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BIO 220

6. Microbial Metabolism

Topics-Oxidation/Reduction reactionsCatabolic/Anabolic reactionsGlycolysisAerobic RespirationAnaerobic RespirationFermentationHydrocarbon Transformation

Oxidation/Reduction Redox reactionsThese terms are discussed more thoroughly in chemistry class.However, as most cellular reactions are Redox reactions, it is difficultto understand Metabolism without a grasp of the meaning of theseterms.

Redox reactions involve the transfer of electrons during a chemicalreaction. In this class we examine redox reactions in very simplifiedterms.

If a molecule gains an electron during a chemical reaction it isreduced. As electrons are negatively charged, a molecule receivingan electron undergoes a charge reduction.

If a molecule loses an electron during a chemical reaction it isoxidized.

General redox reaction

Ae- + B -> A + Be-

In this reaction Ae- loses an electron (is oxidized) while B gains anelectron (is reduced).

2 somewhat confusing terms-

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Oxidizing agent- a molecule which oxidizes another molecule bytaking an electron away from that molecule.

In the above reaction B acts as an oxidizing agent of Ae- by removingan electron from Ae-.

Oxidizing agents become reduced.

Reducing agent- a molecule which reduces another molecule bydonating an electron to that molecule.

In the above reaction Ae- acts as a reducing agent of B by donating anelectron to B.

Reducing agents become oxidized.

Because an electron transfer requires a donor and a receptor,oxidation and reduction always go together.

If we look at the reaction.

C6H12O6 + O2 -> 6 CO2 + 6 H20

we see electrons are transferred from glucose tooxygen.

Thus glucose is the reducing agent for it passes electrons to oxygen.Oxygen is the oxidizing agent for it accepts electrons from glucose.

When viewing redox reactions we like to focus on the transfer ofelectrons. However hydrogen is sometimes added to the oxidizingagent as well to balance the negative charge of the electron. This isseen in the above reaction as oxygen accepts hydrogen (in addition toelectrons) to form water.

Redox PotentialThe change in energy from an electron donor to the electron acceptor

is measured as the Redox Potential or Oxidation/Reduction coupling.Redox Potential is measured in volts. Figure 5.9 depicts an electrontower representing the redox potential between various electrondonors and acceptors.

Metabolism- the totality of chemical reactions within a cell.Comprises both Anabolic (synthesis) reactions and Catabolic(degradation) reactions.

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Heterotrophs extract energy from their environment by utilizingcatabolic pathways. This includes the processes Glycolysis, AerobicRespiration, Anaerobic Respiration and Fermentation. In thesecatabolic processes, complex molecules (such as glucose) are oxidized.

The removed electrons are passed from one molecule to another in aseries of Redox reactions. After each redox reaction the transferredelectron contains slightly less energy. This step-wise removal ofenergy is ultimately used by the organism to generate ATP (Adenosinetri-phosphate). If the energy contained in the electron was liberatedin a single step, the majority of the energy would be released as heatand thus not usable by the cell.

The molecule which finally ends up with the electron is called theTerminal Electron Acceptor. Aerobic Respiration, AnaerobicRespiration and Fermentation all employ different Terminal

Electron Acceptors.

Role of NAD- Nicotinamide Adenine Dinucleotide in catabolism.NADH Cycling

NAD cycles continuously between its oxidized form (NAD+) and itsreduced form (NADH + H+). NADs ability to accept electrons thendonate them to other molecules in specific biochemical pathways iscritical to the cells ability to make ATP from organic molecules(glucose, amino acids, fatty acids).

NAD Redox

NAD+ + 2H + 2e- -> NADH + H+

NAD+is the oxidized formof the molecule.NADH + H+ is the reduced form of the molecule.

Short term and Long term sources of cellular energyCells require a constant source of energy. ATP is a common source ofshort term energy. ATP hydrolysis (ATP-> ADP + Pi) is tremensdously

exergonic ( G =-34 kJ). The cell constantly recycles ADP to make ATP with the ration of ATP:ADP being approximately 1000:1). Thecellular concentration of ATP is typically 2mM.

CoEnzyme A, a molecule involved in the Citric Acid Cycle may serveas a short term supply of energy for anaerobes. Hydrolysis of themolecules thioester bonds yields sufficient free energy for othercellular processes.

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Long Term sources of energy include organic carbon molecules suchas Polyglucose (sugar based), Poly- -Hydroxybutyrate (lipid). Chemolithotrophs use inorganic metals such as elemental Sulfur as asource of long term energy.

Glycolysis

Occurs in the cytoplasm1 glucose broken down into 2 pyruvate2 ATP are spent

4 ATP are generated (Net yield = 2 ATP)2 NADH + H+ are generated

RespirationIn Respiration the 2 molecules of pyruvate generated by

glycolysis are completely oxidized to CO2 in a series of biochemicalreactions known as the Krebs Cycle (Citric Acid Cycle). During theoxidation of carbon compounds in the Krebs Cycle, NAD+ is reduced toNADH + H+ and a related compound FAD is reduced to FADH2. Inaddition to energy storing compounds NADH + H+ and FADH2, the KebsCycle also generates GTP (guanosine triphosphate). GTP is converted

to ATP via substrate level phosphorylation.

Overall each cycle of the Krebs Cycle generates-

4 molecules of NADH + H+

1 molecule FADH21 molecule of GTP (that can be converted later to ATP)

NADH + H+ (and FADH2) participate in further redox reactions,donating electrons to a chain of proteins known as the ElectronTransport Chain (ETC). The passage of electrons along the ETC ischemiosmotically linked to the generation of ATP.

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Free Energy and the ETCIn terms of free energy relative to the terminal electron acceptor, eachprotein in the ETC has slightly less free energy than theprotein preceding it.

Each component of ETC has a higher affinity for electrons thanthe preceding component, with the terminal electron acceptorhaving the highest affinity of all.

The decreasing free energy and increasing electron affinity ofthe ETC components keep the electrons movingunidirectionally down the ETC.

Energy is released with each step in the ETC. This energy is notreleased as heat but transferred to another form of energycalled a Proton Motive Force.

See Figures 5.19 and 5.20.

Chemiosmosis in ProkaryotesElectrons are passed from NADH (and a related molecule FADH2) to aseries of enzyme complexes (ETC) in the Plasma Membrane. Theelectrons are then passed through these enzymes in a series ofoxidation/reduction reactions. As this occurs a H+ gradient isgenerated outside the plasma membrane. Such a gradient containsenergy called a proton motive force. The proton motive force is

utilized to drive the reaction ADP + Pi -> ATP (OxidativePhosphorylation), drive bacterial flagella and move molecules acrossthe plasma membrane.

Brock defines respiration where the terminal electron acceptor is anexogenous molecule.

In Aerobic Respiration the terminal electron acceptor isoxygen.In Anaerobic Respiration the terminal electron acceptor is aninorganic compound other than oxygen (Nitrates, Sulphates

are examples).

Although, aerobic respiration generates more ATP, many of theenzymes in the Electron Transport Chains are the same for bothaerobic and anaerobic respiration. In organisms which can utilize bothpathways, gene expression of enzymes involved in anaerobicrespiration occurs only in the absence of oxygen.

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Chemiosmotic Coupling of the proton motive force to ATPsynthesis

AsH+ ions accumulate in the Periplasmic space, a pH gradient and

electrical gradient develops. Taken together these create a form ofenergy called the proton motive force. H+ ions cannot cross theinner membrane freely, however they are able to pass through aprotein complex called ATP Synthase.

ATP Synthase is composed ofmany protein subunits and has 3main parts:

RotorRodKnob

As H+

ions pass through the ATP Synthase the rotor spins in aclockwise direction which activated the catalytic site in the knob. Theknob portion of the enzyme catalyzes the reaction-

ADP + Pi -> ATP

See diagram on board.

ENERGY YIELD Aerobic Respiration-

Theoretical Yield-1 NADH + H+ entering ETC -> 3 ATP

1 FADH2 -> 2 ATP

Experimental Yield-1 NADH + H+ -> 2.5-2.7 ATP

Other uses for proton motive force besides ATP generation

Maximum theoretical ATP yield from 1 glucose-

Glycolysis-2 ATP

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2 NADH + H+

Pyruvate -> Acetyl CoA

2 NADH + H+

Citric Acid Cycle

8 molecules of NADH + H+

2 molecule FADH22 molecule of GTP

Oxidative Phosphorylation (converts energy in NADH and FADH2 into

ATP)

34 ATP

38 ATP are generated from 1 molecule of glucose.

Fermentation-Fermentation involves the partial breakdown of sugars without theassistance of oxygen. Terminal electron acceptor is an organic (carbonbased) molecule.Fermentation products can be acidic (lactic acid, formic acid) or neutral(acetoin, ethanol). Bacteria can be differentiated by the fermentationpathways they employ.

The Energy Yield of fermentation is much less than obtained via

respiration. Genes involved in fermentation pathways are expressed inanaerobic conditions.

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