Announcements Now is the time to master quiz game and get highest score. I specifically designed levels to help you practice these concepts You can do each level over and over until you understand every answer Honestly, I cannot possibly tell you how much you really really ought to do this. I expect you to have mastered this stuff for the exam after thanksgiving, and I will write all the questions from this perspective

Today’s goal: Finish central metabolism in chemoorganoheterotroph Glucose Glycolysis Pyruvate Citric acid Cycle ATP NAD+ H2O NADH ETS ATP synthase

The TCA cycle (citric acid or Krebs cycle)

Principles of oxidative phosphorylation

Learning outcomes Be able to calculate the free energy of a reaction based on the change in reduction potential Be able to identify potential electron donors and acceptors for metabolism based on reduction potential Apply understanding of reduction potential to determine how electron transfer releases energy in oxidative phosphorylation Explain the elements of proton-motive force and how it powers the ATP synthase and flagella Be able to compare and contrast oxidative phosphorylation and substrate-level phosphorylation Use redox potential to make predictions about how and why oxidative phosphorylation varies, resulting in alternative metabolisms and acclimation to the environment. Compare and contrast aerobic and anaerobic respiration

Reduction potential Fundamental concept affecting everything else you learn today and Thursday.

Reduction potential and relationship to ∆G E = Potential to accept electrons Eo = in standard conditions Eo’ = standard plus pH 7 ∆E = E of acceptor –E of donor ∆Go’ = -nF∆Eo’ n = moles of electrons Faraday’s constant = 96.5 kJ/Vmol

Example:complete oxidation of glucose Between two redox couples: C6H12O6 + 6 O2 → 6 CO2 + 6 H2O C6H12O6 + 6 O2 → 6 CO2 + 6 H2O What is the electron donor? What is the electron acceptor? Redox couple:

We can find E in a redox tower Capacity to donate Capacity to accept

∆E and ∆G for complete oxidation of glucose Between two redox couples: C6H12O6 + 6 O2 → 6 CO2 + 6 H2O ∆Eo’ = E(acceptor) – E(donor) = (O2/H2O) - ([CH2O]/CO2) ∆Go’ = -nF∆Eo’ n = moles of electrons Faraday’s constant = 96.5 kJ/Vmol ∆G = -2895 kJ/mol

We can use the redox tower as a cheat sheet ∆Go’ = -nF∆Eo’ For negative ∆G ∆E should be + or- ? Down the tower or up the tower? In an endergonic reaction, should E increase or decrease? Capacity to donate Capacity to accept

A series of redox reactions in cell Membrane generates proton-motive force How the reservoir above the dam gets filled up with water….

Electron carrier molecules do redox rxns

Arrange carriers in order to get -deltaG NADH Eo’ = -320mV O2 Eo’ = +820 mV Ubiquinone Eo’ = +110mV FMN Eo’ = -190 mV FeS cluster Eo’ = -100 mV Cytochrome c Eo’ = +250 mV

Series of oxid/reduction reactions ensues NADH O2 STEAM >>> STEAM train

Electron carriers are arranged in proteins

Energy release from rxns fuels proton pumps Oxidoreductase: Protein complexes where oxidation/reduction reactions occur Buckets thrown over the reservoir

Quinone pool: Electron transfer and pmf Protons come from cytoplasm

Proton-motive force generation in E. coli

Microbes can change composition of their ETS To modulate their physiology depending on environment. They have preferred ratio of +/- across membrane. Example: acidic environment

What proton-motive force is and how it drives ATP synthesis What is in the reservoir, and how that potential energy is converted into ATP

Potential energy → mechanical → ATP

Proton-motive force Denoted ∆p Includes: ∆pH and ∆ψ (electrical potential) The force exerted by the reservoir

ATP synthase: potential energy >> mechanical

How PMF runs bacteria flagella

What is the 3rd function of PMF in bacteria? Follow-up: Then why might the cell want to use ATP to run ATP synthase backwards?

Anaerobic respiration What it is E of terminal acceptor structures communities Variation in e-donors and acceptors determines G for anaerobes Anaerobes may not use NADH

Anaerobic respiration Electron acceptors other than oxygen Examples include: SO4 Mn Fe NO3 NO2 Uranium, Plutonium, Tc99

ETS complexes vary with donors/acceptors

Analyzing respiration 1. Nature of the acceptor: What is the acceptor reduced to? How many reduction steps occur? 2. What is the electron donor? It’s role in central metabolism? What molecule starts the Electron transport system (ETS)? 3. How does the ETS differ depending on 1 and 2? How many protons pumped? How does this relate to delta G?

Example anaerobic respiration: nitrate ∆Go’ = -nF∆Eo’ How many steps does each acceptor take? Which molecule donates electrons to ETS? Which transfers more electrons? Which has biggest change in reduction potential? Donor = C6H12O6 Pseudomonas NO3- – NO2- – NO – ½ N2O – ½ N2 + 740mV Donor = C6H12O6 E. coli NO3- – NO2- + 420mV Based on above, what do you expect? How does the ETS differ depending on 1 and 2? How many protons pumped? How does this relate to delta G?

Nitrate reduction (denitrification) Do these images match what you might expect? Why or why not? E.coli with oxygen E.coli, no oxygen but nitrate Pseudomonas

Example: sulfate respiration If they catabolize lactate – are they likely to use NADH? E for lactate = - 190mV E for NADH = -320mV Lactate → Pyruvate electrons ? NADH How are electrons donated to the ETS? ? ETS?

Hint: hydrogen accumulates Is hydrogen involved?

Central metabolism layout, sulfate reducers

Electron donor, acceptor, predictions 1. Nature of the acceptor: What is the acceptor reduced to? How many reduction steps occur? 2. What is the electron donor? It’s role in central metabolism? What molecule starts the Electron transport system (ETS)? 3. How does the ETS differ depending on 1 and 2? How many protons pumped? How does this relate to delta G?

Abbreviated model of sulfate reduction Hydrogenase (H2ase): Enzyme splits hydrogen into protons and electrons

This is real life: Just FYI Model for the respiratory chain of D. vulgaris during growth on hydrogen, or lactate, and sulfate. Hydrogenases Formate dehydrogenases Cytochromes Fe-S proteins Cytochromes Hydrogenases Venceslau S S et al. J. Biol. Chem. 2010;285:22774-22783 ©2010 by American Society for Biochemistry and Molecular Biology

What is oxidative phosphorylation?