MM- LECTURE 23-25

Why does this TCA pathway have to operate under these anaerobic pathway:

1) To form precursor metabolites: succinyl coA, oxaloacetate, a-ketoglutarate 2) Operates to make these metabolites in such a way to minimise the production of reducing power to minimise the redox balance problem

Why does E.coli have a hierarchy for expression of different TEAs

1) Uses compounds that release most free energy first and then the least last - if all these options are available to it 2) Hierarchy of control determines which TEA is used first based on which one releases the most energy and has the most positive redox potential first

Basic ETC process

1. NADH > NAD+ 2. 2e- and 2H+ produced 3. Electrons pass through Dehydrogenases (contain flavoproteins and Iron-sulfer clusters) 4. To Quinones 5. To cytochromes (length varies). Cytochrome oxidase/ terminal oxidase turn electrons, protons and oxygen to water (2e- +2H+ O > H20 ) 6) Process is coupled with forcing H+ across the membrane for PMF Process releases free energy: Amount of free energy : delta G = -nF delta Eh [Eh = redox potential difference between the donor and acceptor in the ETC or redox reaction]

Why is the TCA cycle different under anaerobic conditions compared to aerobic conditions

2 limbed pathway as no ETC acting therefore NADH >NAD cannot happen!!! The ability to regenerate NAD becomes a problem, redox balance cannot be maintained, so this process operates to produce minimal excess reducing power NADH/NADPH and has a reductive arm to use up excess NADH

Similarity/difference of terminal oxidases across bacteria

Difference: Have different haems - Apparent diversity of composition of bacterial electron transport chains - particularly at the level of terminal oxidase Similarities: Similar at level of protein sequence - despite the wide variety of terminal oxidases all these enzymes (proteins) belong to the same protein family - known as the haem copper oxidase superfamily

Arc regulon - kinase activity under different oxygen conditions

Kinase activity is at 100% in no oxygen conditions and under 0% when under oxygen conditions

Why does redox balance need to be maintained?

Need to maintain redox balance because the oxidative reaction of glycolysis requires a continuous supply of NAD for that reaction t take place - so in the absence of maintaining this redox balance then glycolysis will stop and carbon utilisation

ETC under low oxygen conditions

New cytochromes are produced when E.coli is switched to low oxygen conditions. -Cytochrome oxidase changes: 1) Cytochrome bO3 oxidase - this pathway potentially still exists 2) New enzyme - cytochrome bd oxidase - is produced, giving a new alternative pathway

NarX NarL system

Process of narX and NarL system (2 component system) 1) Auto phosphorylates - transfers phosphate onto NarL (response regulator) 2) NarL phosphate acts to stimulate the transcription of nitrate reductase

Fnr protein role

Fnr protein is a direct sensor of oxygen concentration

Equation for free energy

Free energy : delta G = -nF delta Eh

Main role of TCA cycle during aerobic growth on glucose

Generation of precursor metabolites and DOES NOT need to operate as a full cycle

Fnr dimer

Active anaerobically Role: Transcriptional regulator - acts to both activate and inhibit transcription of certain genes 1) Inhibits genes encoding cytochrome bd and cytochrome bO3 2) So ensures there is no oxygen utilising terminal oxidases being made anaerobically 3) Activates enzymes such as nitrate reductase (and other reductases e.g. TMAO reductase etc.) and certain enzymes central metabolism stimulates (such as pyruvate formatalyase - part of fermentation pathway of E.coli)

Q:QH2 ratio - aerobic conditions

Aerobic conditions: 1) ETC oxidises XH2 to X: 2) Electrons are transferred along ETC: 3) Q reduced to QH2 4) Electrons passed from QH2 to cyt b03/cyt bd [QH2 becomes Q again] 5) Electrons used by cytoxidases to reduce oxygen to water Q:QH2 ratio: 1) Under aerobic conditions there is a certain level of Q and QH2 in the ETC

Aim of metabolic pathways in combination with ETC

Aim of metabolic pathways in combination with ETC: to produce sufficient energy and sufficient energy and sufficient reducing power for growth

Arc Regulon - study aim

Aim: was to identify an undiscovered regulatory system that somehow controls the switch of E.coli between aerobic and anaerobic conditions

Facultative anaerobe

An organism that makes ATP by aerobic respiration if oxygen is present but that switches to anaerobic respiration or fermentation if oxygen is not present.

Fnr regulon - anaerobic conditions

Anaerobic conditions - formation of active fnr dimer: 1) Nif-S protein and a small tri-peptide called glutofiome work together if provided with substrates (Fe2 and sulphide (S2-)) - can act on the 8-O fnr protein to form a protein that via the 4 cysteine residues (in its N terminal domain) can bind to an electron transfer component called iron sulphur cluster to form a four iron 4 sulphur, iron sulphur cluster 2) The fnr protein dimerises and becomes an active fnr dimer 3) Active fnr protein is stable under anaerobic conditions - In this form protein is active and can act as a transcriptional regulator

Arc proteins - anaerobic conditions

Anaerobic conditions: Cytochromes cannot reduce oxygen to water 1) So higher proportion of QH2 than Q (as electrons aren't passed to oxygen) ArcB senses this change in Q:QH2 ratio and becomes activated Active ArcB: 1) Hydrolyses ATP to ADP 2) Phosphate from ATP hydrolysis is used to phosphorylate the histidine amino acid acid as part of the cytoplasmic domain of the ArcB protein 3) ArcB o transfers this phosphate to its partner protein - ArcA forming ArcA phosphate, this phosphorylation is occurring at the aspartic acid residue of ArcA ArcA-P: 1) ArcA is a DNA binding protein so ArcA phosphate can change gene expression - ArcA is a transcriptional regulator 2) § Inhibits transcriptition of key genes: Binds to promoters of a range of genes that are normally expressed aerobically to stop that expression (including succinate dehydrogenase, a-ketaglutamate DH, cyo ABCDE genes that encode for cytochrome bo3 etc) 3) Switches on expression of certain genes: ArcA activates gene expression of the Cyd genes - these genes encode for the cytochrome bd oxidase (an enzyme required to use oxygen in the low oxygen conditions in E.coli)

Cysteines - ArcB

1) 4 cysteines are crucial to ArcB function (cysteine is an amino acid that has an SH group) 2) ArcB is sensing this change in Q:QH2 ratio - ArcB is sensing the availability of Q (oxidised form) that helps the formation of disulphide bonds and changes the conformation and kinase activity 3) Kinase activity: Leads to formation of ArcA phosphate - and thus activation of transcriptional regulator

Generating a membrane potential

1) A membrane potential is generated when chemical (or light) energy is used to pump protons (H+), Na+, or K+ to the outside of the cell, making the cation concentration (positive charges) greater outside the cell than inside. 2) For example, membrane proteins such as cytochrome oxidases use energy from respiration to pump protons across the cell membrane, and out of the cell, generating a proton gradient. 3) The proton gradient (ΔpH) plus the charge difference (voltage potential) across the membrane form an electrochemical potential. When this electrochemical potential includes a proton gradient, it is also called the proton potential, or proton motive force 4) The energy stored in the proton motive force can be used by specific transport proteins to move nutrients into the cell to directly drive motors that rotate flagella, and to drive the synthesis of ATP by a membrane-embedded ATP synthase

Modifying oxygen pulse technique

1) Above process uses NADH as an electron donor 2) Could use artificial electron donor e.g. DH2 oxidised to D: Can follow electron transfer from point of electron donation to oxygen and measure the proton translocation stoichiometry for this portion of the electron transport chain 3) So can adjust experimental conditions to either get the H:O ratio for the whole pathway or for a portion of the pathway and work out where proton translocation is taking place and its stoichiometry

Fnr regulon activity under different oxygen conditions

1) Active anaerobically 2) Inactive aerobically

Different energy generating pathways

1) Aerobic - presence of oxygen 2) Anaerobic - presence of nitrate but absence of oxygen 3) Anaerobic - absence of oxygen and absence of nitrate

Combining the systems

1) Arc system represses aerobic enzymes and switches on cytochrome bd 2) In anaerobic conditions the fnr suppresses cytochrome bd (as oxygen gets low) and switches on the production of the key reductases 3) If this was the only process then this would be quite wasteful - only want nitrate reductases anaerobically if there is nitrate in the environment etc. 4) Fnr has the potential to stimulate transcription of every anaerobic enzyme - but the specificity is controlled by a third system - e.g. NarX and NarL system for nitrate reductase (other systems exist for other reductases)

Overall control system for nitrate reductase:

1) Arc system switches off aerobic enzymes to get reduction of a nitrate reductase terminated ETC 2) Under anaerobic conditions with nitrate - need to bind the promoter of the nitrate reductase genes 3) Both fnr (which is signalling anaerobic conditions) and NarL phosphate (signalling presence of nitrate ) 4) If both are present then transcription can take place and nitrate reductase is produced and respiratory chain is built

Hierarchal control - expression of TEAs in E.coli

1) Bacterium like E.coli can use multiple different electron acceptors 2) The amount of energy released from these TEAs varies - strict hierarchy [Oxygen then nitrate then DMSO then TMAO then fumarate]

Sources of energy

1) Chemical energy that is contained in the high-energy phosphate bonds in adenosine triphosphate (ATP) 2) Electrochemical energy, that is stored in the form of an electrical potential generated between compartments separated by a membrane. Energy stored by an electrical potential across the membrane is known as the membrane potential (for most cells the membrane potential is more negative inside than outside).

Cytochrome bd oxidase:

1) Cytochrome bd oxidase is made under low oxygen conditions 2) Cytochrome bd oxidase is a quinol oxidase - gets electrons from reduced quinones

Why doesn't the bacteria make bd oxidase under all conditions because of its high oxygen affinity?

1) Cytochrome bd oxidase terminates the pathway with a lower energetic efficiency 2) A pathway that translocate fewer protons contributing less to proton motive force

MacConke medium - aerobic conditions why are colonies red?

1) E.coli forms red colonies 2) Promoter for SDH gene is induced - so betaglactosidase is produced 3) Production of betagalactosidase enables E.coli to break down lactose 4) Despite aerobic conditions some parts of colony become anaerobic and lactose gets broken down by fermentation reactions to form an acid 5) Formation of acid lowers the pH - giving colony 'red' colour

MacConke medium - anerobic conditions why are colonies white?

1) E.coli forms white colonies 2) Promoter of SDH gene is OFF - so no betaglactosidase is produced, lactose isn't broken down and no acid is formed - giving colony a 'white' colour

ETC and the energy released

1) Each component of the ETC comprise a redox couple - which redox potential stays a small difference from its preceding neighbour 2) So electron transport within the pathway is associated with release of small amounts of free energy 3) This packaged release of free energy allows it to be conserved in a way that is useful to the organism 4) This is achieved by proton translocation - occurs by a variety of mechanisms at different points of the ETC 5) Proton translocation mechanisms - all three operate in the mitochondrion , but not all the three mechanisms work in all bacteria all the time [Redox loops, Q cycle, Proton (H+) pumps]

Quinol oxidase role

1) Electron donor to this enzyme is not cytochrome C but instead it is quinol (hence it is called quinol oxidase) 2) This oxidase transfers electrons to oxygen (reducing oxygen to water) 3) Enzyme present in E.coli

Cytochrome B03 oxidase

1) Enzyme present when the bacterium is under high oxygen conditions 2) Low oxygen affinity 3) It oxidises quinols so often called a quinol oxidase (not a cytochrome c oxidase)

Cytochrome bd oxidase

1) Enzyme present when the bacterium is under low oxygen conditions 2) High oxygen affinity

How is redox balance maintained under anaerobic growth?

1) Fermentation reactions are not maintaining redox balance 2) Too much reducing power would be made and cell will not maintain redox balance 3) So there is a change in how the TCA cycle operates

Production of fnr protein

1) Fnr gene and protein it encodes is produced throughout growth under all conditions 2) Fnr gene is transcribed and translated - producing fnr protein (8-O fnr)

Fnr regulon - aerobic low oxygen conditions

1) Fnr protein destabilises - lose some of the iron and sulphur , resulting in a form of fnr that has a 2 iron 2 sulphur cluster 2) This new form is now a monomer (no longer a dimer) ad is inactive as a transcriptional regulator 3) After this first stage this fnr protein can redimerise and be built up again to form an iron sulphur cluster more rapidly and dimerise 4) Can switch between active dimer and inactive monomer (after this first stage)

NarX

1) Histidine kinase 2) Activated by the presence of nitrate in the environment

Paracoccus denitrificans

1) Main pathway is closely related/resembles mitochondria - The study of this ETC of this bacterium led to the endosymbiotic hypothesis 2) Common soil bacteria

Fnr protein

1) N terminus - N terminal domain contains 4 cysteine residues 2) Central domain - dimerization domain 3) C terminus - C terminal h-th domain (helix turn helix domain) Helix turn helix domain is characteristic of DNA binding proteins

Components of the paracoccus denitrificans ETC

1) NADH dehydrogenase (similar to Complex I of mitochondria) 2) Succinate dehydrogenase (similar to complex II 3) Ubiquinol-cyt-c oxidoreductase (similar to complex III of mitochondria) 4) Terminal oxidase - aa3 oxidase is a cytochrome c oxidase (similar to the oxidase of mitochondria known as complex IV)

Process of dentrification

1) NO3- -> NO2- (using nitrate reductases) 2) NO2- -> NO (using nitrite reductase) 3) NO -> N2O (using nitrous oxide reductase) 4) N2O -> N2 (using nitric oxide reductase)

Anaerobic growth on glucose - TCA cycle

1) Not a cycle - operates as a two limbed pathway 2) No a-ketoglutarate dehydrogenase enzyme (converts a-ketoglutarate to succinyl coA) as the gene transcription of this enzyme is inhibited under anaerobic conditions 3) Lower limb: oxidative arm - Forms some reducing power (results in formation of alpha ketoglutarate) 4) Upper limb: reductive arm - Operates with a series of new reactions - which use up reducing power ultimately forming succinyl co-a

E.coli ETC under high oxygen conditions - process

1) Oxidises compounds such as NADH, succinate, glycerol etc. 2) Electrons are transferred along the ETC 3) Quinol oxidase is the terminal oxidase and oxidises quinols and reduces oxygen

Enzymes involved in TCA cycle during anaerobic growth

1) PEP carboxylase 2) Pyruvate formatolyase 3) Asp transaminate 4) Aspartase 5) Fumarate reductase

Proton translocation mechanisms

1) Redox loops 2) Q cycle 3) Proton (H+) pumps

ETC of aerobic TCA cycle

1) Reducing power is generated by glycolysis and other reactions occurring in TCA 2) This reducing power can be oxidised by the ETC aerobically

Fnr regulon

1) Role: positive activator of anaerobic gene expression 2) 30 kd protein (small) 3) One component regulator (ArcA and ArcB are a two component regulator)

List enzymes that Arc A and B mutants lose the ability to repress under anaerobic conditions

1) TCA cycle enzymes: SDH, a-KGDH, pyruvate DH 2) Lactate DH (normally only made aerobically) 3) Cytochrome bo3 (failure to repress expression of cytochrome bo3) - Cytochrome bo3 is encoded by cyoABCDE genes

ETC - anaerobic growth on glucose

1) Terminal electron acceptor: No oxygen and No alternate terminal electron acceptor - so no ETC 2) Fermentation reactions occur (in absence of ETC)

Terminal reductase

1) Terminal reductases (these enzymes can operate under anaerobic conditions) 2) When bacteria grow in anaerobic environments, the terminal electron acceptor is reduced by an enzyme called a reductase

ArcB protein (about and the role)

About: 1) A membrane bound protein - located in cytoplasmic membrane with a large cytoplasmic domain 2) Histidine kinase - as its histidine amino acid of its cytoplasmic domain is phoshorylated Role: 1) ArcB senses the shift from high to low and then onto no oxygen conditions (anaerobic condtions) - doesn't sense oxygen, it senses the ratio of quinone: quinol 2) Upon activation (by change in Q:QH2 ratio) ArcB hydrolyses ATP to ADP 3) Transfers phosphate from ArcB's histidine amino acid to ArcA forming ArcA phosphate (activates ArcA protein)

ArcA protein

About: 1) Repsonse regulator 2) A DNA binding protein so ArcA phosphate acts as a DNA binding protein and changes gene expression Role upon activation into ArcA phosphate: 1) Inhibits transcription of key genes: Binds to promoters of a range of genes that are normally expressed aerobically to stop that expression (including succinate dehydrogenase, a-ketaglutamate DH, cyo ABCDE genes that encode for cytochrome bo3 etc) 2) Switches on expression of certain genes: ArcA activates gene expression of the Cyd genes - these genes encode for the cytochrome bd oxidase (an enzyme required to use oxygen in the low oxygen conditions in E.coli)

What type of reaction happen in addition to regular reactions during aerobic glucose respiration

Anapleurotic reactions occur in these pathways: 1) Generate intermediates that may not be effectively formed when these pathways operate in real world situations 2) Ie Phosphoenolpyruvate > Oxaloacetate is catalysed by phosphoenolpyruvate carboxylase is an anapleurotic reaction to ensure there is enough oxaloacetate formed as its not directly formed through the cycle (half cycle)

ArcA and ArcB genes

ArcA and ArcB genes encode proteins called ArcA and ArcB proteins

What happens when there is no oxygen left for bacteria?

Can use an anaerobic electron transport chain - anaerobic respiration Process: 1) Transfers electrons to quinones of the electron transport chain - generating a quinone pool 2) Electrons are transferred to an appropriate terminal reductase to allow anaerobic respiration to take place as long as the substrate for that reductase is present in the environment E.g. nitrate reductase - if nitrate is available in anaerobic conditions then the bacteria can use this nitrate as an electron acceptor E.g. dimethyl sulfoxide (DMSO) Components: Dehydrogenases: can oxidise molecules, produce molecules generated by metabolism

Arc A and Arc B mutants

Common property: expressing aerobic genes/enzymes anaerobically These enzymes are normally expressed aerobically, repressed anaerobically - but this repression is lost in arcA and arcB gene mutants

Components of cytochrome bd oxidase

Components: 1) Contains different cytochromes/ haems : B558, B595 and BD (cytochrome B D) 2) Contains no copper groups 3) So not a member of the haem copper oxidase family - one of only bacteria to not be a part of this protein family, must have evolved differently and distinctly

Arc regulon - method of study

Constructing gene fusion 1) Searched for global regulatory system - constructed a gene fusion for the SDH gene 2) Fuse promoter of SDH gene (gene they wish to study) to a LacZ (reporter gene) 3) Amount of beta galactosidase transcribed is dependent on transcription of promoter of SDH (pSDH) Testing gene fusion 1) Grew E.coli in anaerobic conditions (no oxygen) 2) Measured betagalactosidase activity - levels of lacZ protein 3) Found 70 units of B-gal activity under anaerobic conditions vs 700 units of B-gal activity under aerobic conditions 4) Gene fusion was expressing aerobically and repressed 10 fold anaerobically Used a selective medium - MacConkey agar to demonstrate whether an E.coli is expressing lacZ or not 1) Aerobic conditions on this MacConkey medium: E.coli forms red colonies 2) Anaerobic conditions on this MacConkey medium: E.coli forms white colonies

Difference between the cytochrome oxidases E.coli uses under different oxygen conditions:

Cytochrome bd oxidase: 1) Activity is inhibited by 50% by 2 mM of KCN (cyanide inhibits respiration by binding or competing for binding to the oxygen binding site in this type of enzyme) 2) Km for oxygen (affinity of enzyme for its substrate) ; 0.24 - 0.38 micromolar , very high oxygen affinity 3) Low energetic efficiency (h+/2e- = 2) and can maintain redox balance (NADH>NAD) Cytochrome b03 oxidase: 1) Activity is inhibited by 50% by 0.01 mM of KCN - Inhibited by much lower concentration of cyanide - suggests that b03 oxidases active site is different 2) Km for oxygen: 2.9 micromolar - has a higher km and so lower affinity (oxygen) than cytochrome bd 3) High energetic efficiency (h+/2e- = 4).

Change in oxygen concentration affect on cytochrome composition [E.coli]

Cytochrome composition of our bacterium has changed as we have changed oxygen concentrations in the growth environment: These changes reflect major change in composition of the aerobic respiratory chain High oxygen conditions: Cytochrome b03 oxidase (seen as one peak) Low oxygen conditions: 1) Generating growth conditions: using chemostat of shaking very slowly at 50 rpm 2) Additional peaks in absorbance spectrum - characteristic of the production of a new type of cytochrome, called cytochrome bd oxidase

Cytochrome terminal oxidase

Cytochrome oxidase for terminal electron acceptor

E.coli ETC under high oxygen conditions - components

Dehydrogenases: 1) NADH dehydrogenase 2) Succinate 3) Glycerol Quinones: 1) UQ/MK Cytochromes: 1) Cytochrome b03 oxidase (different to cytochrome aa3 oxidase) - it oxidises quinols so often called a quinol oxidase (not a cytochrome c oxidase) 2) Terminal oxidase: quinol oxidase looks differently to other terminal oxidases

Different components of the ETC

Dehydrogenases: LHS 1) E.g. NADH oxidation - uses NADH dehydrogenase 2) Dehydrogenases are common enzymes t mitochondria, bacteria ETCs 3) Dehydrogenases comprise of: Flavour proteins (Fp) AND Iron sulphur (FeS) proteins Quinones: MIDDLE Cytochromes: RHS 1) At end of pathway - at terminal electron acceptor - the cytochrome is specifically cytochrome terminal oxidase

E.coli's prefered energy generating pathway

E.coli favours particular energy generating pathways: 1) Energy yield varies between these processes - so E.coli prefers particular energy generating pathway 2) Bacteria must sense the availability of oxygen, nitrate and bring about regulatory changes because of this sensing to construct the most appropriate energy generating pathway (coordinated by global regulatory systems)

How is redox balance maintained in aerobic E.coli ETC

ETC provides a way of re-oxidising NADH to NAD (ETC couples this with oxygen reduction to water)

Eh

Eh = redox potential difference between the donor and acceptor couple in the ETC or redox reaction (NADH+NAD and O2 and H20)

Energetic efficiency

Energetic efficiency = How bacteria phenotypically modify their ETC composition in response to environment: For ETC in a bacterium (oxidising NADH) - what is the energetic efficiency? How many protons does it translocate when electrons are transferred to oxygen or alternative electron acceptor?

Paracoccus denitrificans - cytochrome aa3 oxidase

Enzyme is called cytochrome c oxidases - as they oxidise cytochrome C 1) Electron donor for cytochrome aa3 oxidase is cytochrome C (Takes reduced form of cytochrome c and oxidises it, transferring electrons into the enzyme) 2) Enzyme has 2 types of electron transferring groups: Haem a and Haem a3 & CuA site (contains 2 Cu atoms) and CuB site (contains 1 Cu atom) 3) Electrons are transported through the pathway: Electrons transferred through the Heam a and 2x copperA centres to two more centres: Haem A3 site and single copper B site 4) Electrons transferred onto oxygen HaemA3/CopperB site is the oxygen binding site (haem-copper binuclear centre). Haem and copper come together VERY CLOSE to bring about oxygen reduction 4) REDUCTION: oxygen accepts 4 electrons and is reduced to water (2H20) Haem copper binuclear centre: 1) Haem group and copper sites are close to each other 2) It is the site where oxygen is found and reduced Energy released during transfer of electrons 1) This energy released leads to proton translocation

Components of the aerobic TCA cycle and products formed

Enzymes involved: 1) Pep carboxylase - responsible for conversion of PEP (phosphoenolpruvate) into oxaloacetate 2) Other enzymes are operating during TCA cycle to convert different metabolites into one another What products are formed? 1) TCA cycle in E.coli operates primarily to form three precursor metabolites: oxaloacetate, succinyl coA, a-ketoglutarate 2) TCA cycle generates reducing equivalents of NADH and FADH2 - required to transfer electrons to the ETC

Availability of oxygen (or other TEA) regulates energy generating pathways - e.g. TEA

Excess energy cannot be used or stored - no purpose in making excess energy Central metabolic pathways operate differently depending on the environment e.g. availability of oxygen (Aerobic conditions or anaerobic conditions 1) Aerobic conditions; truncated cycle that operates to meet the cells needs because of metabolites reducing power and energy 2) Anaerobic conditions: maintaining redox balance is difficult due to no ETC and repression of dehydrogenase - pathway is modified to minimise the production of reducing power

E.coli and respiration

Facultative anaerobe: Can obtain energy using aerobic respiration or anaerobic respiration/fermentation (in absence of oxygen)

Electron transport chain

Final stage in the flow of electrons from organic molecules (carbon) under aerobic conditions to oxygen.

Oxygen pulse technique

Grow bacteria aerobically Make a kind of preparation from bacterial cells - e.g. right side out vesicle (RSV) 1) Break bacterial cell open (using chemical approach) under certain conditions that breaks cell wall and cytoplasmic membrane 2) Fragments of cytoplasmic membrane reform and fold - forming small right sided vesicles (intact membrane spheres) 3) Can prepare these RSV (vesicles) in such a way as to trap certain chemicals within them e.g. prepare these vesicles so they have a certain concentration of NADH within them Move vesicles into anaerobic conditions (no oxygen) 1) Without oxygen so ETC of RSV cannot function Place vesicles in a lightly buffered medium Add a known amount of oxygen 1) ETC now able to function in this vesicle - as oxygen is an electron acceptor ETC functions: 1) Oxidation: NADH oxidised to NAD 2) Electrons are transferred along ETC 3) Reduction: oxygen is reduced to water by accepting electrons from ETC 4) Energy release due to proton translocation Result of electron transfer - energy release 1) Proton translocation: protons translocated outside of vesicle Measure external pH change via [H+] : [O] ratio using a sensitive pH electrode

Energetic efficiency of the paracoccus denitrificans ETC

H+ : O ratio of 10 (similar to mitochondria) 1) 4 protons translocated in early part by redox loop 2) 4 protons translocated in central part by Q cycle mechanism 3) 2 protons translocated by cytochrome oxidase (final part) by proton pump mechanism

E.coli ETC under high oxygen conditions - energetic efficiency

H+ : O ratio of 4-6

Why does cytochrome change when bacteria are switched from high oxygen to low oxygen?

High oxygen: 1) Only upper pathway is operating using only cytochrome b03 oxidase (low affinity for oxygen) 2) Electron transport route from NADH to oxygen gives an H+ : O ratio of 5-6 3) As concentration of oxygen drops in environment this bacteria starts to work less well Change : 1) Bacteria senses change in oxygen concentration in environment and produces cytochrome BD oxidase (has a much higher affinity for oxygen) Low oxygen: 1) Bacteria will tend to use the lower pathway - using an enzyme with very high oxygen affinity to scavenge the last molecules in the environment

Fnr regulon - Aerobic high oxygen conditions

If aerobic conditions persist: 1) If concentration of oxygen persists (or gets higher) then another 2 irons and 2 sulphurs are removed - reforming the 8-O fnr protein 2) Monomer degrades to form the 8-O enzyme cannot easily switch back to active dimer, need to reform the active dimer all over again

Paracoccus nitrificans - pathway under anaerobic conditions:

It is a facultative anaerobe - so able to produce anaerobic respiratory chains Components - pathway also contains terminal reductases (these enzymes can operate under anaerobic conditions) 1) 3 types of nitrate reductases, a nitrite reductase, nitrous oxide reductase, nitric oxide reductase 2) Enable paracoccus to carry out the process of denitrification - can reduce nitrates to nitrite, nitrite to nitrous oxide (NO), nitrous oxide to

Arc regulon : ArcB senses ratio of Q:QH2 [no oxygen, low oxygen, high oxygen]

No oxygen: 1) Anaerobiosis 2) 4 cystines (4 CSHs) 3) 100% kinase activity - maximum efficiency of phosphorylating itself Low oxygen: 1) Aerobiosis 2) Oxygen available - by operation of ETC the oxygen will attempt to oxidise QH2 to form more Q (changing the Q:QH2 ratio) 3) So quinone can directly oxidise the SH groups of the cysteine - this oxidation leads to the formation of a disulphide bond 4) This disulphide bond changes the conformation of protein in the cytoplasmic space 5) Now only 15% kinase activity (Reduced kinase activity) High oxygen: 1) Increase in level of Q - Q:QH2 ratio changes 2) Increased level of Q leads to oxidation of the two other cysteines - resulting in formation of 2 disulphide bonds in the cytoplasmic domain 3) This change in conformation results in 0 kinase activity

Role of the ETC in aerobic TCA cycle

Producing energy 1) Most energy is produced via glycolysis (ATP released when PEP is converted to pyruvate) Maintaining redox balance 1) Provides a way of re-oxidising NADH to NAD (ETC couples this with oxygen reduction to water) 2) Need to maintain redox balance because the oxidative reaction of glycolysis requires a continuous supply of NAD for that reaction t take place - so in the absence of maintaining this redox balance then glycolysis will stop and carbon utilisation 3) Maintaining redox balance is straightforward - due to ETC operation

Cytochrome C oxidase

Protein complex that serves as the final electron carrier in the respiratory chain; removes electrons from cytochrome c and passes them to O2 to produce H2O.

How do you quantify energetic efficiency

Quantifying energic efficiency: using the H+ : 2 electron ratio OR H+ : O ratio 1) Quantifies how many protons are being translocated for every two electrons that are transferred through the pathway to oxygen 2) How many protons are translocated for every oxygen atom reduced

Quinol oxidase

Quinol oxidase often called cytochrome B03 oxidase: It oxidises quinols so often called a quinol oxidase (not a cytochrome c oxidase) Components: 1) Contains cytochromes - cytochrome b/heam b , cytochrome O or O3 and contains copper 2) Cytochrome O3 and copper form a haem copper binding centre that is chemically/closely similar in the cytochrome C oxidase of paracoccus

RSV

RSV: RIGHT SIDE OUT VESICLE 1) Contains all components (e.g. ETC) of cytoplasmic membrane in the same orientation in which they were placed in original bacterial cell 2) No cell wall (for gram negative bacteria there is no outer membrane) - avoiding issues with permeability of chemicals we wish to use which would be affected by having whole cell

Difference/similarity between bacterial energetic pathway and mitochondrial energetic pathways

Similarity: 1) Both contain similar complexes/components/enzymes of pathways E.g. Structure of cytochrome aa3 oxidase is similar to complex IV of mitochondria - the core catalytic subunit is highly conserved Difference: Bacteria have alternative pathways they can adopt and use depending on the environment they encounter E.g. paracoccus denitrificans bacteria have 3 different oxidases (cbb3 oxidase and cytochrome c peroxidase, aa3 oxidase) - 3 alternative ways of sending electrons to oxygen as apart of electron transport chain (depending on environment) Bacteria are capable of changing their electron transport chain composition

Advantages of dentrification

So can convert nitrate to N2 - environmental benefit particularly in nitrate polluted soils/water

Aerobic growth on glucose - TCA cycle

TCA cycle operates in a truncated form under aerobic conditions: Full cycle not necessary as sufficient energy if being generated by preceding reactions (such as glycolysis and pentose phosphate pathway) to support growth and support the incorporation of carbon being provided into cell material

Cytochrome C

The enzyme to which electrons are transferred in complex III of the electron transport chain.

Proton motive force

The potential energy of the concentration gradient of protons (hydrogen ions, H+) plus the charge difference across a membrane.

ArcA and arcB genes operate as a...

Two-component regulatory system. ArcB is a histidinekinase (sensor) ArcA is a response regulator (work with sensors, to sense environmental signal and translate it to an inner cell signal). They do this in response to a shift between aerobic and anaerobic conditions.

Purpose of oxygen pulse technique:

Want to determine the proton stoichiometry of its ETC - want to determine the proton transport efficiency (energetic efficiency)

Discovering ARC mutants

White colonies formed: E.coli are carrying SDH lacZ gene fusion on MacConke agar in anaerobic conditions - these gene fusions are not switched on (anaerobically) In some white colonies there would be a red sector after 5 days 1) White part: phenotype is lac- (not making beta-galactosidase 2) Red part: phenotype is lac+ (part of cell is making beta-galactosidase) Cells in red sector: 1) Cells in that red sector have now switched on the SDH lacZ gene fusion under anaerobic conditions, lost ability to repress expression of that gene 2) Red sector could have only occurred due to a secondary mutation - which has resulted in the loss of repression of Lac SDH gene promoter Mutant selection procedure: 1) Needed to find a mutant in a gene that might be affecting expression of target gene 2) Took the cells and obtain a tiny sample of cells from these red sectors 3) Grow these cells (From red sector) 4) Purify these cells into single colonies 5) Study - mutants were termed arc mutants (aerobic respiration control mutants) Result: 1) Identified mutations in two genes Arc A gene and Arc B gene 2) These mutants with these mutated genes had lost the ability to repress succinate dehydrogenase under anaerobic conditions but also had lost the ability to repress a wide range of other anaerobic genes and enzymes