Lecture 9 - respiration without oxygen

Question 1

Glycolysis and TCA generate mainly

Answer 1

NADH by specific oxidation reactions

Question 2

SLP

Answer 2

ATP from cytoplasmic reactions

Question 3

OXPHOS

Answer 3

ATP from reducing power transducer into H+

Question 4

Key features of ETCs

Answer 4

membrane, oxidative membrane complexes, electron carriers, reductive membrane complexes

Question 5

Most bacteria are metabolically flexible

Answer 5

Diverse organic and inorganic electron donors

OXygen and anaerobic electron acceptors (faculatative anaerobe)

Fermentation as terminal, sustained mode of energy generation

Can change lots of genes especially in the ETC as shown in the image (donors on left, acceptors on right), E.Coli can make up various different quinones which can link up various types of electron transport chains and can also use various kinds of electron acceptors

Enables adaptation to a much wider range of electron acceptors

Question 6

When oxygen is limiting,

Answer 6

alternative ETC configurations are used

Question 7

Branched oxidase paradigm

Answer 7

Low affinity with more PMF OR high affinity with less PMF

Bacteria often make 2+ complexes for terminal electron reduction e.g.
Complex III and IV: often exists as a super complex, cyt c tramped in complex
Cytochrome bo3: does the same job as III-IV, but one complex and no cyt c needed
Cytochrome bd: pumps 3 fold less H+ but works at low oxygen
Pathogens and facultative anaerobes use cyt-bd during microaerobiosis

Low oxygen conditions - complexes do not really have a high enough affinity when oxygen becomes limited with it so complex III and IV often is instead cytochrome. Bc1aa3, you can get a lot of protons (6H+/2e-) out of this electron transport pathway - lots of protons pumped so more PMF but very low affinity

High affinity, less PMF cytochrome bd oxidase can be used under hypoxic conditions - less oxygen therefore have a small redox potential gap which is why we do not end up getting to pump as many protons

Question 8

Only low affinity oxidase

Answer 8

Mycobacterium leprae: leprosy

Very inflexible, oxygen not too high to too low

Infection best on extremities, ideal oxygen (typically will be obligate aerobes or inflexible organisms)

Quite rare

Only complex III and IV, not cytochrome bd enzyme which is the enzyme for the high affinity

Question 9

Both low and high affinity oxidases

Answer 9

Most cultured bacteria i.e. E. coli

Obligate aerobes : survive low oxygen (truly anaerobic causes it to die off for example the TB microbe

Faculative anaerobes: maximise energy

Question 10

Only high affinity oxidase

Answer 10

Many lactic acid bacteria i.e. enterococcus

Normally do not use ETC, just SLP

Can help opportunists be infectious - pathogens actually use this oxidase to generate enough ATP for infection processes whereas with using less efficient pathways they just can’t maintain the levels of energy production

Typically anaerobe side, usually fermenters or perform anaerobic respiration - encode the cytochrome bd enzyme so if there is an influx of oxygen they can take it up and get a bit of extra energy production out of it

Question 11

Anaerobic electron acceptors

Answer 11

Usually highly prevalent but less redox potential difference = less energy release

Nitrate = highly prevalent nitrogen source in the environment

Fumarate = highly prevalent in cell as it is a TCA intermediate but less energy

Fumarate has the smallest difference in redox potential, if you are encoding these genes you will know that they will always be there so it is a very nice last resort anaerobic respiration strategy

Question 12

Respiratory nitrate reductase

Answer 12

For nitrate

Instead of our terminal reductase which was complex II and IV or cytochrome bd instead have this

Dissimilatory nitrate reduction is a process for energy conservation, in which nitrate is used as an electron acceptor in the (near) absence of oxygen. Converts nitrate to nitrite
Not directly for biomass production (c.f. assimilatory)
This protein is not a proton pump but you still get 15 ATP out of using this enzyme and a reasonable amount of PMF is still being formed because a method of proton translocation called the redox loop is used which is essentially, instead of pumping protons inside one complex, we are translocating protons based on their acid base chemistry on electron carriers

Used in environments with high e- donors but low oxygen conditions
Marine sediments, human GI tract, thermal vents

Not a proton pump, but uses redox loop to make PMF
Process = get a donor and take the electrons odd it and it goes on to our electron transferring cofactor e.g. quinone which accepts the electrons and transports it but whenever these molecule are getting reduced they inherently have to take up protons to balance the reaction and that’s why when we go from quinone to quinol we go from Q to QH2 because we are picking up 2 protons and then this quinone can then move to some other complex and if it so happens to end up on the other side of the membrane then essentially it gives its electrons to the other complex and it will inherently release its protons and now it is one the periplasmic side of the membrane so release protons here, this is called a redox loop because then it comes back around and does the whole thing again, not within complexes it is between complexes

Question 13

Redox loop process for respiratory nitrate reductase

Answer 13

Process = get a donor and take the electrons odd it and it goes on to our electron transferring cofactor e.g. quinone which accepts the electrons and transports it but whenever these molecule are getting reduced they inherently have to take up protons to balance the reaction and that’s why when we go from quinone to quinol we go from Q to QH2 because we are picking up 2 protons and then this quinone can then move to some other complex and if it so happens to end up on the other side of the membrane then essentially it gives its electrons to the other complex and it will inherently release its protons and now it is one the periplasmic side of the membrane so release protons here, this is called a redox loop because then it comes back around and does the whole thing again, not within complexes it is between complexes

Question 14

Fdn-Nar: the prototypical redox loop

Answer 14

Formate dehydrogenase is the oxidative complex

Menaquinone is the electron carrier

Nitrate reductase is the reductive complex

Formate is produced during the fermentation in E.coli
Detoxify formate, increase energy gain
Formate is essentially being used as our electron donor (coming from inside the cell during fermentation)

Electrons are dumped onto the quinone and reduce it at the cytoplasmic face which inherently takes up protons and it goes to the nitrate reductase which has its binding site at the periplasmic face and so it has to release its protons into the periplasmic space and so we get the generation of a PMF by this mechanism - so essentially if you make any two combinations of complexes that have opposing quinone binding sites then you are going to get this happening however if the quinones are on the same side of the membrane then it does not work as no gradient is generated

Question 15

Fumarate reductase (Frd)

Answer 15

We get more ATP from Fdn-Nar

Widespread and dependable as fumarate is part of the citric acid cycle

Reverse reaction of complex II
Doesn’t (usually) make PMF. Last ditch option for OXPHOS
In many cases it does not have the opposing quinone binding sites therefore does not have redox loop occurring therefore no PMF forming (just has cytoplasmic binding sites)

PMF can come from oxidative complexes, allows continuation of other functions of OXPHOS
May be relying on other proton pumping complexes e.g. NADH dehydrogenase linked to this enzyme

Question 16

Non-proton pumping ETCs

Answer 16

Bacteroides fragilis. Model anaerobe

NQR - sodium translocating NADH dehydrogenase. Make a membrane potential

NDH2 - type II NADH dehydrogenase. Non-proton pumping

NDH2—> FRD = no PMF

Question 17

Why make ETC with no theoretical gains?

Answer 17

Alternative roles
Redox balance - maintain NADH/NAD+ to promote catabolic processes - e.g. NDH2
Radical detoxification - cytochrome bd can detoxify hydrogen peroxide, NO etc., more beneficial to still use it than just switching to a nitrate reductase which has less of this role
Protect other enzymes - nitrogen fixing enzymes are inactivated by oxygen —> use oxidases, acts like a buffer in some scenarios to protect other enzymes

Question 18

Proton back pressure

Answer 18

PMF is a chemical product of proton pumping reaction

Subject to chemical equilibrium chemistry

High PMF - equilibrium shifts to substrates, no longer spontaneous

Called “backpressure” and shows need for homeostatic balance

Too much PMF is a bad thing…
If you are generating lots of PMF in your reactions then you get a very acidic periplasm with lots of protons and when you have a lot of products it shifts the equilibrium to the substrates which makes the reaction less favourable so the forwards direction does not want to happen, same with protons, concept of back pressure
If you have lots of protons there it is like pumping a bike tire - when it gets to a point the pressure pushes back on you and its the same idea as proton back pressure that essentially shuts down proton pumps so if you have too much PMF because of an inhibitor being added or because there is no oxygen therefore no terminal electron acceptor left for example you have all these protons and kind of get stuffed up because then the proton pumps get shut down and there are not any enzymes to deal with this

Question 19

Catabolic:anabolic balance and ETCs

Answer 19

Non-proton pumping ETCs have increased flexibility to deal with environmental stressors when ATP demand is low

Low biomass synthesis = low ATP demand

Increased ATP = more biomass and can cause accidental exit from persistence
Persister cells have extreme problems with redox balance and back pressure because basically their only choice if they accidentally have too many protons is to make ATP by funnelling through ATP synthase

Alternative strategies include ATP hydrolysis, transporters that use H+

Question 20

Different ETC configurations are reflected in differences of central carbon metabolism …

Answer 20

Due to direct (FADH2) or indirect (NADH) links to citric acid cycle 
Produces necessary substrates or avoids them during reductive stress 
Focus for this module is on TCA cycle, but same is true for glycolysis and other pathways

Question 21

TCA rerouting

Answer 21

Producing NADH, FADH2 is not always desirable
Backpressure or lack of a terminal electron receptor

Alternative pathways that avoid NADH production

Used under hypoxia or antibiotic resistance

E.g Mycobacterium tuberculosis

Question 22

Reverse TCA (rTCA)

Answer 22

TCA operates in reverse direction (reductive rather than oxidative)
Green sulphur bacteria, ε-proteobacteria
Many enzymes replaced, especially those performing decarboxylation

CO2 is fixed from the atmosphere without needing photosynthesis

Chemoautotrophic growth

Deep see hydrothermal vents
Low light, plenty of inorganic carbon
rTCA highly prevalent
Thought that rTCA preceded photosynthesis in evolution

Question 23

TCA half cycles (forwards and backwards)

Answer 23

Reductive branch AND oxidative branch

Allows the production of key precursors while fumarate used as terminal electron acceptor

Genome typically lacks ⍺-KG dehydrogenase or it is not expressed anaerobically

e.g. E.coli, Helicobactor pylori

Question 24

No/limited TCA cycle

Answer 24

Facultative/obligate anaerobes that are normally fermenters

ETC used to regenerate NADH, gain extra energy, avoid toxic end products

Opportunistic pathogens can use extra energy for infection

e.g. enterococcus, streptococcus

Question 25

Fermentation maintains redox balance when …

Answer 25

ETCs fail, but fermentation end products need to be secreted