrome c, this additionally results in the vectorial transport of protons from the matrix to the intermembrane space. The functional unit of Complex III is a dimer. Each monomer consists of three proteins central to the action of the complex: cytochrome b, cytochrome c1, and the Rieske iron-sulfer protein.

Here is the general reaction scheme showing the transfer of electrons that undergo a process known as the Q cycle. Utilizing this picture and the one above it we can describe the process. The path of electrons through complex III is shown by blue arrows. The movement of various forms of ubiquinone is shown with black arrows. In the first stage (left), Q on the N side is reduced to the semiquinone radical, which moves back into position to accept another electron. In the second stage (right), the semiquinone radical is converted to QH2. Meanwhile, on the P side of the membrane, two molecules of QH2 are oxidized to Q, releasing two protons per Q molecule (four protons in all) into the intermembrane space. Each QH2 donates one electron (via Rieske Fe-S center) to cytochrome c1, and one electron (via cytochrome b) to a molecule of Q near the N side, reducing it in two steps to QH2. This reduction also consumes two protons per Q, which are taken up from the matrix (N side). Reduced cyt c1, passes electrons one at a time to cyt c1, which dissociates and carries electrons to complex IV. If I were to explain this to a third grader, a QH2 will donate 1 electron each to cytochrome b and Rieske Fe-S center as well as transferring protons to the intermembrane space. Then the cytochrome b will eventually transfer the electrons to a Q to reduce it to Q⋅ (semiquinone radical). While the electrons from Rieske Fe-S center will transfer electrons from cytochrome c1 and eventually to the water-soluble cytochrome C (THAT IS 1 CYCLE). Then again for the second cycle another QH2 will transfer the electrons the same way as before while donating two protons to the intermembrane space (P side). However, reduction of the semiquinone radical requires two protons from the matrix (N side) in order to produce Ubiquinol (QH2)! Overall, we can think of it 2 QH2 are needed to fully reduce a Q to QH2 as well as reduce two cytochrome c, while adding 4 protons to the intermembrane space and 2 protons are consumed from the matrix side. +¿ ¿ +¿ →Q+ 2cyt c (reduced)+ 4 H P Q H 2 +2 cyt c (oxidized)+ H N¿

Complex IV: Cytochrome c oxidase

Cytochrome oxidase transfers electrons from the reduced form of cytochrome c to O2, the final electron acceptor. It contains many subunits, however three subunit proteins (I, II, III) are

critical to electron flow. Electron transfer begins with reduced cytochrome c. Two molecules of cytochrome c will donate an electron to the binuclear center CuA. From here, electrons pass through heme a to the Fe-Cu center (heme a3 and CuB). Oxygen now binds to heme a3 and is reduced to its peroxy derivative (seen on right) by two electrons from the Fe-Cu center. Delivery of two more electrons from cytochrome c, for a total of four electrons, converts the peroxy to two molecules of water, with consumption of four “substrate” protons from the matrix. At the same time, two protons are pumped from the matrix for each pair of electrons passing through complex IV. The reduction of O2 to H2O requires 4 electrons or 2 pairs of electrons. + ¿+ H 2 0 1 ¿ +¿+ O2 → 2 cyt c(oxidized)+2 H P 2 2 cyt c (reduced)+ 4 H ¿N + ¿+2 H 2 0 + ¿ + O 2 → 4 cyt c (oxidized)+4 H ¿P 4 cyt c(reduced)+8 H N¿ Highly exergonic btw like -200 Gibbs. We will see that it is conserved though!

EXTRA STUFF for complex IV ● Reduction of O2 is through redox centers that carry only 1 electron at a time. o Sometimes a small fraction of oxygen intermediates escapes as reactive oxygen species (ROS) that can damage cellular components unless eliminated by defense mechanisms. ● How do I stop the stupid ROS??? o Our boi superoxide dismutase will turn ROS into H2O2, then glutathione peroxidase will then render H2O2 harmless. ▪ Glutathione reductase will regenerate the reduced form of glutathione peroxidase by using nicotinamide nucleotide transhydrogenase (mitochondria) or NADPH (cytosol) ATP synthesis and proton-motive force (basics) ● The energy released from the transfer of electrons is conserved (stored) in the proton gradient, this is termed proton-motive force which has two components

1) The chemical potential energy due to the difference in concentration of a chemical species (H+) in the two regions separated by the membrane 2) The electrical potential energy that results from the separation of charge when a proton moves across the membrane without a counterion. ● The proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motive force, which will power ATP synthesis!

According to the chemiosmotic model, the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane—the proton motive force—drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore in ATP synthase. Oxidation and phosphorylation are obligately coupled! ● Chemiosmotic coupling refers to the obligate connection between mitochondrial ATP synthesis and electron flow through the respiratory chain o Neither of the two processes can proceed without the other. ● Because substrate oxidation drives ATP synthesis, inhibitors of electron transfer block ATP synthesis. ● Conversely, inhibition of ATP synthesis blocks electron transfer in intact mitochondria! o We will explore specific inhibitors later, but we can still look at what occurs through uncoupling of the proton gradient. Uncoupling and the proton gradient ● As we explained above, a structurally intact membrane to maintain a membrane potential is essential for oxidative phosphorylation. ● The electron transport proteins must span the inner membrane o Asymmetrically oriented to allow proton transport in one direction.

o For example, complex IV binds to cytochrome c only in the intermembrane space side. ● Some chemical compounds cause uncoupling without physically disrupting mitochondrial structure. o These compounds will permit electron transport along the respiratory chain to O2 however no ATP will be synthesized o Ex include 2,4-dinitrophenol (DNP) and carbonylcyanide-ptrifluoromethoxyphenlhydrazone (FCCP) ▪ These uncouplers are weak acids with hydrophobic properties that permit them to diffuse readily across the mitochondrial membranes.

After entering the matrix in the protonated form, they (DNP) can release a proton, thus dissipating the proton gradient. Resonance stabilization delocalizes the charge on the anionic forms, making them sufficiently hydrophobic to diffuse back across the membrane, where they can pick up a proton and repeat the process. (We will return to this to look at the clinical manifestations)

Structure of ATP synthase contains two functional domains

● The two functional domains of ATP synthase are F1 (a peripheral membrane protein) and Fo (o denoting oligomycin-sensitive, is integral to the membrane) o The two F1 and Fo functional domains are connected by the γ (gamma) subunit ● Each enzyme has three active sites located on the three β (beta) subunits o Each β (beta) subunit is distinct in that each subunit interacts differently with the γ (gamma) subunit (as we will see through conformational changes) F1 subunit catalyzes the formation of ATP through three different conformations ● F1 is a hexamer that is arranged in three αβ dimers, these dimers can exist in three different conformations o Open conformation in which the β-subunit is empty and can either bind or release ▪ β-empty conformation o Loose conformation in which the β-subunit has ADP and Pi associated with it ▪ β-ADP conformation o Tight conformation in which the β-subunit catalyzes ATP formation and binds product ▪ β-ATP conformation

In this view from the N (matrix) side, the proton-motive force causes rotation of the central shaft —the γ subunit—which comes into contact with each αβ subunit pair in succession. This produces a cooperative conformational change in which the β-ATP (TIGHT) is converted to the β-empty (OPEN), and ATP dissociates; the β-ADP site (LOOSE) is converted to the β-ATP (TIGHT) conformation, which promotes condensation of bound ADP + Pi to form ATP; and the β-empty (OPEN) becomes the β-ADP (LOOSE), which loosely binds ADP + Pi entering from the solvent. This model requires that two of the three catalytic sites alternate in activity; ATP cannot be released until ADP + Pi are bound at the other. ● Recap the conformational changes in a single 120° rotation of the γ subunit o TIGHT goes to OPEN o LOOSE goes to TIGHT o OPEN goes to LOOSE ▪ Reversing this process will lead to ATP hydrolysis instead of synthesis

● Once the γ subunit goes through 360° rotation 3 ATP molecules would have been produced ● The αβ subunits do not rotate and will remain stationary

Evidence of rotation through an experiment where genetically altered α3β3γ subunits were engineered to contain a run of His residues that allowed for attachment to a glass slide that allowed the movement of the γ subunit to be visualized because of ATP hydrolysis. The hydrolysis of a single ATP powered the rotation of the γ subunit 120°. Fluorescence of the actin filament allowed researchers to visualize the rotation following addition of ATP and its hydrolysis by the αβ catalytic subunits, the actin molecule rotated, which proved that the γ subunit itself was rotating.

How does proton flow through the Fo complex produce rotary motion?

A proton enters from the intermembrane space into the cytoplasmic half-channel to neutralize the charge on an aspartate residue in a c subunit. With this charge neutralized, the c ring can rotate clockwise by one c subunit, moving an aspartic acid residue out of the membrane into the matrix half-channel. This proton can move into the matrix, resetting the system to its initial state ● The c ring contains c subunits that have a critical Asp (or Glu) residue at about the middle of the membrane. o Protons cross the membrane through a path made up of both a and c subunits ▪ The a subunit has a proton half-channel leading from the P side to the middle of the membrane, where it ends near the Asp residue.

The proton-motive force energizes active transport

● The inner mitochondrial membrane has many transporters or carriers to enable the exchange of ions or charged molecules between the mitochondria and cytoplasm o Includes guys like adenine nucleotide translocase, and phosphate translocase ● Adenine nucleotide translocase (ATP-ADP translocase) enables the exchange of cytoplasmic ADP for mitochondrial ATP. o ADP must enter the mitochondria for ATP to leave (ATIPORT!) o The translocase is powered by the proton-motive force ● The phosphate translocase, promotes SYMPORT of one H2PO4- and one H+ into the matrix. o This transport process is favored by the transmembrane proton gradient. o Essential for oxidative phosphorylation (provides phosphates) ● ATP synthasome is the term to describe a complex of ATP synthase with ATP/ADP and phosphate translocase

Malate-Aspartate Shuttle

This shuttle for transporting reducing equivalents from cytosolic NADH into mitochondrial matrix is used in liver, kidney, and heart. In step (1) NADH in the cytosol enters the intermembrane space through openings in the outer membrane, then passes two reducing equivalents to oxaloacetate, producing malate. Then (2) malate crosses the inner membrane via the malate transporter. (3) In the matrix, malate passes two reducing equivalents to NAD+ and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol. (4) Oxaloacetate is first transaminated to aspartate, and (5) aspartate can leave via the gluatmate0aspartate transporter (6) Oxaloacetate is regenerated in the cytosol, completing the cycle, and glutamate produced in the same reaction enters the matrix via the glutamate0aspartate shuttle Glycerol Phosphate Shuttle

This alternative m...