Metabolism Lectures

1. Metabolism Lectures Outline: • Part I: Fermentations • Part II: Respiration • Part III: Metabolic Diversity Learning objectives are: • Learn about anaerobic respiratory metabolisms. • How can an inorganic compound be use as an energy source.

2. low Eo’ electron flow hi Eo’ Respiration Review 2H+ H+ H+ 3H+ per Electron Tower Q cyto O2 4H+ 2 H2O 2NADH 2NAD+ 2H+ H+ H+ ADP ATP

3. Anaerobic Respiration • Anaerobic metabolism is of clinical importance: • Deep tissue infections can lead to abscess formation, foul-smelling pus, and tissue destruction • Uses inorganic and organic molecules other than oxygen as terminal electron acceptors • Extensive list of electron acceptors • oxyanions, metals, metal oxides, organic acids, inorganic • Energy and carbon sources are diverse

4. Metabolic Classification Based on Oxygen Concentrations • Points of reference: • Atmospheric oxygen is ~21% (v/v) (or 2.1 x 105 parts per million) • Low solubility in water: up to 14 parts per million (T and P dependent) • Remember metabolic classifications: • Strict aerobe (non-fermentative, respires oxygen) • Strict anaerobe (sensitive to oxygen) • Facultative anaerobe: (fermentative and/or respiration) • Microaerophilic (or microaerophile) • 40:1 anaerobes to facultative anaerobes in human feces

5. Diversity of electron acceptors for respiration • Organic compounds: • Eg. fumarate, dimethylsulfoxide (DMSO), Trimethylamine-N-oxide (TMAO) • Inorganic compounds: • Eg. NO3-, NO2-, SO42-, S0, SeO42-, AsO43- • Metals: • Eg. Fe3+, Mn4+, Cr6+ • Minerals/solids: • Eg. Fe(OH)3, MnO2 • Gasses: • Eg. NO, N2O, CO2 Why is there so much diversity? How can prokaryotes accomplish this?

6. Cyt b, Fe/S, FAD fumarate Fumarate reductase Cyt b, Fe/S, Mo DMSO MQ UQ DMSO reductase Cyt b, Fe/S, Mo TMAO TMAO reductase Cyt b, Fe/S, Mo NO3- Nitrate reductase Answer: Electron donor modules Electron acceptor “modules” Dehydrogenase: Lactate Succinate Formate NADH Glycerophosphate Hydrogenase

7. Modularity of electron transport chainswhat do most of these have in common?

8. Example 1.Nitrate reduction

9. Figure 24.19 Nitrogen cycle 78% N2

10. Nitrate reducing bacteria • Contribute to denitrification (removal of ?) • Beneficial process for sewage treatment plants Nitrogenous waste good food for algae • Dissimilatory nitrate reduction widespread in microbes • Used for making energy via oxidative phosphorylation • Nitrate is a strong oxidant similar to oxygen • Some microbes can take Nitrate all the way to Nitrogen gas: • Pseudomonas stutzeri • E0’ +0.74 V compared to +0.82 for 1/2O2/H2O • How many electrons are used from NO3- to N2?

11. Dissimulative NO3- reduction

12. Denitrification by Pseudomonas stutzeri

13. Denitrification by Pseudomonas stutzeri • Four terminal reductases • Nap: Nitrate reductase (Mo-containing enzyme) • Nir: Nitrite reductase • Nor: Nitric oxide reductase • N2or: Nitrous oxide reductase • All can function independently but they operate in unison

14. Dissimilatory nitrate reduction: Biochemistry • Electron donor: lactate, formate, H2, others • Uses special dehydrogenases for these. • Enzymes are membrane-bound • Periplasmic nitrate reductases (NapA) contains a molybdenum cofactor • Coupled to the generation of PMF • ATP synthesized by oxidative phosphorylation

15. Nitrate vs. oxygen vs. denitrification respiration oxygen nitrite denitrification

16. example Nitrate (NO3-) ?NAD+ ?e- Nitrite (NO2-) ?NADH How much energy is made by reducing nitrate to nitrite with NADH? What’s reduce and oxidized? ?ATP Determining oxidation state of N and # of electrons: Nitrate= N(x) + 3O2- x + 3(-2) = -1  x= Nitrite= N(x) + 2O2- x + 2(-2) = -1  x= We only need to oxidize ______ NADH for this: NADH + H+ NAD+ + 2H+ + 2e- Find ∆Eo’ of nitrate/nitrite and NAD+/NADH Use Nernst Eq to find ∆Go’

17. Example 2.Arsenate reduction Eo’+0.139 V Arsenate  arsenite

18. Arsenic respiring bacteria and human health • Arsenic is mainly a groundwater pollutant • Affects ~140 million people among ~70 countries • Arsenate (As[V]): • Like phosphate: H2AsO4- • Affects ATP synthesis • Arsenite (As[III]): H3AsO3 • More toxic than As(V) • Binds proteins • Causes DNA damage • Microbes respire arsenate and make arsenite • Medical Geology problem http://phys4.harvard.edu/~wilson

19. Respiring AsO43- Shewanella sp.strain ANA-3 Dividing cell 2As(V) Lactate Acetate + CO2 2As(III) As2S3 Respiring O2 Isolation of strain

20. Example 3. Iron

21. FeIII-oxide Iron oxide reducing bacteria 2Fe(II) 2Fe(III) • Examples: • Geobacter, Shewanella, Rhodoferrax • How do they do it? OM c-heme Q CM QH2 NADH2 NAD+

22. Chemolithotrophy and Oxidation of Inorganic Molecules • A pathway used by a small number of microorganisms called chemolithotrophs • Produces a significant but low yield of ATP • The electron acceptor is commonly O2, some others include sulfate and nitrate • The most common electron donors are hydrogen, reduced nitrogen compounds, reduced sulfur compounds, and ferrous iron (Fe2+)

23. Geological, biological, and anthropogenic sources of reduced inorganic compounds supporting chemolithotrophs Typical habitats of chemolithotrohs: Near the interface of oxic/anoxic conditions

24. Energy yields from various inorganic electron donors:

25. Oxidation of sulfur-compounds • E.g.: Sulfur oxidizing Thiobacillii • Thiobacillus thiooxidans Thiobacillus ferrooxidans • Produces sulfuric acid (H2SO4) • Acidification of soil • Dissolution of minerals, e.g. CaCO3

26. Lecture Summary • Anaerobic respiration • Alternative terminal electron acceptors are used • ATP generated by oxidative phosphorylation • Often not as energetically favorable as oxygen respiration • Anaerobic electron transport chains are branched • Ecologically and medically significant • In some cases toxic metals are used as electron acceptors • Chemolithotrophy • Energy sources are reduced inorganic compounds • Chemolithotrophs often live near redox gradients where there is a mixture of reduced and oxidized chemicals.