Lecture 10 - Bacterial ATP synthesis and how it became the newest target-space for antibiotics Flashcards
Terminal electron acceptors are used by
availability and energy gain e.g. oxygen -> nitrate -> fumarate - as determined by redox potentials
Cytochrome bd oxidase =
Cytochrome bd oxidase = high affinity for oxygen but less PMF
Nitrate =
Nitrate = used by nitrate reductase, redox loop generates PMF
Fumarate =
Fumarate = used by fumigate reductase, no PMF is made (smaller reduction potential difference, does not do the redox loop)
Other roles of PMF
Redox balance, radical detoxification, enzyme protections
In this case, PMF back pressure promotes non proton pumping configurations
Central metabolism is reconfigured in analogous ways
TCA rerouting, reverse TCA, TCA half cycles or no/limited TCA encoded
Fumarate reductase is the ….
least efficient enzyme at generating a proton motive force
Proton motive force
An electrochemical gradient
Pump protons and make our periplasmic space more acidic - pH gradient of protons that has energy stored in it since they want to diffuse back but they are prevented by the membrane
Energy stored in a build up of H+ and other ions in the periplasm
Drives many transporters. Uptake carbon/energy sources
Required for high efficiency ATP synthesis
Even fermenters need a PMF
Some bacteria use sodium ions instead of protons (they need specialised proteins to do this)
Back pressure
Cellular respiration and ATP synthesis occur in concert - tightly coupled
Back pressure = overload of protons in the periplasmic space, can feedback and inhibit proton pumps and this is why bacteria encode some non-protein pumping enzymes but normally under homeostatic conditions we have a fine balance between putting a lot of PMF that is enough to drive ATP synthesis as well as pumping it at the same time
Catabolism:anabolism and proton backpressure govern…
Catabolism:anabolism and proton backpressure govern all steps:
Central carbon metabolism
ETC activity
ATP synthesis
If these links are broke for any reason the cell is called an uncoupled cell
Cell exhausts energy currencies to try and restore PMF
F1F0 ATP synthase
2 major components
F1F0 ATP synthases found in mitochondria, chloroplasts, and bacteria. They are also known as F1F0 ATPases because they can catalyze ATP hydrolysis. The mitochondrial F1 component appears as a spheri- cal structure attached to the mitochondrial inner membrane surface by a stalk. The F0 component is embedded in the membrane. ATP synthase is on the inner surface of the plasma membrane in bacterial cells. F0 participates in proton movement across the membrane. F1 is a large complex in which three a subunits alternate with three b subunits. The catalytic sites for ATP synthesis are located on the b subunits. At the center of F1 is the g subunit. The g subunit extends through F1 and interacts with F0.
ATP synthase as the method from both utilising protons as well as producing the ATP and ATP feedsback into catabolic processes and these catabolic and anabolic processes make NADH…
Chemomechanical coupling
F1 subunit
Cytoplasmic
Binds ADP + Pi and converts to ATP - forms the phosphate high energy bond required to make ATP
Not in the membrane
Soluble fraction - can be knocked off and be soluble in the cytoplasm i.e. can be dissolved
F0 subunit
Membrane bound region
Motor
Able to rotate
Turbine is driven by H+ flow
Rotates central stalk in F1
Flow rotates the subunits and the rotation of the central stalk actually causes conformational changes which is called chemomechanical coupling because there is chemical energy from the proton motive force and then being transduced to mechanical energy in the stalk
Key subunits of ATP synthase
Catalytic subunits …
β - Binds ADP+Phosphate—> ATP
𝛾 - Rotates, induces conformational change
Ion binding subunits …
a-Take H+ and passes to c subunit
c-Rotated and releases H+ in cytoplasm
C-subunit has the proton and rotates it before releasing it into the cytoplasm
Other subunits have roles in regulation or complex stability
Catalytic subunits of ATP synthase
β - Binds ADP+Phosphate—> ATP
𝛾 - Rotates, induces conformational change
Ion binding subunits of ATP synthase
a-Take H+ and passes to c subunit
c-Rotated and releases H+ in cytoplasm
C-subunit has the proton and rotates it before releasing it into the cytoplasm
Other subunits of ATP synthase
Other subunits have roles in regulation or complex stability
Three major conformations of ATP synthase
Three major conformations: Open = Nothing bound Loose = ADP+Pi bound Tight = closed, forms ATP (when the stalk rotates it is closed) Three major steps in rotation Open - loose - tight - open etc 3 x 120 degree steps
Open configuration
Nothing bound
Loose configuration
ADP+Pi bound
Tight configuration
Closed, forms ATP (when the stalk rotates it is closed)
Binding change mechanism
Three major conformations:
Open = Nothing bound
Loose = ADP+Pi bound
Tight = closed, forms ATP (when the stalk rotates it is closed)
Three major steps in rotation
Open - loose - tight - open etc
3 x 120 degree steps
May be 6 or more subsets
May vary between organisms
Conformational change that causes something to happen but what is unique about this is that it is driven by its mechanical process
Textbooks = 3.3 H+ to 1 ATP made Reason = mitochrondrial enzyme has 10 c-subunits = 10H+ (how many protons you can bind per rotation) for full rotation = 3 ATP from 10 H+ However = Different organisms have different amounts of c-subunits. Anywhere from 2.7-5 H+ per ATP
Sodium motive force
Advantageous when passive proton leak/loss is high - some anaerobes
High temperature (makes plasma membrane more fluid and can cause things to passively diffuse more efficiently) or pH (alkaline environment)
e.g. Clostridium paradoxum
Group of organisms that tends to do this and might find that some organisms have this advantage to grow in high temperatures or alkaline environments by switching to a sodium motive force
Used by some pathogens
Fusobacterium nucleate: inhabits gums, periodontal disease
SMF and PMF at same time to get the benefits of both worlds
(Partially) vibrio cholera: cholera, free-living phase -> pH stress
PMF
PMF = c-ring glutamate binds H+ (functional group on amino acid binds protons)
SMF
SMF = Na+ coordinated in c-ring pocket by glutamate and others (instead larger space/cavity so less steric hinderance to allow the sodium to come and be bound more traditionally)
While PMF or SMF is essential for all organisms…
The ETC and F-type ATP synthase are not
Fermentation
synthesis of ATP exclusively by substrate level phosphorylation
Lower ATP yield compared OXPHOS
Used if OXPHOS not possible
Some bacteria are obligate fermenters - produce no OXPHOS genes, always ferment
Most pathogens use OXPHOS, but some exceptions (e.g. strep sore throat - streptococcus pyogenes)
Nowhere near as good as respiration
Pyruvate converted to reduced end products
e.g. alcohols and carboxylic acids
Avoids TCA cycle
“Last resort” strategy to maintain redox balance
End products are toxic - secreted. Not viable in very long term
Uses simpler enzymes - less cost to make (no complexes etc.)
End products dependent on the bacteria and the conditions
Pathway role - consume only NADH (generating NAD+ for catabolic reactions) … what are the end products
Lactate
Ethanol
2-propanol
Butanol (net)
Pathway role - produce ATP only (for anabolic reactions or for powering reactions that need ATP for them) …what are the end products
Acetate
Pathway - consume NADH and produce ATP (this occurs at the same time) … what are the end products
Propionate
Lactic acid fermentation
Homolactic = Streptococcus thermophilus and Lactobacillus delbrueckii. Only lactate —> yoghurt
Consuming only NADH and under these conditions their main priority is maintaining redox balance which is very industrially important for example with yoghurt
Heterolactic = Leuconostoc mesenteroides. Lactate, ethanol and acetate —> sauerkraut
Lactate is produced with other compounds
Mixed acid fermentation
Lots of different acids are produced
E. Coli normally uses all pathways simultaneously (simultaneous to try and get a many benefits and to be as diverse as possible so then you are not producing one toxic by-product, you can spread the load a little bit)
End products vary according to environmental conditions - still some OXPHOS linked steps
Alcohol fermentation
Usually ethanol is the majority product
Zymomonas mobilis - higher production than brewer’s yeast but fouls beer due to H2S etc. (makes taste funny and smell bad)
Interest in production of bioethanol (for fuel mostly)
How do we use this module’s information to design better antibiotics?
Fermentation could be a drug target
Cases study - Mycobacterium tuberculosis
Exemplary persistent pathogen - high drug discover efforts
Long treatment timeframe (at least 6 months but up to three years)
High incidence in developing countries approximately 1.5 million deaths per year
Bedaquiline and TB
Bedaquiline discovery 2005 - ATP synthase inhibitor
Reduction in treatment time frame from 6 to less than 2 months with combination therapy
ATP synthase c-ring identified as the target (stops protons form binding to the c-ring by bed aquiline binding there instead)
Effects on proton back pressure and catabolic:anabolic balance suggests upstream respiratory enzymes are also good targets BUT, bedaquiline was an uncoupler which means that it turned ATP synthase into a hole for protons and caused protons to flow through unregulated which is bad (acts like DNP)
Multitherapy with TB
Dealing with respiratory flexibility
Branched oxidase paradigm - TB has complex III/IV and cyt-bd
Block both oxidases = rapid death
NADH buildup very lethal due to lack of fermentation pathways in TB
Dual inhibition = completely cleared in mice
Multitherapy are essential for killing persisters, prevents rerouting
Block cyt-bd - good at controlling back pressure so when it is removed it causes back pressure which inhibits maybe type 1 NADH dehydrogenase, proton pumping complexes, NADH is going to go through the roof, central metabolism is going to shut down which will cause a lot of problems that can lead to cell death.
All forms of persistance cause a short to …
forms of energy metabolism that permit survival without growth
Environmental persistance
A population-level response to limited nutrient conditions
Antibiotic persistance
Is a non-heritable physiological change in a few cells that allows them to survive
stringent response, SOS response and toxin-antitoxin systems are some mediators of
antibiotic persistance
stationary phase / nutrient deprivation is one way that…
environmental and antibiotic persistance are linked
Glycolysis and TCA cycle produce NADH for
aerobic respiration
TCA takes …
3C products and performs oxidations
Beta oxidations and transamination can enter
fatty acids or amino acids into glycolysis or TCA cycle
Aerobic ETC produces enough …
PMF for high ATP synthesis
Oxygen is the terminal electron acceptor means
aerobically
Nitrate or fumarate are some…
anaerobic electron acceptors
Whether it is an aerobic or anaerobic ETC
the ETC follows the same general structure either way
Why is oxygen a better terminal electron acceptor than nitrate and fumarate ?
Molecular oxygen is a high-energy oxidizing agent and, therefore, is an excellent electron acceptor.
These molecules have a lower reduction potential than oxygen; thus, less energy is formed per molecule of glucose in anaerobic versus aerobic conditions.
anaerobic respiration: metabolic reactions and processes that take place in the cells of organisms that use electron acceptors other than oxygen
Why fumarate makes less PMF than nitrate?
Fumarate has a lower reduction potential than nitrate
Terminal oxidases such as complex III/IV pump
protons
ETC used to make
PMF
NADH/FADH2 comes from …. (aerobically)
TCA
OXPHOS is more efficient than
fermentation
SLP and PMF are
essential for growth regardless of pathway
Fermentation doesn’t require
Complex enzymes
Fermentation produces
toxic byproducts which are bad in the long term
