Cellular Respiration and ATP Production

Cellular Respiration

• Series of catabolic reactions converting food molecule energy into ATP energy.

ATP Production

• Occurs through substrate-level phosphorylation and oxidative phosphorylation.

Substrate-Level Phosphorylation

• An organic molecule directly transfers a phosphate group (Pi) to ADP to make ATP.
• It produces only a small amount of the total ATP generated in cellular respiration.
• Takes place during glycolysis and the citric acid cycle.

Oxidative Phosphorylation

• Major ATP producer (88%) and occurs in stage 4 of cellular respiration.
• ATP is produced using energy from electrons transferred through the electron transport chain (ETC).
• Coupled with chemiosmosis.

Energy in Glucose

• Stored in chemical bonds, specifically C-H and C-C bonds.
• These bonds hold high-energy electrons released during cellular respiration.

Energy Release from Bond Conversion

• Converting C-C and C-H bonds into C-O and H-O bonds releases energy.
• C-O and H-O bonds are more stable with less energy than C-C and C-H bonds.
• The process involves oxidation, where the carbon atom loses electrons.
• Products such as CO2 and H2O become more stable than original reactants.
• The energy difference is released in the form of ATP.

Electron Transfer to Oxygen

• Electrons are not directly passed to O₂ to prevent too much energy release which could damage the cell.
• First, they are transferred to NADH and FADH₂, which carry them to the electron transport chain (ETC).

Redox Reactions

• Oxidation involves the loss of electrons (LEO).
• Reduction involves the gain of electrons (GER).
• Electron transfers always occur between atoms.
• Electron transfer happens spontaneously from less to more electronegative atoms.
• Oxidation and reduction are coupled reactions.
• Reduced forms have more chemical energy than oxidized forms.
• Nicotinamide adenine dinucleotide (NAD) and Flavin adenine dinucleotide (FAD) are two important electron carriers.

NAD+

• Most commonly used electron carrier in cellular respiration.
• NAD+/NADH picks up/drops off 2 electrons and is diffusible.
• FAD/FADH2 also carry pairs of electrons.

Oxidation States of Carbon

• Carbon's oxidation state depends on electron sharing, particularly with electronegative oxygen.
• Carbon loses electrons to oxygen during respiration, increasing its oxidation state from 0 (glucose) to +4 (CO₂).
• Gradual oxidation releases energy captured as ATP.
• Food molecules with C-C and C-H bonds have high potential energy because electrons are not tightly held by oxygen.
• Food molecules are excellent sources of energy transferred to oxygen during respiration.

Four Stages of Cellular Respiration

• Glycolysis, pyruvate oxidation, citric acid cycle, oxidative phosphorylation

Glycolysis

• Takes place in the cytoplasm.
• Glucose is broken down into pyruvate, ATP, and reduced electron carriers.
• Glucose is only partially oxidized and some energy held in its chemical bonds is released.
• Oxygen is not consumed, making it anaerobic.
• The net yield per mole of glucose is 2 pyruvate, 2 ATP, and 2 NADH.

Glycolysis steps

• Carbon backbone, phosphorylation, oxidation state

Carbon Backbone

• Glucose (6C) splits into two 3C molecules (G3P) and ends as pyruvate (3C).

Phosphorylation

• 2 ATP is used and 4 ATP are made, with a net gain of 2 ATP.

Oxidation State

• Step 6 is the only step involving oxidation (NADH formation).
• Pyruvate is partially reduced and can be further oxidized in the Krebs cycle.

Pyruvate Oxidation

• This occurs in the mitochondria and converts pyruvate (from glycolysis) in acetyl-CoA, which enters the Krebs cycle.
• Produces reduced electron carriers (NADH) and CO2 in the mitochondrial matrix.

Pyruvate Processing

• Each 3-carbon pyruvate undergoes decarboxylation, NADH formation, and coenzyme A attachment.
• Decarboxylation removes one carbon from pyruvate, forming CO₂ (waste product).
• Electrons lost during this process are donated to NAD+, reducing it to NADH.
• Coenzyme A (CoA) then binds to the remaining 2-carbon molecule, forming Acetyl-CoA.

Acetyl-CoA synthesis

• Products are 2 NADH, 2 CO2, and 2 Acetyl-CoA.

Citric Acid Cycle

• Completes glucose breakdown by oxidizing Acetyl-CoA into CO₂, and generates NADH, FADH₂, and ATP.
• Occurs in the mitochondrial matrix.
• The cycle begins with Acetyl-CoA and regenerates oxaloacetate.
• Chemical potential energy stored in acetyl-CoA is then moved into NADH and FADH2.
• All six carbons from initial glucose are oxidized to CO₂, which is released as waste, dismantling the glucose skeleton completely.

Products per Acetyl-CoA turn

• 3 NADH (electron carrier).
• 1 FADH₂ (electron carrier).
• 1 ATP (energy).
• 2 CO₂ (waste product).

Products at the End of the Citric Acid Cycle

• 2 ATP.
• 6 NADH.
• 2 FADH2.
• 4 CO2.

Oxidative Phosphorylation

• ATP is produced using energy from electron carriers through the electron transport chain in the inner mitochondrial membrane.
• Oxygen is required as the final electron acceptor and this is the final stage of cellular respiration.
• Electron transport chain, chemiosmosis

Electron Transport Chain

• Creates a proton gradient.

Chemiosmosis

• (ATP synthesis) uses the gradient to make ATP.

Electron Transport Chain (Details)

• Series of protein complexes in the inner mitochondrial membrane that takes electrons from NADH & FADH₂ to oxygen (O₂).
• ETC complexes increasing electronegativity, transfers electrons, and pumps protons with four large protein complexes (I to IV) in the inner mitochondrial membrane.

Electron Transport Chain (steps)

• NADH and FADH₂ donate electrons to the ETC.
• Electrons move through Complexes I-IV, releasing energy.
• Proton (H⁺) ions are pumped into the intermembrane space, creating a proton gradient.
• At Complex IV, electrons combine with O₂ and H⁺ to form water (H₂O).
• O₂ is the final electron acceptor, making this process aerobic.

Proton Movement

• Facilitated diffusion back into matrix via the ATP synthase drives formation of ATP from ADP + Pᵢ.

Proton Gradient

• Protons flow down concentration gradient and power ATP synthase, converting one energy form into energy in ATP bonds.
• It is a source of potential (stored) energy.

ATP Synthase

• An enzyme complex in the inner mitochondrial membrane.
• Uses energy from the proton gradient (H⁺) created by the electron transport chain (ETC) to synthesize ATP.
• Potential energy stored in the proton gradient is converted to mechanical energy (kinetic) as protons move down their concentration gradient through ATP synthase.
• Changes in conformation of the subunit in the mitochondrial matrix catalyze the reaction to converting ADP and Pᵢ to ATP.

Flow of Energy in Cellular Respiration

• Glucose transforms to electron carriers then to a proton gradient and ultimately to ATP.
• A single glucose molecule produces 32 ATP molecules.

Energy Observations

• To determine energy content, count C-H, C-O, and C=O bonds.
• The more C-H and C-C bonds, the higher the energy of the molecule.
• C=O bonds are not high-energy bonds.
• Energy content order, from highest to lowest: alcohol > carbonyl > carboxylic acid.

Cloning

• Cloning uses bacteria to make numerous copies of DNA.
• Molecular cloning involves copying a gene or DNA fragment, inserting it into a host organism (like bacteria) for research.
• Organismal cloning creates a genetically identical copy.

Plasmids

• Bacterial 'mini-chromosomes'.
• Small, circular pieces of double-stranded DNA in bacteria and eukaryotes.
• In laboratories, plasmids act as carriers, or vectors, to insert foreign DNA into bacteria.
• Bacteria that take up plasmids are transformed.
• Transformed bacteria express plasmid genes.
• The plasmid independently replicates and has its own origin of replication (Ori).
• Under the right situations plasmids are transferred between cells.
• Certain cells carry genes that provide special properties or selective advantages: antibiotic resistance or ability to metabolize a food source.

Inserting New Genes Into Plasmids

• Extra DNA is inserted into plasmids using restriction enzymes.
• Enzymes cut DNA at specific motifs to add a DNA piece.
• Bacteria have a defense system that uses restriction enzymes to cut up viral DNA before taking over the cell.
• A bacterium protects DNA by adding methyl groups on As and Cs.
• Restriction enzymes cut DNA in a zigzag, leaving unpaired nucleotides that makes it easy for segments to stick.

Cloning Steps

• The plasmid and target DNA are cut with restriction enzymes.
• The new gene is inserted and sealed with DNA ligase.
• Recombinant plasmids are introduced into bacteria via transformation.
• Transformed bacteria are selected using antibiotic resistance.
• Insertion confirmed with PCR or DNA sequencing.

Determining if Bacteria Have Been Transformed

• (Most common) Antibiotic selection is used with a selectable marker gene for ampicillin resistance.
• The plasmid carries a gene for antibiotic resistance (e.g., ampicillin resistance).
• Bacteria are grown on agar plates with the antibiotic.
• Only transformed survive because they have resistance, while non-transformed die.

Using Transformed Cells

• Extract the plasmid to sequence the cloned fragment.
• Extract/produce protein to analyze biochemically or use in biochemical/industrial applications.

DNA Cloning Summary

• DNA fragment of interest: to be cloned needs DNA ligase that repairs the cuts.
• Transformed cells can grow without ampicillin, but cells may lose the plasmid without selective pressure.

Lab Experiment: Transformation with pGLO

• Green fluorescent protein (GFP) derived from jellyfish makes transformed bacteria glow under UV light.
• Ampicillin resistance gene (bla) with the β-lactamase enzyme provides resistance to ampicillin

Regulatory system (araC)

• Controls GFP expression under right conditions.

Restriction Enzyme Digestion of Plasmid DNA.

• Enzymes recognize base sequences in DNA and cleave the sugar-phosphate backbone in or near the sequence.
• Functions in an environment found in the bacterium, with the correct pH, salt, and temperature.