MICRB 265 topic 5

What is the difference between SLP and oxidative phosphorylation?

SLP: metabolic reaction/substrate provides energy to drive ATP production

OxPhos: using the ETC, oxidation (eletron transfer reactions) generate a proton motive force that powers the ATP synthase

What is photophosphorylation?

Capturing light energy to create a proton motive force that drives phosphorylation (of ADP)

Cell resp net reaction and free energy change

Glucos + 6O2 --> 6CO2 + 6H2O

-2895 kJ

What organic molecules can chemoorganotrophs use?

Prefer glucose, but can use dissacharides or other monosaccharides that feed into glycolysis or the CAC

the ATP in glycolysis is made via

SLP (at 1,3-BP to 3-P-glycerate and from PEP to pyruvate)

Glycolysis net reaction

Glucose + 2Pi + 2ADP + 2NAD+ → 2pyruvate + 2ATP + 2NADH + 2H+ + 2H2O

What types of organisms are more likely to use fermentation?

Static, simple organisms (don't need much E or oxygen, just to restore redox balance from NADH made in glycolysis)

CAC is in the

mitochondrial matrix for eukaryotes, the cytoplasm for prokaryotes

Why is the CAC a hub for metabolism?

Organic molecules feed into the CAC to be used for energy, and they exit to provide key anabolic intermediates

Not always aerobic, variations on exact cycle

Pyruvate is converted into ________ to enter CAC

Acetyl Co-A

Pyruvate + NAD+ + CoA --> Acetyl Co-A + NADH + CO2

CAC begins when citrate is made from

acetyl CoA (2C) condensing with oxaloacetate (4C)

NADH vs NADPH

NADPH has phosphate group and tends to work in anabolism rather than ETC

Isocitrate to a-KG step can use NAD+ or NADP+ depending on the species

CAC net reaction

Acetyl CoA + 2 NAD+ + NADP+ + FAD + Pi + ADP + 2H2O --> 2CO2 + CoA + 2NADH + NADPH + FADH2 + ATP + 2H+

(plus one more Co2 and NADH from pyruvate to ACoA)

Two turns per glucose

Reduce lots of electron carriers!

ETC is in

Cytoplasmic membrane for prokaryotes, inner mitochondiral membrane for eukaryotes

Depending on species/conditions, terminal electron acceptors can also be

NO3-, SO42-, multiple

Electrons are passed from ________ in increasing order of reduction potential

NADH/FADH2 --> flavoproteins --> Fe/S proteins -> quinones --> cytochromes --> O2

Iron-sulfur proteins can be in what two types of clusters? Affects what?

Fe2/S4 OR Fe4/S4 --> affects reduction potential and oxidation state of iron, affects how it interacts with the protein

Fe/S clusters are

Metal cofactors used by many different proteins involved in electron transfer (proteins maintain proper chemical environment for ions to give and take e-)

Quinones are

Small membrane-soluble (hphob) molecules (NOT PROTEINS) that can shuttle electrons between carriers (often link Fe/S proteins to cytochromes)

Quinones (ubiquionone) shuttle ____ e- from Fe/S cluster in photosystems to cytochrome C

2 electrons at a time

Cytochromes contain what prosthetic groups?

Heme (iron coordinated within organic molecule)

Different proteins/different heme groups can change reduction potential of cytochrome

_______ are typically the last carrier before the terminal acceptor in the ETC

cytochromes

Terminal electron acceptors are _____ quickly and must form _______

Used up (hence we breathe); must form non-toxic product or be removed/processed immediately

NADH produces ______ H+ pumped, FADH2 provides ____

10 (4 at complex I), 8 (2 at complex II)

NADH is a better electron donor (more negative reduction potential) so more energy is released

How many H+ pumped out gives the drive to make one ATP

About 3.3

This is an average number, not the amount actually flowing back in through the ATP synthase

How is the ATP synthase reversible? How do fermenters use this?

Can hydrolyze ATP which provides the energy to pump protons back out and restore the gradient

In fermentation, the ATP synthase is used to create a proton motive force as an energy supply to drive other reactions

Aerobically one glucose yields about how many ATP

38

Chemoorganotrophs convert fatty acids to ______ using _____

Acetyl CoA, ß oxidation

Chemoorganotrophs convert amino acids to ______

TCA/CAC/Krebs intermediates like pyruvate

Anaerobic terminal electron acceptors in the ETC can be

Nitrate, sulfate

Some microbes can switch between acceptors depending on availability

E. coli is _______robe

Facultative anaerobe -- assembles different ETCs depending on conditions

E. coli can use _______ anaerobically

Nitrate or dimethyl sulfoxide in ETC (not as good as O2 but better than just glycolysis/fermentation), or fermentation as last resort if not available

E. coli don't have _____ in ETC

Complex III, and the final one can change depending on what acceptor to use

Fermentation restores redox balance by

Oxidizing electron carriers (move electrons to fermentation products) and excreting the products

Heterofermentative lactic acid fermenters vs homo

Het = generate mix of lactose and other lactose products - can be useful to avoid lactate/lactic acid fermentationm (low pH can damage cell)

Lactic acid fermentation equation and enzyme

2 pyruvate + 2 NADH --> 2 lactate (excrete) + 2 NAD+

Lactate dehydrogenase

Lactic acid fermentation used for

yogurt, kimchi, sauerkraut

Ethanol fermentation equation and enzyme

1. 2 pyruvate ---(pyruvate decarboxylase)--> 2 acetaldehyde + 2 CO2 (excreted)

2. 2 acetaldehyde + 2 NADH ---(alcohol DH)--> 2 ethanol (excreted) + 2 NAD+

Ethanol fermentation used by

yeast (Saccharomyces cerevisiae) and some bacteria

Ethanol fermentation used to

Make alcoholic drinks

Baking (CO2 raises dough and alcohol will be evaporated off)

Naturally carbonate drinks before forced carbonation

Diversity in fermentation

Fatty acids, amino acids, purines/pyrimidines can all be fermented

Different fermentation products can be make (e.g. mixed acid fermentations - acetate, lactate, succinate, formate, ethanol: prevent accumulation of these metabolites)

Overall common theme is that you generate an E-rich bond that drives ATP synthesis (PEP), donate e- to metabolite (pyruvate/acetaldehyde), and excrete it to get rid of electrons and get redox balance (ethanol/LA)

Only _______ are chemolithotrophs

Prokaryotes (euks need organic food)

Chemolithotrophs can be found

Anywhere with reduced inorganic compounds, but commonly are extremophiles (because no organic sources since other stuff is killed)

Inorganic e- donors include

H2S, H2, Fe2+, NH4+

T/F: chemolithotrophs are anaerobic

F: can be either (many use O2 in ETC)

T/F: chemolithotrophs are mostly autotrophs

True: make their own food by fixing CO2 into organic molecule. REQUIRES LOTS OF NADH (REDUCING POWER) for biosynthetic reactions to allow this

Complex II aka

succinate DH (oxidises succinate to fumarate, generate FADH2)

Common sulfur electron donors (diff E'º and oxidation states)

H2S, elemental S, thiosulfate (S2O3 2-), sulfite (SO3 2-)

Elemental S can be stored in sulfur granules as an energy/electron reservoir)

Note that ACCEPTOR doesn't have to be sulfur just because the food source is (organisms adapt to use what is available, so donors and acceptors can be anything)

Final oxidation product of sulfur donors usually

sulfate

Oxyenic bacteria

Make O2 as biproduct of photosynthesis (e.g. cyanobacteria, algae (eukaryotes)

Anoxyenic bacteria

More ancient than oxygenic phototrophs

Green sulfure bacteria, phototrophic purple bacteria

T/F: phototrophs can be auto or heterotrophs

True, but mostly are autotrophs (use photosynthesis)

Rarely they can get carbon from organic molecules but use light energy to power ETC (photoheterotrophs)

Photosynthetic reaction centres

Complexes of proteins & pigments where electrons are excited and transferred to the ETC

What uses chlorophylls

oxygenic phototrophs

Look like heme groups, but have Mg instead of Fe

What uses bacteriochlorophylls

anoxygenic phototrophs

Look like heme groups, but have Mg instead of Fe

Antenna pigments

Complexes of bacteriochlorophylls/chlorophylls embedded in the membrane that capture light energy and transfer to reaction centre (optimize light capturing)

P870

Bacteriochlorophyll in purple bacterior that absorbs light E and goes from weak to strong e- donor

Donates electrons to a quinone where they travel down an ETC and make ATP (P870* then goes back to normal)

Cyclic photophosphorylation

Electrons at the end of the ETC (at cytochrome C2) are taken up by the bacteriochlorophyll (no terminal electron acceptor) and returned back to the first quinone

Happens not in everything but in purple bacteria and some others

Cytochrome C2 can donate backwards because P870 isn't a very good electron donor normally (positive ish Eº')

______ allows different phototrophs to coexist in same habitat

Different pigments with different absorption ranges -- allow organisms to take in a wide spectrum of light from the sun

Pigments can allow you to use light others can't

Q-type reaction centre

Electrons from transferred from reaction centre to quionones

Other anoxygenic bacteria use FeS type

FeS type reaction centres are _____

More efficient at capturing E (Fe/S clusters are stronger electron donors - electrons release more E as they're passed on (more negative reduction potential)

Phototrophs make ______ to use as reducing power in driving metabolism

NADH or NADPH

Electrons for phototrophs usually come from

External donor like H2S (enter quinone pool)

If anoxygenic phototroph lacks an electron donor with negative enough reduction potential to pass external e- to NAD+, what does it do?

It has to use reverse electron transport (use proton motive force to drive electrons in opposite direction of ETC, e.g. from quinones to NAD+) so that it can get a source of reducing power for biosynthesis (NADH)

Chloroplasts in eukaryotes evolved from

Cyanobacteria engulfed

PSI aka

P700 - Fe/S type

PSII aka

P680 - Q type

Photosystems are in the

Cytoplasmic membrane for cyanobacteria, thylakoid for eukaryotes (in stacks)

Photosynthesis for oxygenic phototrophs starts at

PSII: excited by light, transfers electrons to quinone pool in system and down ETC (PSI, NADP+)

Pumps out some protons

What happens when light hits PSII (P680)?

P680 donates electrons to the quinone pool and becomes very electropositive

Can take electrons from water to restore electrons (split water into 4H+ + O2)

Ultimate electron donor in photosynthesis of oxygenic phototrophs

water (sulfur compounds for anoxgenic bacteria like purple bacteria e.g. H2S)

Order of electron flow in oxygenic photosynthesis once light excites PSII

Quinone pool --> cytochromes --> Fe/S clusters in PSI where it gets reexcited by sunlight, NADP+ --> NADPH --> used to drive biosynthetic reactions like CO2 fixation

Ultimate electron acceptor in oxygenic photosynthesis

CO2

Do anoxygenic microbes use water as electron source?

No - that means they'd produce oxygen

T/F: most chemolithotrophs and phototrophs are heterotrophs

False: most are autotrophs and need to fix CO2 as organic molecules

For every 6 CO2, the calvin cycle produces

2 glyceraldehyde 3-phosphates that combine into fructose 6-phosphate

First step in calvin cycle (enzyme)

Rubisco: combines CO2 with ribulose 1,5-bisphosphate --> make 3-PG (6C)

Carboxylation

Whole cycle multiplied by 6 (so 6 R15BP and 6CO2)

_____ is the enzyme that does the key carboxylation in the calvin cycle

RuBisCo

6 x 5C + 6 x CO2 --> 12 x 6C (G3P) ------> siphon two off to do what?

Use two glyceraldehyde 3-phosphates to make fructose 6-phosphate --> feeds into glycolysis; highly useful for metabolism

Calvin cycle uses up _____ NADH or NADPH and _____ ATP per 6 CO2 that enter

12, 18

For every 36C into calvin cycle ______ get drawn off for biosynthesis

6 C (2 x GA3P)

T/F: only eukaryotes and archae use calvin cycle

F: phototrophic bactera, most chemolithotrophic bacteria, algae, some archae

Not the only way to fix carbon tho

Effect of N2 triple bond

Very stable, metabolically useless for most organisms even though it is abundant

Needs to be fixed

Nitrogenase

Converts N2 to NH3, which is metabolically useful

N2 + 8H = 2NH3 + H2

Cells can use NH3 to build

Nitrogen containing molecules like nucleic acids and proteins

Diazotrophs

Bacteria and archea that make nitrogenase and fix N2 into NH3

E.g. cyanobacteria, Rhizobia, some archael metahnotrophs

Nitrogenase is made of which two proteins

Dinitrogenase and dinitrogenase reductase

Nitrogenase uses what cofactor

Fe/Mo cofactors

Challenging chemistry = require metals

Flow of electrons in the nitrogenase reaction:

From some metabolite (pyruvate) --> Fe/S proteins like flavodoxin --> go to dinitrogen reductase --> dinitrogenase to N2

How many ATP to make 2 NH3 from N2 and 8H+

16 ATP (2 per electron added)

(6 electrons added to Ns plus 2 to H+ -- we don't know why we need to use 8 and produce H2)

Electrons are transferred to dinitrogenase one at a time

How many electrons consumed by nitrogenase

8

Not 6 (reason unknown)

Gluconeogenesis is like

Reverse of glycolysis