Early bioenergetics:

1

• Early bioenergetics
• First life about 3.8 billion years ago.
• Earliest replicating system probably an RNA
  molecule.
• - RNA world located in rock pores around volcanic
  springs.
• Lived on redox contrast of more oxidised
  atmosphere/ocean more reduced fluids in contact
  with volcanic magma.
• Dissolved sulphate provide oxidation power for
  organisms to react against hydrothermal fluids,
  eg. hydrogen methane.
• ATP addiction probably emerged from the RNA
  world.
• Ribozymes (catalytic RNA) can polymerise RNA-NTP
  molecules (NTP nucleotide tri-phosphates).
• 14 nucleotide addition with 97 fidelity was
  demonstrated.
• Early metabolism of RNA world based on an NTP.
• Triphosphate bond releases 10 kcal/mol is
  stable (khydrolysis
• 10-10/min).

2

• Cellular respiration
• Glycolysis
• - Breaks down glucose to pyruvic acid.
• Three main pathways for pyruvic acid
• - Aerobic oxidation and citric acid cycle
• Breaks down pyruvic acid to produce NADH.
• - Anaerobic alcoholic fermentation (eg. yeast).
• Break down of pyruvic acid to produce ethanol.
• - Anaerobic homolactic fermentation.
• Break down of pyruvic acid to lactate.

3

• Glycolysis
• With a few exceptions, living things all
  metabolise glucose with
• identical pathways.
• - In animals glucose arises in the blood from the
  break down of of polysaccharides or
  noncarbohydrate sources.
• Glucose enters the cell's cytosol via a specific
  carrier.
• The enzymes of glycolysis are located in the
  cytosol.

4

• Basic overview of glycolysis
• Glycolysis converts glucose to two C3 units
  (pyruvate)
• Ten enzymes catalyse the reactions.
• First stage (5 reactions) in which glucose
  phosphorylated to yield two molecules of a
  triose glyceraldehyde 3-phosphate.
• Two molecules of ATP consumed.
• - Second stage (5 reactions) converts this triose
  to pyruvate.
• Four molecules of ATP produced.
• - Net result is to produce two ATP and two NADH.

5

• Recycling NAD
• NAD must be recycled (used as an oxidising
  agent in glycolysis)
• - Aerobic oxidation.
• NADH acts as an electron donor to the
  respiratory chain.
• - Homolactic fermentation.
• NADH used to reduce pyruvate to lactic acid.
• - Alcoholic fermentation.
• Pyruvate decarboxylated to produce
  acetaldehyde, and NADH reduces this to ethanol.

6

• Citric acid cycle
• First step
• Conversion of pyruvate to Acetyl-CoA
  (acetyl-coenzyme A).
• Produces CO2 and one NADH (from NAD).
• Consumes CoASH (the coenzyme)
• Citrate synthasis
• - Condenses Acetyl-CoA to yield citrate (gives
  the cycle its name).
• - Recovers CoASH.

7

• There are eight enzymatically catalysed steps in
  the cycle.
• The overall reaction is
• 3 NAD FAD GDP Pi acetly-CoA
• ? 3 NADH FADH2 GTP CoA 2 CO2
• Proposed by Krebs in 1937, often called the
  Kreb's cycle.

8

• Respiratory chains
• Accept electrons from an upstream donors.
• Catalyse the transfer of electrons to a terminal
  electron acceptor.
• Harvest the energy released to pump protons.
• - Proton gradient harvested by ATPsynthase.

9

• Mitochondria
• The workhorse'' of the cell.
• - Typically 0.7 to 1.0 mm long.
• - Outer membrane has porins allowing the free
  access of small particles, metabolites etc.
• Inner membrane an energy-transducing membrane.
• - About 500 mg/ml of this membrane is membrane
  proteins.

10

• Mitochondrial respiratory chain
• Complex I
• - Transfers e- from NADH to quinone pool pumps
  H.
• Complex II
• - Transfers e- from succinate to quinone pool.
• Complex III
• - Transfers e- from quinol to cyt. c pumps H.
• Complex IV
• - Accepts e- from cyt. c, reduces O2 to H2O
  pumps H.
• Complex V
• - Harvests H gradient regenerates ATP

11

• Simplified bacterial respiratory chain
• The E. coli respiratory chain is simpler than
  the mitochondrial.
• - Lacks Complex III and cytochrome c.
• - Terminal electron acceptor takes electrons from
  quinol directly.
• Contains analogues to Complex I and Complex II.
• Complex IV analogue is Quinol oxidase.
• - Accepts e- from quinol, reduces O2 to H2O
  pumps H.
• ATP-synthase

12

• Anaerobic bacterial respiratory chain
• P. denitrificans' respiratory chain may also
  donate electrons to nitrate (NO32-) or nitric
  oxide (NO).
• Nitrate reductase.
• Accepts electrons from quinol directly.
• Nitrate (NO32-) is reduced to nitrite (NO2-).
• Nitric oxide reductase.
• - Nitric oxide (NO) is reduced to nitrous oxide
  (N2O).

Two additional soluble proteins - Nitrite
reductase catalyses NO2- ? NO. - Nitrous oxide
reductase catalyses N2O ? N2. Process called
denitrification''.
13

• Complex I of the mitochondrial respiratory chain
  
• Glycolysis produces 2 molecules of NADH, the
  Krebs cycle another six, for each molecule of
  glucose consumed.
• Complex I (NADH-UQ oxidoreductase) catalyses the
  2 e- transfer
• from NADH to ubiquinone.
• Em,7 of the NAD/NADH couple is - 320 mV.
• Em,7 of the ubiquinone/ubiquinol couple is 60
  mV.
• 380 mV of energy believed used to pump 4H/2e-.

14

• Very large (about 750 kDa the large rybosome
  subunit).
• Contains 41 subunits.
• Bacterial analogues have only 14 subunits.
• Its redox centres (except one flavin FMN, 4Fe/S)
  are eight iron-sulphur (2Fe/2S) complexes.
• Cannot be studied by optical techniques.
• Requires less powerful electron spin resonance
  techniques.
• Only a low-resolution electron microscopy
  structure.
• - Shows an L-like shape.
• No clue how it pumps protons.

15

• Other methods of delivering e- to ubiquinone
• Succinate dehydrogenase (complex II)
• - Transfers electrons from succinate.
• - Part of the TCA cycle.
• ETF-ubiquinone oxidreductase (water soluble).
• - Has a surface which can dip into the membrane
  which contains a ubiquinone binding site.
• s,n glycerophosphate dehydrogenase.
• - Apparently similar to ETF-ubiquinone
  oxidreductase.
• All three are flavin proteins (FAD of FMN
  prosthetic groups) with Em,7 0 mV.
• Since the Em,7 of the ubiquinone/ubiquinol
  couple is 60 mV, there is no net proton
  translocation.
• Feed electrons into the respiratory chain from
  flavin linked step of fatty acid oxidation.

16

• Complex II of the mitochondrial respiratory
  chain
• Complex II (succinate dehydrogenase) catalyses
  the 2 e- transfer from succinate to ubiquinone.
• Em,7 of the Fumarate/succinate couple is 30
  mV.
• Em,7 of the ubiquinone/ubiquinol couple is 60
  mV.
• No excess energy available to pump H.

17

• Structure of Complex II
• Solved by X-ray diffraction.
• - Packs as a homo-trimer (not a functional trimer
  however).
• - Total molecular weight 360 kDa.
• - Four sub-units (SdhA, SdhB, SdhC, SdhD).
• Quinone binding site on cytoplasmic side.
• Redox transfer chain identified
• - Formed by FAD, 2Fe-2S, 4Fe-4S, and 3Fe-3S
  clusters.
• - Extends 40 Å from succinate to quinone binding
  sites.
• - All distances between centres lt14 Å limit for
  electron transfer.
• Heme b is not in the pathway.
• - Provides a handy place to store unwanted''
  electrons.
• - Prevents FAD from reducing O2 when quinone
  deficient.

18

• Summary Lecture 3
• Glycolysis degrades glucose into two pyruvic
  acid molecules.
• Citric acid cycle produces three NADH and two
  CO2 molecules for each pyruvate consumed.
• NAD recycled for glycolysis by Complex I of
  respiratory chain.
• Delivers e- into the respiratory chain.
• Other pathways (eg. through complex II) for
  introducing e- into the respiratory chain.
• No X-ray structure exists for complex I but an
  X-ray structure for complex II was solved.
• More structural details are known of the
  functional mechanisms of complex III, complex IV
  and complex V.