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. Respiration Review 2NADH Q 2NAD+ 2H+ H+ O 2 4H+ 2 H 2 O H+ per cyto low E o ’ electron flow hi E o ’ ATP ADP 3H+ Electron Tower

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 10 5 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. NO 3 -, NO 2 -, SO 4 2-, S 0, SeO 4 2-, AsO 4 3-  Metals: –Eg. Fe 3+, Mn 4+, Cr 6+  Minerals/solids: –Eg. Fe(OH) 3, MnO 2  Gasses: –Eg. NO, N 2 O, CO 2 Why is there so much diversity? How can prokaryotes accomplish this?

6. Dehydrogenase: Lactate Succinate Formate NADH Glycerophosphate Hydrogenase MQ UQ fumarate Cyt b, Fe/S, FAD Fumarate reductase Cyt b, Fe/S, Mo DMSO DMSO reductase Cyt b, Fe/S, Mo TMAO TMAO reductase Cyt b, Fe/S, Mo NO 3 - Nitrate reductase Electron donor modulesElectron acceptor “modules” Answer:

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

8. Example 1. Nitrate reduction

9. Figure 24.19 Nitrogen cycle 78% N 2

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 –E 0 ’ +0.74 V compared to +0.82 for 1/2O 2 /H 2 O –How many electrons are used from NO 3 - to N 2 ?

11. Dissimulative NO 3 - reduction

12. Denitrification by Pseudomonas stutzeri

13.  Four terminal reductases –Nap: Nitrate reductase (Mo-containing enzyme) –Nir: Nitrite reductase –Nor: Nitric oxide reductase –N 2 or: Nitrous oxide reductase  All can function independently but they operate in unison

14. Dissimilatory nitrate reduction: Biochemistry  Electron donor: lactate, formate, H 2, 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. How much energy is made by reducing nitrate to nitrite with NADH? Nitrate= N(x) + 3O 2-  x + 3(-2) = -1  x= Nitrite= N(x) + 2O 2-  x + 2(-2) = -1  x= Determining oxidation state of N and # of electrons: ?e- Nitrate (NO 3 - ) Nitrite (NO 2 - ) ?NADH ?NAD+ example ?ATP We only need to oxidize ______ NADH for this: NADH + H + NAD + + 2H + + 2e- What’s reduce and oxidized? Find ∆Eo’ of nitrate/nitrite and NAD+/NADHUse Nernst Eq to find ∆Go’

17. Example 2. Arsenate reduction Arsenate  arseniteE o ’+0.139 V

18. http://phys4.harvard.edu/~wilson Arsenic respiring bacteria and human health  Arsenic is mainly a groundwater pollutant  Affects ~140 million people among ~70 countries  Arsenate (As[V]): –Like phosphate: H 2 AsO 4 - –Affects ATP synthesis  Arsenite (As[III]): H 3 AsO 3 –More toxic than As(V) –Binds proteins –Causes DNA damage  Microbes respire arsenate and make arsenite  Medical Geology problem

19. Shewanella sp. strain ANA-3 2As(V) 2As(III) As 2 S 3 Lactate Acetate + CO 2 Dividing cell Isolation of strain Respiring O 2 Respiring AsO 4 3-

20. Example 3. Iron

21. 2Fe(III) 2Fe(II) NADH2NAD+ c-heme QH 2 Q CM OM Fe III -oxide Iron oxide reducing bacteria  Examples: –Geobacter, Shewanella, Rhodoferrax  How do they do it?

22. Chemolithotrophy and Oxidation of Inorganic Molecules A.A pathway used by a small number of microorganisms called chemolithotrophs B.Produces a significant but low yield of ATP C.The electron acceptor is commonly O 2, some others include sulfate and nitrate D.The most common electron donors are hydrogen, reduced nitrogen compounds, reduced sulfur compounds, and ferrous iron (Fe 2+ )

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 (H 2 SO 4 ) –Acidification of soil –Dissolution of minerals, e.g. CaCO 3

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.