Cellular Respiration & Energy Flow

Energy Flow in Cellular Respiration

• Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell
• Electrons from reduced fuels are transferred to reduced cofactors NADH or FADH2
• Oxidative phosphorylation uses the energy from NADH and FADH2 to make ATP

Chemiosmotic Theory

• Formation of ATP from ADP and Pi is thermodynamically unfavorable
• Energy to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient
• Electron transport releases energy to transport protons against the electrochemical gradient

Chemiosmotic Energy Coupling and Membranes

• A proton gradient is needed for ATP synthesis, established across a membrane impermeable to ions
• Plasma membranes in bacteria, inner membranes in mitochondria, and thylakoid membranes in chloroplasts
• Membranes contain proteins that couple the downhill flow of electrons in the electron-transfer chain with the uphill flow of protons
• Membranes contain a protein that couples the downhill flow of protons to the phosphorylation of ADP

Mitochondrial Membranes

• The outer membrane is relatively porous and allows passage of metabolites.
• The intermembrane space (IMS) has a similar environment to the cytosol, with a higher proton concentration (lower pH)
• The inner membrane is relatively impermeable, with a proton gradient and the electron transport chain complexes, convolutions called cristae increase the surface area
• The matrix is the location of the citric acid cycle and parts of lipid and amino acid metabolism and a lower proton concentration (higher pH)

Electron Transport Chain

• There is a series of electron carriers with multiple redox centers

Electron Transfer Order

• The order of transfer of electrons depends on reduction potential
• AG = -n F Δεo
• NADH + H+ +1/2 O2→ NAD+ + H2O
• ΔG = - 2×96500 × {0.817- (-0.32)} ≈ - 220 kJ/mol

ETC Co-factors

• Coenzyme Q, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), porphyrin, and iron-sulfur clusters are co-factors

NADH

• The TCA cycle and fatty acid β-oxidation provide electrons for NAD+ reduction
• Only two electrons can be taken up or released at a time

FMN and FAD

• Can be covalently bound to proteins
• Act to distribute electrons. Accept two electrons from carriers unstable with single electrons, then donate one electron at a time to acceptors that can only accept single electrons

Cytochromes

• Cytochromes are one-electron carriers
• They have iron-coordinating porphyrin ring derivatives
• Cytochromes a, b, and c differ by ring additions

Iron-Sulfur Clusters

• Iron-sulfur clusters are one-electron carriers
• Contain cysteines and equal numbers of iron and sulfur atoms

Coenzyme Q (Ubiquinone)

• Accepts electrons readily
• It is a lipid-soluble conjugated dicarbonyl compound
• Ubiquinone picks up two protons to give an alcohol, or ubiquinol Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side to the other
• Transfers electrons from Complexes I and II to Complex III

Protein components of mitochondrial respiratory chain

• Complex I (NADH dehydrogenase): 850 kDa, 45 (14) subunits, prosthetic groups FMN and Fe-S
• Complex II (Succinate dehydrogenase): 140 kDa, 4 subunits, prosthetic groups FAD and Fe-S
• Complex III (Ubiquinone: cytochrome c oxidoreductase): 250 kDa, 11 subunits, prosthetic groups Hemes and Fe-S
• Cytochrome c: 13 kDa, 1 subunit, prosthetic group Heme, moves between Complexes III and IV as a freely soluble protein
• Complex IV (Cytochrome oxidase): 204 kDa, 13 (3-4) subunits, prosthetic groups Hemes, CUA and CUB

NADH: Ubiquinone Oxidoreductase (Complex I)

• Is one of the largest macro-molecular assemblies in the mammalian cell
• It has over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes
• It's NADH binding site is on the matrix side
• A noncovalently bound flavin mononucleotide (FMN) accepts two electrons from NADH.
• Iron-sulfur centers pass one electron at a time toward the ubiquinone binding site

NADH: Ubiquinone Oxidoreductase as Proton Pump

• Two electrons from NADH are transferred to ubiquinone
• Protons are transferred from the matrix (N) to the intermembrane space (P)
• Four protons are transported per one NADH: NADH + Q + 5H+N = NAD+ + QH2 + 4 H+P
• Reduced coenzyme Q picks up two protons
• Protons are transported by proton wires (a series of amino acids)

Complex I and Redox State

• Electron flow changes the redox state of the protein complex, inducing conformational changes
• Long-range conformational changes alter the transmembrane helices. The oxidation/reduction cycle of coenzyme Q is linked to this process
• Quinone binding induces a conformational change needed for its reduction
• When QH2 is formed and released, all 4 H+ are pumped in one stroke from a single subunit

Succinate Dehydrogenase (Complex II)

• It does not transport protons
• Accepts two electrons from succinate
• Electrons are passed, one at a time, to ubiquinone
• It contains iron-sulfur centers to QH2.
• It's the same enzyme in TCA

Complex II and Electron Transfer

• Distance for the electron to travel is 40 Angstroms
• Fe-S cluster centers are less than 14 Angstroms apart
• 1 electron goes from FADH2 to Fe-S to coenzyme Q at a time
• 2 Hydrogens go to Q from water
• Role of heme is unknown, one hypothesis is it acts as an electron sink to prevent the formation of reactive oxygen species

Ubiquinone: Cytochrome c Oxidoreductase (Complex III)

• Complex III reduces two molecules of cytochrome c using two electrons from QH2
• It conttains iron-sulfur clusters, cytochrome b, and cytochrome c
• Four additional protons are translocated to the intermembrane space
• Translocation is facilitated by via the Q-cycle

The Q Cycle

• Four protons are transported across the membrane per two electrons which reach cytochrome c
• Two of the four protons come from the first incoming QH2
• Electrons travel on split pathways to the P and N side
• The Q cycle explains how two additional protons are picked up from the matrix
• One second molecule of QH₂ becomes oxidized
• Protons release into the IMS A molecule becomes reduced regenerating a QH2, picking up protons from the matrix side. Thus a net transfer of four protons passes per reduced coenzyme Q

Cytochrome c

• A soluble heme-containing protein in the intermembrane space
• Heme iron can be either ferrous (Fe3+, oxidized) or ferric (Fe2+, reduced)
• Second mobile electron carrier, (ubiquinone moves through the membrane)
• Cytochrome c carries a single electron from the cytochrome bc1 complex to cytochrome oxidase
• Ancient protein that is highly conserved

Cytochrome Oxidase (Complex IV)

• Mammalian cytochrome oxidase is a membrane protein with 13 subunits
• Contains two heme groups: a and a3
• Contains copper ions: Сид (two that accept electrons from cyt c) and CuB (bonded to heme a3, forming a binuclear center)

Cytochrome Oxidase (Complex IV) and Electrons

• Four electrons are used to reduce one oxygen molecule into two water molecules
• Four protons are picked up from the matrix in theis process
• An additional four protons are passed from the matrix to the intermembrane space.

Summary of Electron Flow

• For Complex I to Complex IV yields: 1NADH + 11H+(N) + ½O2 yields NAD+ + 10H+(P) + H2O

Summary of transport

• For Complex I to Complex IV: 1NADH + 11H+(N) + ½O2 yields NAD+ + 10H+(P) + H2O
• For Complex II to Complex IV: FADH2 + 6H+(N) + ½O2 yields FAD + 6H+(P) + H2O
• The difference in the number of protons transported reflects differences in ATP synthesized.

Respirasome

• Respirasomes are usually made of complexes I, III, and IV Complex II does not interact and moves freely in the membrane (TCA enzyme)

Chemiosmotic Model for ATP Synthesis

• Electron transport sets up a proton-motive force (PMF)
• A direct pump removes H from Q and forms water
• Energy of proton-motive force drives synthesis of ATP

Calculating G

• The pH of the matrix is 7.8 while is 7 in the intermembrane space
• The transmembrane potential drops by 0.15 V
• ΔG = 8.314 ×298 ×ln (1.6 ×10-8 / 10-7 ) + (+1)×96500 ×(-0.15) = -19000 J/mol = -19 kJ/mol

40 For 10 protons passing back into the matrix, -190 kJ/ mol is released. The oxidation of NADH results in 220 kJ/mol of free energy. The pumping of protons has captured the free energy.

ATP-coupled electron transport in mitochondria

• ADP and Pi alone are by themselves not enough to drive ATP synthesis
• CN- (cyanide) prevents electrons passing to O2 through binding to complex IV, therefore halting O₂ consumption and ATP synthesis
• Uncouplers (like DNP) halt ATP synthesis but do not affect O₂ consumption, by freely allowing protons to cross the membrane

Mitochondrial ATP Synthase Complex

• Contains two functional units: F1 and F0

1. F1 is a soluble complex in the matrix that individually catalyzes the hydrolysis of ATP
2. F0 is an integral membrane complex that transports protons from IMS to matrix, dissipating the proton gradient, therefore energy is transferred to F1 to catalyze phosphorylation of ADP

F1 - Synthesizing ATP

• A hexamer is arranged from three αβ dimers
• Dimers can exist in three different conformations:
  • Open: empty
  • Loose: binds ADP and Pi
  • Tight: catalyzes ATP formation, and binds product

Coupling Proton Translocation to Synthesis

• Proton translocation causes a rotation of the F0 subunit and the central shaft γ
• This causes a conformational change within all the three αβ pairs
• The conformational change promotes condensation of ADP and P₁ into ATP

Transport of ADP and P₁ into the Matrix

• Translocation of a fourth proton per ATP is required to facilitate cotransport of substrates into and products out of the mitochondria

ATP by Oxidation of Glucose

• In prokaryotes, organelles do not segregate machinery, so all electron carriers can directly feed into the electron-transport chain In eukaryotes, segregation prevents NADH from the cytosol from directly entering the electron-transport chain at Complex I
• NAD+ pools don't directly cross mitochondrial inner membrane
• Electrons from NADH from the cytosol are fed into the mitochondria with two methods: malate-aspartate shuttle and glycerol-3-phosphate shuttle

Regulation of Oxidative Phosphorylation

• Primarily regulated by substrate availability of NADH and ADP/Pi
• Controlled by inhibitor of F1 (IF1)
  • Prevents hydrolysis of ATP during low oxygen
  • Only active at lower pH, when electron transport stalls (i.e., low oxygen)

Inhibition of OxPhos is used for:

• Feedback loops of accumulation of NADH
• Feedback inhibition cascade up to PFK-1

Uncoupling Synthesis from ETC

• Uncoupling protein 1 (UCP-1) uncouples synthesis generate heat
• Seen hibernating animals and babies

Explanation of energy flow in cellular respiration. Covers reduced fuels, electron transfer to NADH/FADH2, and ATP production via oxidative phosphorylation. Also describes the chemiosmotic theory and the role of membranes.