Test I Flashcards
- Describe how the proton motive force is generated and how ATP is produced from PMF.
When e-are transported through an e- transport chain, protons are extruded to the outside of the membrane forming a proton motive force. The production of ATP is linked to the establishment of the PMF
- e- transport carriers are orientated so that separation of protons from e- occurs across the membrane during the transport process
- 2 e- plus 2 protons, removed from substances such as NADH are transported though the chain by specific carriers
- Protons are released in the environment
- Results in slight acidification of the external surface of the membrane
- At the end if the ETC, e- are passed to the terminal e- acceptor –O2 in aerobic respiration, reduced to H20.
- To reduce O2 to H2O, protons are needed from cytoplasm, originate from the dissociation of water- H+ and OH-
- Use of H+ to reduce O2 and extrusion of H+ to environment causes OH- to accumulate on inside of membrane. They cannot diffuse through membrane as they are carged.
- Net results of e- transport is generation of a pH gradient and an electrochemical potential across the membrane. Inside is electrically negative/alkaline, outside being positive/ acidic.
- ATP production from aerobic respiration is called oxidative photophosphorylation and is carried out by a complex of proteins called ATP synthase.
- Complex is made up of 2 subunits, F1 and F0. H+ are driven back through the membrane from the outside by the PMF, go through F0 and place pressure (or torque) on F1
- A molecule of ADP + Pi binds to F1 and where torque is released, energy is free to power to formation of ATP through the bonding of ADP and Pi
- Describe in detail how ATPase works for ATP production and why cells may use their ATPase in reverse.
ATPase (complex V) is a large membrane enzyme complex that catalyzes conversion of PMF into ATP and has two parts:
- Multi-subunit headpiece of alpha and gamma called F1 on cytoplasmic side of membrane.
- Proton-conducting channel called F0 that spans membrane.
- F1/F0 complex catalyzes a reversible reactions between ATP and ADP + Pi
- Proton movement through subunit a of F0 drives rotation of c protein, generating a torque that is transmitted to F1 by gammaE subunit
- Energy transferred to F1 through coupled rotation of gammaE subunits
- Cause conformational change in beta subunits, a form of potential energy tapped to make ATP
- Possible because conformational change to beta subunits allows for sequential binding of ADP + Pi to each subunit
- Conversion to ATP occurs when beta subunits return to original conformation
- The primary function of b2 subunits of F1 is to prevent alpha and beta subunits from rotation with gamma-eipsilon so conformational changes in beta can occur. 3-4 protons consumed by ATPase per ATP produced.
Why ATPase is reverse?
- ATP hydrolysis can be used to create a H+ gradient by the reversal of the ion flux
- F1 rotary motors works in the forward motion to hydrolyse ATP and to drive the F0 motor n reverse to create a H+ gradient
- The generation of a H+ gradient can then be used to maintain ionic balance, as well as for active transport to drive substrate accumulation.
- Bacteria play key roles in the biogeochemical cycling of carbon. Describe the redox cycle, key processes, and types of microorganisms involved.
Photosynthesis and respiration
Photosynthesis: CO2 + H2O –> (CH2O) + O2
Respiration: (CH2O) + O2 –> CO2 + H2O.
Major end products CH4 and CO2
-All nutrient cycles are coupled
-CO2 taken up by photosynthetic organisms and used to make organic molecule.
-Carbon atoms are released as CO2 respiration.
Autotroph- capture CO2 and use it to make organic compounds
Heterotrophs=consume the organic compounds
- C and N cycles. The rate of CO2 fixation is controlled by size of photosynthetic biomass and available nitrogen.
- Most methanogens reduced CO2 to CH4 with H2 as an e- donor; some can reduce other substrates to CH4 (e.g. acetate). Methanogens team up with partners (syntrophs) that supply then with the necessary substrates
- CH4 is formed from reduction of CO2 by H2 supplied by syntrophic bacteria; these organisms depend on H2 consumption to balance their energetics.
- Acetogenesis is another H2 consuming process
- Acetogens can ferment glucose and methyoxylated aromatic compounds.
Describe the diversity of pigments and membrane systems used in bacteria to utilize light as an energy source. How do some bacteria adapt to life at low light intensity?
Photosynthesis= process that converts light energy to chemical energy.
-Different species have different pigments so that unrelated organisms can co exist in an environment, each using wavelengths of light the other is not using.
Pigments
- Chlorophyll and bacteriochlorophyll
- Carotenoids
- Phycobilisomes
Membrane systems
- Oxygenic photosynthesis
- Anoxygenic photosynthesis
Low light intensities
- Light harvesting or antenna chlorophylls
- Chlorosomes
- Phycobilisomes
Light sensitive pigments
Chlorophylls and Bacteriochlorophylls
- Chlorophyll a is the main one, absorbs read and blue light and transmits green
- Bacteriochlorophyll a absorbs between 800-925 nm, others absorb in the regions of the visible and IR spectrum
- Found in photosynthetic membranes where the light reactions of photosynthesis are carried out.
Carotenoids
- Hydrophobic pigments embedded in the membrane
- Absorb light in the blue region
- Can transfer energy to the reaction centre but also functions as a photoprotective agent. Carotenoids quench toxic oxygen species produced form photooxidation caused by bright light.
Phycobilisomes
- Very efficient energy transfer form biliproteincomplex to chlorophyll a, allows for growth of cyanobacteria at low intensities
- Phycobilisomes content increases in cells as light intensity decreases
- Accessory pigments allow the organism to capture more of the available light
- Cyanobacteria contain phycobiliproteins, their main light harvesting pigments are red of blue. Absorb light in the range of 550-650 nm.
Membrane systems used in bacteria to utilize light as an energy source.
Oxygenic Photosynthesis
- Z scheme
- Contains PSI (P680) and PSII (P700)
- Starts with PSII, P680 absorbs light and water is split into oxygen and hydrogen with an e- donated to P680.
- e- travels through PSII where is donates it to the protein plastocyaninin and then the e- gets donated to PSI which leads to the reduction of NADP+
- Not a closed circuit, needs an exogenous e- donor
- Transfer of e- from acceptor in PSII to PSI generates a PMF where ATP can be made= non cyclic photophosphorylation
- If sufficient reducing power us present, ATP can also be produced when e- travel from ferredoxin to cytochrome bf complex from which e- transport returns the e- to P700, this flow generates a membrane potential and synthesis of ATP- cyclic photophosphorylation.
Anoxygenic Photosynthesis
- Light excites P870 to P870*
- At the higher state, a cascade happens where the energy gets passed to Bchl then Bph and through a series of e- carriers in the process
- This is coupled with the transfer of protons across the membrane creating the PMF→ drives ATPase making ATP.
- The e- returns the P870 at the end of the chain so to can be used again once light excites the reaction centre.
Location of photosynthetic pigments
- In prokaryotes, chloroplasts are absent. Photosynthetic pigments are integrated into internal membrane system
- In organisms e.g. cyanobacteria, these pigments are found in chloroplast.
How do some bacteria adapt to life at low light intensity?
- Reaction centres surrounded by more numerous light harvesting or antenna chlorophylls, which funnel light energy to the reaction centre. At low light intensities, this allows the capture and use of photons that would otherwise be insuffiecient to drive the reaction centre alone
- Green sulfur bacteria contain chlorosome. Giant antenna system, Chlorosomes absorb low light intensities and transfer the enrgy to bacteriochlorophyll a in the reaction centre in cytoplasmic membrane
- Phycobilisomes, very efficient transfer from the biliprotein complex to chlorophyll a, allows for growth of cyanobacteria at low light intensities. Phycobilisome content increases in cells as light intensities decrease. Accessory pigments allow the organism to capture more of the available light
Anoxygenic photosynthesis
- Light energy is captured and converted to ATP, without the production of oxygen.
- Light converts a weak e- donor, p870 into a very strong e- donor, p870*. Its is excited by the absorption of light. It becomes more willing to give up that e-.
- When at the higher state, a cascade happens where the energy gets passed to Bchl then Bph and through a series of e- carriers, transferring e- in the process.
- Coupled with the transfer of protons across the membrane that ultimately gives you energy → Proton motive force.
- The e- return to p870 at the end of the chain so it can be used again once light excites the reaction centre.
ATP generation
- Formation of the PMF generated by proton extrusion during e- transport and the activity of ATPase in coupling the dissipation of the proton motive force to ATP formation
- This method of making ATP is called cyclic photophosphorylation as e- move around a closed circle. No net gain or loss of e-
CO2 fixation
- Autotrophic growth requires reducing power (NADH of NADPH) and ATP so that CO2 can be reduced to the level of cell material
- Reduced substances (e.g. H2S or S2O32-) are oxidised by c type cytochromes and e- end up in the quinone pool
- E0’ of quinone is not negative enough to reduce NAD+ directly so e- from quinone pool must be forced backwards against the thermogradient to reduce NAD+ to NADH, a process called reverse electron flow which is driven by ATP.
- e- are coming from external e- donors.
- Not very efficient as you need to spend more energy to make more energy to reduce CO2.
The Calvin cycle
- Requires NAD(P)H, ATP and two key enzymes (RubisCo, Phosphoribulokinase)
- First step in CO2 reduction is catalysed by RubisCO
- RubisCO catalyzes formation of two molecules of PGA from Ribulose biphosphate
- PGA is then phosphorylated and reduced to a key intermediate of glycolysis, glyceraldehyde 3-phosphate
- Final step is phosphorylation of ribulose 5-phosphate with ATP by Phosphoribulokinase which like RubisCO is unique to the Calvin cycle.
1. Light independent reactions are initiated; CO2 is fixed from an inorganic to an organic molecule
2. ATP and NADPH are used to reduce 3-PGA into G3P. ATP and NADPH are converted to ADP and NADPH
3. RuBP is regenerated, which enables the system to prepare for more CO2 to be fixed.
The Reverse Citric Acid Cycle,
- In the green sulfur bacterium Chlorobium, CO2 fixation occurs by a reversal of steps in the citric acid cycle (Krebs cycle)
- Chlorobium contains two ferredoxin-linked enzymes that catalyse the reductive fixation of CO2 into intermediates of the citric acid cycle, most other reactions are catalysed by enzymes working in reverse of the normal oxidative direction of the cycle
- Reverse TCA utilises numerous ATP molecules, H2 and CO2 to generate acetyl CoA. This process requires a number of reduction reactions using various carbon compounds
- Enzymes unique to the reverse TCA that function in catalysing these reactions include: ATP citrate lyase, fumarate reductase and ferredoxin-dependent 2-oxoglutarate synthase
- ATP citrate lysase cleaves citrate into acetyl-CoA and oxaloacetate.
The Hydroxypropionate Cycle, and comment on the other three.
Used by Chloroflexus (a green non sulfur phototroph) grows autotrophically with either H2 or H2S as electron donors
-Two molecules of CO2 are reduced to glyoxylate.
The others:
Reductive Acetyl CoA Pathway
-Key enzyme:CO dehydrogenase
CO2 + H2 → CO + H20
-Noncyclic pathway fixing two CO2 molecules
-One C02 is reduced to a methyl group bound to a tetrahydropterin
-The other CO2 is reduced to CO bound to nickel in the reaction centre of CO dehydrogenase, which also acts as an acetyl CoA synthase
-Releasing the combination of the CH4 and CO as acetyl-CoA
HP/HB and DC/HB cycles
- Known as 4-hydroxybutyrate cycles
- In both cycles, acetyl-CoA and 2 inorganic carbons are converted to succinyl CoA
- DC/HB is anaerobic, HP/HB is aerobic
- Describe how the concept of chemolithotrophy emerged from the studies of sulfur bacteria by the great Russian microbiologist Sergei Winogradsky.
Winogradsky studied sulfur bacteria Beggiatoa and showed they were only found in waters rich in H2S, as the H2S dissipated, sulfur bacteria were no longer present. So Winogradsky suggested that their development was dependent on the presence of H2S. when sulfur bacteria Beggiatoa filaments were starved of H2S, they lost their sulfur granules, which were rapidly restored if a small amount of H2S was added. So Winogradsky concluded H2S was being oxidised to elemental sulfur. He showed that when sulfur granules disappeared, sulfate appeared in the medium.
H2S → S0 → SO4-
Because these organisms seemed to require H2S for development in the springs, he postulated that this oxidation was the principal source of energy for these organisms - origin of chemolithotrophy.
Discuss how chemolithotrophic aerobic H2-oxidizing bacteria use H2 as an energy source and fix CO2.
Most H2 bacteria can also grow as chemoorganotrophs. When growing chemolithotrophically, they fix CO2 by the Calvin cycle.
6H2 + 2O2 + CO2 → (CH2O) + 5H20
-Grow under microaerobic conditions, as hydrogenases are oxygen sensitive enzymes
-Nickel also required for chemolthotrophic growth as virtually all hydrogenases contain Ni2+ as a metal cofactor.
Acetogenesis
Acetogenesis and methanogenesis, strictly anaerobic prokaryotes can use. Co2 is the e- acceptor in energy metabolism, H2 is a major e- donor for both. Both result in the generation of an ion gradient either H+ or Na+ which drives ATPase.
Homoacetogens carry out the reaction
4H2 + H+ + 2HCO3- → CH3COO- + 4H2O
-In addition to H2, e- donors include a variety of C1 compounds, sugars, organic and amino acids etc.
-Homoacetogens convert CO2 to acetate by the acetyl coA pathway.
-Homoacetogens can grow at the expense of the reactions of the acetyl coA pathway
-ATP synthesis is during the conversion of acetyl coA to acetate and via sodium motive force. An input of ATP is initially needed to make a Na+ motive force and therefore more energy
Methanogenesis
Acetogenesis and methanogenesis, strictly anaerobic prokaryotes can use. Co2 is the e- acceptor in energy metabolism, H2 is a major e- donor for both. Both result in the generation of an ion gradient either H+ or Na+ which drives ATPase.
- Production of methane is carried out by anaerobic Archaea called methanogens
- Methanogenesis occurs through a series of reactions involving novel coenzymes
- Those involved in carrying C1 units from initial substrate Co2 to final product CH4 and those that function in redox reactions to supply electrons necessary for reduction of co2 to CH4.
Methane is produced by 3 major pathways
- Reduction of CO2
- Fermentation of acetate
- Using methyl substrates, reduced using an external donor.
- Discuss in detail the biochemistry of methanogenesis from CO2
Generally H2 dependent, but formate, CO, and organic compounds such as alcohols can supply electrons for CO2 reduction
Steps in CO2 reduction:
1. CO2 is activated by methanofuran containing enzyme and reduced to formyl
2. Formyl group is transferred to methanopterin, dehydrated and reduced to methylene, then to methyl
3. Methyl group is transferred to coenzyme M
Methyl-CoM is reduced to methane by methyl reductase system, in which F430 and CoB are involved. F430 removes CH3 group from CH3-CoM, forming a Ni2+-CH3 complex, which is reduced by electrons from CoB generating CH4 and a disulfide complex of CoM and CoB (CoM-S-S-CoB). Free CoM and CoB are regenerated by reduction of this complex with H2
Summary
- Reduction of Co2 to formyl
- Reduction of formyl to methylene and then methyl
- Reduction of methyl groups to methane.
- Methanogens can utilize three main groups of substrates for the production of methane: CO2, methyl compounds, and acetate.
Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.
Methyl Compounds
Methanogens use CO2 as an electron acceptor in energy metabolism, with H2 a major electron donor. Methanogenesis occurs through a series of reactions involving novel coenzymes. Two classes of coenzymes are involved; one class carrying C1 unit from the initial substrate CO2 to final product CH4, and the other class functioning in redox reactions to supply electrons necessary for reduction of CO2 to CH4.
Methyl Substrates CH3OH + H2 → CH4 + H2O (-131 kJ) Or in the absence of H2 4CH3OH → 3CH4 + CO2 + 2H20 (-319 kJ) e,g, methanol
-Even without H2 it can still be done
- Methanogens can utilize three main groups of substrates for the production of methane: CO2, methyl compounds, and acetate.
Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.
Acetotropic substrates
CH3COO- + H20 → CH4 + HCO3- (-31 kJ)
e.g. acetate, pyruvate
Acetate is first activated by acetyl-CoA, which can interact with carbon monoxide dehydrogenase (CODH) of the acetyl-CoA pathway.
The methyl group of acetate is transferred to the corrinoid enzyme to yield CH3-corrinoid and then goes through the CoM mediated terminal step of methanogenesis.
Those type of bacteria have small diversity but they can produce a lot of methane for what they are
- Methanogens can utilize three main groups of substrates for the production of methane: CO2, methyl compounds, and acetate.
Discuss the biochemistry of each of these pathways for methanogenesis and comment on their environmental significance.
Co2 type substrates
CO2 + 4H2 → CH4 + 2H2O (-131 kJ)
e.g. CO2(e- derived from H2, alcohols, pyruvate), formate, CO
- The reduction of CO2 to CH4 is generally H2-dependent, but formate, CO, and organic compounds such as alcohols can supply electrons for CO2 reduction.
- First, CO2 is activated by methanofuran-containing enzyme and reduced to formyl. The formyl group is then transferred to methanopterin, dehydrated and reduced to methylene, then to methyl.
- The methyl group is transferred to coenzyme M and methyl-CoM is reduced to methane by the methyl reductase system in which F430 and CoB are involved.
- F430 removes CH3 group from CH3-CoM, forming a Ni2+-CH3 complex, which is reduced by electrons from CoB generating CH4 and a disulfide complex of CoM and CoB (CoM-S-S-CoB).
- Free CoM and CoB are regenerated by reduction of this complex with H2
CO2 is abundant, it’s easily obtainable in majority of environments
- Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations:
(i) homofermentative and heterofermentative lactic acid fermentation,
Lactic acid can be produced during fermentation into two pathways: Homofermentative and Heterofermentative.
- Homofermentative is the process of producing lactic acid in a single yielding pathway.
- Homofermentative lactic acid bacteria contain aldolase and produce a molecules of lactate from glucose by the glycolytic pathway.
- Heterofermantative produces additional products mainly ethanol and CO2.
- The reason for this is it lack aldolase and cannot easily breakdown fructose biphosphate to triose phosphate.
- To achieve redox balance it must go through the process below.
Glucose 6-phosphate (oxidation) -> Phosphogluconic acid (Decarboxylated) -> Pentose phosphate (convert to) -> Triose phosphate & Acetyl phosphate. Key enzyme: Phosphoketolase.
Ethanol is roduced from acetyl phosphate. CO2 will be observed.
Glucose → Lactate + ethanol + CO2 + H+ + ATP
- Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations:
(ii) mixed acid fermentation by enteric bacteria,
This is the process in which acids are generated from fermentation of sugars through glycolysis.
- The acids produced in this process are acetic, lactic and succinic acids and the process produces additional substances such as Ethanol, CO2 and H2.
- It is also able to generate other neutral products e.g. Butanediol.
- The process produces more CO2 than mixed-acid fermenters. Therefore, the process does not acidify its environment, this means that the organisms’ are unable to tolerate more acidic environment.
- Sugars are common substrates in microbial fermentations. Describe two of these three common fermentations: (i) homofermentative and heterofermentative lactic acid fermentation,
(iii) butyric acid fermentation by Clostridium species.
This process produces butyric acid.
- The early stage of the fermentation, butyrate and acetate are produced but as the pH drops it affects the synthesis of acid that will result in accumulation of acetone and butanol.
- Acid production will only continue if the media is buffered to keep it neutral. In relation to this idea, lowering the pH will trigger de-repression of genes for solvent production
