BIO131 final PART 1

• Mitosis

  • Prophase

    • This is the stage in which mitosis formally commences

      • Chromosomes condense out of chromatin into their classical shapes.

      • All the new DNA made in S of interphase condenses into a duplicate set of chromosomes Condensins (large protein complexes) assist this

        • Condensed chromosomes look like lowercase Greek letter chi (X, χ) – this is a pair of identical sister chromatids joined by a centromere

    • Nucleoli break down

      • No more ribosomes (thus proteins) can be made. Cell is now focussed on division and day-to-day metabolism stops

    • Centrosomes (replicated in interphase) move apart

      • Each centrosome (microtubule organisation centre) is made up of two centrioles at 90°. They move to opposite poles of the cell and microtubule activity at poles increases

    • Microtubules reorganise

      • In interphase these act like scaffolding

        • They now break down into radial arrays of (short) aster microtubules which make up asters centred on each centrosome. Longer sets of spindle microtubules extend across the cell and form the basis of the mitotic spindle

  • Prometaphase

    • Nuclear envelope breaks down into vesicles

    • Kinetochores form 

      • Disc-shaped pads that bind to centromere on each chromosome

        • Have protein extensions that allow binding to microtubules (look a bit like Command™ strips by 3M)

    • Kinetochore microtubules extend from centrosomes and bind to kinetochores

      • Chromosomes can be seen visibly ‘juddering’ in the cell as this happens

    • Polar microtubules are formed, extending from centrosomes – the mitotic spindle is now complete

  • Metaphase

    • Chromosomes align along the equator of the cell forming the so-called ‘metaphase plate’ 

      • (Misleading – it’s just an imaginary line, not a physical barrier)

        • Chromosomes are pulled back and forth until they form a perfect line.

    • Cell cannot enter anaphase until every chromosome pair is attached to a kinetochore microtubule and aligned along the equator of the cell

  • Anaphase

    • The protein securin blocks anaphase from starting until everything is ready, and the anaphase-promoting complex destroys it, allowing this to proceed.

    • Anaphase A: 

      • Kinetochore microtubules pull on the chromatids pulling them apart and moving them towards the poles

      • Chi-shape is lost

    • Anaphase B: 

      • Microtubules move towards the middle of the cell and push against one another, pushing chromatids nearer to the poles.

  • Telophase

    • Many aspects of prometaphase and prophase are now reversed – nuclear envelopes reassemble around each set of chromatids at the poles of the cell

    • Once nuclear envelopes re-form, the chromatids condense into chromatin and the nucleoli reform

      • Mitotic spindle is disassembled

    • Cytokinesis occurs

      • A myosin and actin ring around the equator of the cell contracts and splits the cell into two daughter cells

• Meiosis

  • Overview

    • Interphase – same as mitosis BUT only G1 and S phases occur

    • Meiosis I – first phase of meiosis:

      • Prophase I

      • Metaphase I

      • Anaphase I

      • Telophase I

    • Meiosis II – second phase:

      • Prophase II

      • Metaphase II

      • Anaphase II

      • Telophase II

  • Mitosis VERSUS meiosis

    • Mitosis would be:

      • 1 cell with 46 → 1 cell with 92 → 2 cells with 46 in each

    • Meiosis would be:

      • 1 cell with 46 → 2 cells with 23 in each → 2 cells with 46 in each → 4 cells with 23 in each

        • Red arrow = meiosis I, violet arrows = meiosis II

  • Meiosis I

    • Prophase I

      • Crossing-over events occur but otherwise the same

      • as in mitosis

      • Leptonene phase 

        • In which pairs of sister chromatids are copied and then become tightly associated (so much so, they look like a single thread in the nucleus)

        • Some elements of the synaptonemal complex assemble between each pair of chromatids (proteins that connect the chromatids along their length)

      • Zygotene phase 

        • Homologous chromosomes (pairs of sister chromatids!) now line up next to one another

      • Pachytene phase 

        • Homologous recombination occurs by crossing-over 

        • Chiasmata (“marks of χ” sing. chiasma) form at contact points. 

          • Chromatids exchange information – results in chromatids that are still complete and same size but have swapped information with the other of same size (crossing over!!)

      • Diplotene phase 

        • Synaptonemal complex degrades. 

        • Chiasmata DO NOT degrade!

    • Metaphase I, anaphase I, telophase I

      • Happens much like mitosis but with chromosomes at the poles of the cell.

        • Note it is not single chromatids at each pole now but pairs of them: chromosomes

      • Sometimes the two daughter cells produced will have a little rest before proceeding after telophase I – this iscalled interphase II or interkinesis

  • Meiosis II

    • Happens much like mitosis, but for each of the two daughter cells made in meiosis I

29 Nov 2024: Vitamins

• Defintions and groupings

  • Vitamins

    • Organic molecules needed in small amounts for the proper functioning of an organism

  • Fat soluble vitamins

    • Vitamin A, vitamin D, vitamin E, vitamin K

  • Water soluble vitamins

    • B complex vitamins (9×), vitamin C

  • Provitamins 

    • Precursors to vitamins themselves

  • Vitamers 

    • Different forms of a vitamin (‘vitamin isomers’)

• Vitamin A: fat soluble

  • All vitamers are isoprenoids

    • ‘All-trans’ versions are usual in diet etc and only change to cis when in use

      • Essential vitamin of all Chordata.

  • Vitamers

    • Retinoic acid, retinaldehyde (retinal), retinol, retinyl esters.

  • Provitamins

    • β-carotene, α-carotene, γ-carotene etc, obtained from plants and retinyl esters from meats

  • Retinaldehyde (retinal) is used in the eye to form rhodopsin, needed

  • in low-light vision

    • Also made in some Archaea such as Halobacterium spp. for light-driven salt pumps/proton pumps based on retinal

  • Retinyl palmitate [ester of palmitic acid and retinol] and carotenes are main forms in our diet, converted to retinol in small intestine

    • Retinol is the storage form

  • Retinoic acid is synthesised from retinol in male gonads (needed for

  • sperm generation) and in embryos (regulates brain development)

    • Used as pharmaceutical (Tretinoin®) for reversing photoaging of skin, treatment of acne (makes keratinocytes shed) and some cancers

  • Sources:

    • Cod liver oil (0.3 g/kg retinyl palmitate)

    • Sweet potato (9 mg/kg carotenes)

    • Carrot (8 mg/kg carotenes)

  • Supplement is usually β-carotene as retinol etc are toxic if too much is taken orally

    • Hypervitaminosis A can kill (cf. toxicity of Ursus maritimus (polar bear) Phipps liver - 6 g retinol/kg)

  • Hypervitaminosis A

    • Xerophthalmia

      • Thickening of surface layers of the eye.

    • Keratomalacia

      • Similar with inability to synthesise specialist tissues of eye surface – opaque cornea develops

    • Nyctalopia (night blindness) 

      • Lack of retinaldehyde (and thus rhodopsin) in retina

    • Photophobia 

      • Aversion to bright light owing to lack of light-absorbing rhodopsin in retina

    • Weight loss

    • 500,000 children go blind each year worldwide from hypovitaminosis A 

      • A GM product ‘golden rice’ was developed to prevent this by providing β-carotene to rice-dominant diets. Has never been used owing to groups like Greenpeace who oppose GM crops

• Carotenoids in the Aves

  • Dietary uptake of carotenoids by the Aves comes from all 3 Domains of Life (Bacteria, Archaea and Eukarya) that they eat

    • EXAMPLE: Phoenicopterus spp. (greater flamingos) live in hypersaline lakes – their red-pigmented feathers have been shown to contain:

      • Cacterioruberin – produced by extremely halophilic Archaea of the Class Halobacteria in the Phylum Methanobacteriota such as Halococcus spp. and Halobacterium spp.

      • Echinenone – produced by Bacteria viz. members of the Phylum Cyanobacteriota (marine biologists will know this pigment from the orange interiors of the Echinoidea of the Metazoa!)

      • Astaxanthin – produced by Eukarya viz. members of the Phylum Chlorophyta of the Viridiplantae. This is used widely as a food colouring (E161j)

• Vitamin D: fat soluble

  • All are secosteroids (‘broken’ steroids)

  • Vitamers

    • Ergocalciferol (D2), cholecalciferol (D3), 22- dihydroergocalciferol (D4), sitocalciferol (D5). (D1 isn’t a thing!)

      • Produced (D3) in the stratum basale layer of the skin of Mammalia by a photodependent pathway (λ = 290-315 nm). D2 is produced in Fungi in same way

  • Regulates intestinal calcium uptake

  • Hypovitaminosis D is not uncommon in people:

    • Living in countries far from the equator;

    • With Fitzpatrick Type V and VI skin;

    • With very low body fat;

    • Whose skin seldom sees direct sunlight without SPF;

      • Standard domestic window glass absorbs a lot of 290-315 nm light, butnot all of it! so yes, you do still need to wear SPF indoors!

  • Sources:

    • Cod liver oil (25 μg/kg)

    • Mushrooms (110 μg/kg if UV-exposed,3 μg/kg otherwise)

    • Canned tuna (68 μg/kg)

  • Hypovitaminosis D

    • Rickets 

      • Bone softening in children. Called osteomalacia in adults

    • Osteoporosis

      • Bones become porous and fragule

    • Clinical depression and clinical anxiety 

      • Common symptoms of early stages of hypovitaminsis D

    • Schizophrenia 

      • not necessarily a symptom but many patients therewith have low serum vitamin D levels. Cause or effect?

    • Muscular pain

    • Muscle twitching

• Vitamin E: fat soluble

  • Eight vitamers

    •  4 are tocopherols, 4 are tocotrienols. Names are prefixed with α, β, γ, δ 

      • Supplements usually use α-tocopheryl acetate as it’s more stable in long-term storage

  • Potent antioxidants (aka reducing agents) 

    • Heads can donate electrons and hydrogens to oxidising agents e.g. reactive oxygen species (ROS). Important in handling oxidative stress

  • Protects cell membranes

  • Hypovitaminosis E

    • Causes neurological damage (as ROS can’t be stopped!) but VERY rare – only occurs if dietary lipid uptake is not functioning.

  • Sources:

  • Wheat germ oil (1.5 g/kg)

  • Oily fish (15 mg/kg)

  • Rapeseed oil (175 mg/kg)

• Vitamin K: fat soluble

  • Two vitamers

    • Phylloquinone (K1) and menaquinone (K2) – menaquinones (MKs) are respiratory chain quinones in many Bacteria and Archaea e.g. MK-4, MK- 7 – we obtain them from our gut microflora, like Escherichia coli, which leaks them into the gut

      • K1 is found in photosynthetic electron transfer chain in the Viridiplantae and green leaves are best dietary source

  • Hypovitaminosis K 

    • Causes issues with blood clotting and with proper Ca metabolism

  • Needed for enzymes that carboxylate glutamine residues in some proteins to form carboxyglutamate

    • Key in blood clotting cascades in Homo sapiens subsp. sapiens L.

    • Some evidence of a positive effect on bone density, particularly if taken with vitamin D3 (“vitamin D + K2” mixed supplements are now abundant!)

  • Various rodenticides work by preventing vitamin K being recycled – organisms bleed to death

  • Sources:

    • K1 – dark green leafy vegetables (4-5 mg/kg)

    • K2 – fermented soy beans e.g.

      • 納豆 (natto, 10 mg/kg)

      • Goose meat (0.3 mg/kg)

    • Our gut Bacteria make it for us!

• Vitamin C: water soluble

  • 2 vitamers:

    •  L-ascorbic acid (L-ascorbate) and dehydroascorbic acid (dehydroascorbate)

      • Oxidised form is L-ascorbic acid, dehydroascorbate must be reduced back at the expense of e.g. glutathione.

        • There are synthetic variations

          • Ex. 3-O-ethyl ascorbate used particularly in skincare/haircare etc as more stable.

  • Potent antioxidant (reducing agent)

    • Key in handling oxidative stress within the cytoplasm or in extracellular fluids (cf. vitamin E which does same in membranes)

    • Used in vitro in culinary settings to stop oxidation of o-quinones by air in cut apples!

  • Enzyme cofactor 

    • Ex. hydroxylases involved in collagen biosynthesis

  • Sources:

    • Rosehips (4 g/kg)

    • Blackcurrents (2 g/kg)

    • Kale (1.2 g/kg) 

    • Citrus fruit (0.3-0.5 g/kg)

  • Hypovitaminosis C

    • Overarching condition is scurvy but not all symptoms are always seen at once

      • Bleeding gums

      • Rashes

      • Fatigue

      • Generalised muscle pains from lack of carnitine production

      • Anemia from low erythrocyte production.

      • Easy bruising

      • Wounds not healing

      • Neuropathy

      • Jaundice

      • Dry mouth, eyes and other orifices – dry mouth leads to dental caries, mouth ulcers etc

      • Personality changes

      • Edema

• Thiamine (B1): water soluble

  • Various enzyme cofactors are made from it 

    • ex. ThPP in the pyruvate dehydrogenase complex (link reaction).

  • Essential for many Bacteria

  • Hypovitaminosis B1 is beri-beri

  • Sources:

    • Grains

    • Meat

    • Yeast

      • Yeast extract (Marmite) was sent by Red Cross to POWs in WW2 on rice-only diets in Far East to provide

• Riboflavin (B2): water soluble

  • Enzyme cofactors are made from it 

    • Ex. flavins FMN/FMNH2 and FAD/FADH2

  • Essential for many Bacteria

  • Hypovitaminosis B2 is ariboflavinosis

  • Sources:

    • Milk

    • Eggs

    • Legumes

    • Meat

  • Can be visually seen excreted in urine if there is an excess

• Niacin (B3): water soluble

  • Enzyme cofactors are made from it 

    • Ex. NAD+ and NADP+

  • Vitamers

    •  Niacin (nicotinic acid), niacinamide (nicotinamide), niacinamide riboside

  • Essential for many Bacteria

  • Hypovitaminosis B3 is pellagra

  • Sources:

    • Red fish

    • Meat

    • Grains

• Pantothenic acid (B5): water soluble

  • Provitamin

    • Panthenol

  • Enzyme cofactors are made from it  

    • Ex. coenzyme A (CoA).

  • Essential for many Bacteria

  • Hypovitaminosis B5 is almost unheard of

  • Sources:

    • Milk

    • Eggs

    • Potato

    • Tomato

    • Oats

• Pyridoxal 5ʹ-phosphate (B6): water soluble

  • Enzyme cofactor itself in many enzymes

  • Vitamers: 

    • pyridoxine (PN), pyridoxal (PL), PL 5-phosphate, PN 5-phosphate, pyridoxamine (PM), PM 5-phosphate and others…

  • Essential for many Bacteria

  • Hypovitaminosis B6 causes skin and neurological issues

  • Sources:

    • Meat

    • Grains

    • Red fish

• Biotin (B7): water soluble

  • Enzyme cofactor itself in many carboxylases

  • Regulates gene expression in some ways

  • Essential for many Bacteria

  • Hypovitaminosis B7 causes rashes, hallucinations etc

  • Sources:

    • Meat

    • Eggs

    • Fish

    • Legumes

• Folic acid, folate (B9): water soluble

  • Involved in DNA biosynthesis

  • Essential for many Bacteria

  • Hypovitaminosis B9 

    • Causes anaemia and B12 uptake issues as well as neural tube development issues during pregnancy

  • Sources:

    • Seeds

    • Legumes

    • Grains

    • Green vegetables

• P-aminobenzoic acid (B10): water soluble

  • Key in folate synthesis in Viridiplantae and Bacteria

  • Essential for many Bacteria

  • Hypovitaminosis B10 is unheard of, as a rule

    • Gut Bacteria make it on our behalf

      • Probably not a vitamin for Homo sapiens subsp.sapiens

• Cobalamins (B12): water soluble

  • Involved in: 

    • Amino acid biosynthesis, fatty acid biosynthesis and DNA biosynthesies

  • Cofactor in many methyltransferases and isomerases

  • Vitamers: 

    • Cyanocobalamin, methylcobalamin (MeB12), hydroxycobalamin, adenosylcobalamin (adoB12)

      • MeB12 and adoB12 are the active forms in the Mammalia – the other forms are converted once ingested

        • MeB12 is found in the cytosol; adoB12 is found in mitochondria

  • Structure based on a corrinoid ring with bound Co+ ion (cf. the porphyrin ring with bound Fe2+ in heme)

  • Essential for many Bacteria BUT only routes of production in Nature are by (other) Bacteria and Archaea

    • Herbivores obtain it only from Bacteria on the surface of plants

  • Hypovitaminosis B12

    • Causes anaemia, fatigue, joint pain, reduced heart function, depression and psychosis but is relatively rare in most people

      • Usually caused by medication inhibiting uptake

        • Can be supplemented by intramuscular injection or transdermal patch if gastric absorption is failing

  • Sources:

    • Meat

    • Fish

    • A few algae do accumulate it but are not reliable dietary sources. Note “Spirulina” 

      • (Really a trade- name for 2 species of the “Cyanobacteria”) don’t make it – they make pseudovitamin B12 which humans cannot use at all

2 Dec 2024: Bioenergetics: central concepts

• Energy as a concept

  • Energy (E) is the capacity to do work, be that mechanical or chemical

  • Work (W) is energy transferred to or from an object by a force

  • W and E are measured in joules (J, 1 J = 6.24 × 1018 eV)

    • Historically, the calorie and kilocalorie were used – latter still appears on food packaging, alongside kJ values.

      • [1 cal = 4,184 J; 1 kcal = 4.184 J]

  • In thermodynamic use, we will often given e.g. J/mol of something or kJ/mol of something

  • First law of thermodynamics:

    • Energy cannot be made or destroyed, only moved around, often changing form

  • Second law of thermodynamics:

    • Energy tends towards spatial homogeneity/entropy

• Coupling-the theory

  • Term was intended to evoke the coupling of gears 

    • Ex. by the chain on a bicycle – work done at the pedals translates to work done by the back wheel

  • Exergonic processes in which energy is released to the Universe can be coupled to endergonic processes that draw energy from the Universe

  • No such thing as 100% efficient coupling – in the bicycle, some energy is always lost to friction (heat, sound) etc.

    • Same is true in biochemical coupling

  • Examples:

    • Heterotrophic metabolism – use of multicarbon compounds

      • Oxidation of a hexose sugar is exergonic

        • C6H12O6 + 6O2 → 6CO2 + 6H2O, 

          • ΔGº = -2,996 kJ/mol hexose oxidised

      • Formation of ATP is endergonic

        • ADP3- + H2PO4- → ATP4- + H2O , 

          • ΔGº = +38 kJ/mol ATP4- produced

      • Oxidation of hexose sugars can be coupled to the synthesis of ATP to provide the energy needed

    • Autotrophic metabolism – use of CO2

      • Production of a hexose sugar is endergonic

        • 6CO2 + 6H2O → C6H12O6 + 6O2, 

          • ΔGº = +2,874 kJ/mol hexose produced

      • Hydrolysis of ATP is exergonic

        • ATP4- + H2O → ADP3- + H2PO4-, 

          • ΔGº = -38 kJ/mol ATP4- hydrolysed

      • Production of hexose sugars can be coupled to the hydrolysis of ATP to provide the energy needed

• Adenosine nucleotides

  • So-called “energy currency”

  • Form varies with pH 

    • At physiological pH, “ATP” is really ATP4-; “ADP” is really ADP3- and “AMP” is really AMP2-, and to complicate things further, all of them are found as various magnesium ion pairs – as such, we condense it all to “ATP” (etc) for simplicity!

  • Similarly, we simplify all the different ions that orthophosphoric acid (H3PO4)

    • Can make at physiological pH to “Pi” and all the different ones pyrophosphoric acid (H4P2O7) can make simplify to “PPi”.

  • Two key hydrolysis routes for ATP:

    • ATP + H2O → ADP + Pi

    • ATP + H2O → AMP + PPi

• ATP biosynthesis

  • Gradient-coupled phosphorylation (“oxidative phosphorylation”) 

    • Uses energy from a hydrogen ion gradient and/or sodium ion gradient (Δp and ΔNa+) to fuel formation from ADP/Pi

      • Can be coupled to:

        • Aerobic respiration – uses O2 as the terminal electron acceptor (reduced to H2O)

        • Anaerobic respiration – uses things other than O2 as the terminal electron acceptor.

          • Examples – sulfate (SO42-, reduced to H2S); fumarate (reduced to succinate); nitrate (NO32-, reduced to N2); uranyl ions (UO22+, reduced to U4+)

            • ***Anaerobic respiration using organic terminal electron acceptors is not fermentation!

  • Gradients are formed by respiration or photolithoautotrophy and ATP biosynthesis is ONE of the things that can consume these gradients

  • Substrate-level phosphorylation

    • Happens in the various glycolytic pathways and in/linked to Krebs’ cycle – formation during enzyme-catalysed reactions

      • One example of this in which respiration is not involved is fermentation, in which e.g. Embden-Meyerhoff glycolysis occurs but the pyruvate is oxidised to lactate to regenerate NAD(P)+ and the only ATP made is during enzyme-catalysed reactions. No gradients are involved!!! 

        • Examples:

          • Lactic fermentation (pyruvate oxidised to lactate in muscle cells or bacterial cells in the vaginal cavity or during Sauerkraut production)

          • Alcohol fermentation (pyruvate oxidised to acetic acid and ethanol in various Fungi)

        • ***Fermentation is not respiration!

• Gradient-coupled phosphorylation

  • Catalysed by a membrane bound enzyme that phosphorylates ADP at the expense of a flow of ions down an ion gradient across a membrane

  • H+-translocating two-sector ATPase (EC 7.1.2.2) is the most common – there are Na+-translocating ones in marine Bacteria and many gut pathogens

    • Former consume Δp, latter use ΔNa+ 

    • “ATP synthase”

      • ADP3+ + 4H+(out) + H2PO4- → ATP4+ + 4H+(in) + H2O

  • Membrane involved is inner membrane of mitochondria or Gram- stain-negative Bacteria (ions build up between the membranes) OR plasma membrane (i.e. the only membrane!) in Gram-stain- positive Bacteria or in Archaea (ions build up in membrane invaginations)

    • Respiration and photolithoautotrophy exist to build the ion gradient needed for this to function!

    • Not all organisms use H+ - almost all marine Bacteria and Archaea and many gut pathogens of the Chordata use Na+ instead for ATP biosynthesis – different synthase, different respiratory chain enzymes!

  • ΔNa+ evolved LONG before proton-motive force Δp

• H+-translocating two-sector ATPase

• Energy in chemical reactions

  • Exergonic reactions 

    • Have negative Gibbs energy changes (ΔG), thus release energy to the Universe whilst favouring proceeding

  • Endergonic reactions 

    • Have positive Gibbs energy changes (ΔG), thus obtain energy from the Universe whilst favouring not proceeding

      • Cf. activation energies covered in Enzymes and Enzymology – they apply – even exergonic reactions have an activation energy!

• Redox reactions

  • Respiratory chains and the electron transfer chains in photolithoautotrophy are just a series of redox reactions

  • Reduction is gain of electrons

    • A reducing agent wants to reduce other things, therefore it becomes oxidised itself

  • Oxidation is loss of electrons

    • An oxidising agent wants to oxidise other things, therefore it becomes reduced itself

  • An antioxidant is just a reducing agent!

  • Always a relative concept – what is a reducing agent when faced with one potential electron donor may not be able to reduce another. There is a degree of “how reducing something is” – the redox potential of a given redox couple

• Redox couples and half reactions

  • One of the redox reactions you have done in this module is the reaction of reducing sugars (reducing agents, electron donors) and Sumner’s reagent - 3,5-dinitrosalicylic acid (DNS) - (oxidising agent, electron acceptor)

    • D-(+)-glucose + 3,5-dinitrosalicylic acid → D-gluconic acid + 3-amino-5-nitrosalicylic acid

  • We can split this into two half reactions so we can see the electrons:

    • 3,5-dinitrosalicylic acid + ε- → 3-amino-5-nitrosalicylic acid

      • ΔG° = +30.9 kJ/mol DNS reduced

    • D-(+)-glucose → D-gluconic acid + ε-

      • ΔG° = -611.3 kJ/mol D-(+)-glucose oxidised

  • Therefore for the whole reaction:

    • D-(+)-glucose + 3,5-dinitrosalicylic acid → D-gluconic acid + 3-amino-5-nitrosalicylic acid

      • ΔG° = -580.4 kJ/mol D-(+)-glucose oxidised

• Photolithoautotrophs

  • Examples:

    • Almost all of the Viridiplantae; the Rhodophyta; in the Bacteria, Chromatium spp., Allochromatium spp., the “Cyanobacteria” and many others!

  • Use photon-excitable electron transfer chains to generate NAD(P)H and Δp and the latter is used to make ATP.

  • Energy source: 

    • Electromagnetic radiation (visible light + IR radiation)

  • Electron donor: 

    • Inorganic (H2O in the Viridiplantae, Rhodophyta and “Cyanobacteria”, which is oxidised to O2; H2S in Chromatium spp., which is oxidised to SO42- - many other sulfur species are used – thiosulfate, elementary sulfur, polythionates…)

  • Carbon source: 

    • CO2/DIC

• Chemolithoautotrophs

  • Examples:`

    • In the Bacteria: Nitrosomonas, Aquifex, Thiobacillus, Acidithiobacillus, Halothiobacillus, Guyparkeria, Annwoodia, Thermithiobacillus…

  • Use direct donation of electrons to respiratory chains to generate both NAD(P)H and Δp and the latter is used to make ATP

  • Energy source: 

    • Energy conserved from inorganic chemical oxidations

      • Ex. H2S → SO42-; H2 → H2O

  • Electron donor: 

    • inorganic (very diverse – ammonia, nitrate, thiosulfate, hydrogen sulfide, dimethylsulfide, arsenite, ferrous iron, elementary sulfur…)

  • Carbon source: 

    • CO2/DIC

• Chemoorganoheterotrophs

  • Examples

    • the Metazoa; in the Bacteria: Escherichia, Pseudomonas, Bacillus, Geobacillus, Mycobacterium, Chlamydia…

  • Use indirect donation of electrons via NADH to respiratory chains to generate Δp and the latter is used to make ATP

  • Energy source: 

    • Energy conserved from organic chemical oxidations

      • Ex. hexoses → CO2 in the Metazoa – beyond that is very diverse,

        • benzene → CO2, paracetamol → CO2, plastics → CO2

  • Electron donor: 

    • Organic (hexoses in the Metazoa and diverse beyond that)

  • Carbon source: 

    • Organic

      • Same substance is the energy source, electron donor and carbon source

  • Oxidation of carbon/energy source to CO2 during ex. the glycolytic pathways and Krebs’ cycle for sugars – dissimilation (catabolism) which generations NAD(P)H (and sometimes trivial amounts of ATP by substrate-level phosphorylation)

  • NADH is consumed at the respiratory chain to generate Δp, which is in turn consumed to generate ATP

  • Oxidation of carbon/energy source to 3PGA during e.g. the glycolytic pathways then assimilation (anabolism) of 3PGA into biomass at the expense of NAD(P)H and ATP

  • D-(+)-glucose catabolism in the Metazoa:

    • C6H12O6 + 30ADP + 30Pi + 30H+ + 6O2 → 6CO2 + 30ATP + 36H2O

    • Coupling the oxidation of the sugar to the synthesis of ATP

2 Dec 2024: Metabolic pathways

• Metabolic pathways: terminology

  • Catabolism

    • Oxidation of energy sources to ultimately generate ATP/[H]

    • “Simplifying molecules, energy released to the universe”

  • Anabolism

    • Uptake of carbon compounds into biomass at the expense of ATP/[H] 

    • “Complexifying molecules, taking up energy from the universe”

  • Metabolism

    • The sum of anabolism and catabolism

  • [H] 

    • Reducing equivalents – quite old-school shorthand for NADH, NADPH, FADH2, FMNH2, cyt c(red) etc etc without being specific! Therefore, very useful and quite lazy! Don’t confuse with [H+]

• Metabolic pathways: conservation

  • Integrated networks of chemical reactions – many are coupled

  • Cccurately replicated in progeny of a cell and progeny of an organism

  • Many are highly conserved across all Domains of Life

    • Whilst e.g. a single Escherichia coli cell (quite a metabolically basic organism) has >3,000 different biochemical reactions and there are millions of species on Earth with thousands of different reactions, biochemical unity underpins diversity: 

      • Core metabolites common to most organisms across Life amount to about 100 molecules

        • This is why the Eukarya are not very diverse metabolically

        • Very few reaction types 

• Overview of chemoorganoheterotrophy

  • Fatty acid degradation

    • • Catabolism breaks down diverse and complex macromolecules

        • Ex. fatty acids, sugars, proteins, nucleic acids… etc INTO common intermediates

          •  Ex. acetyl-CoA, pyruvate, intermediates from Krebs’ cycle…

    • • Anabolism uses common intermediates

        •  Ex. acetyl-CoA, pyruvate, intermediates from Krebs’ cycle to make diverse and complex macromolecules

          •  Ex. fatty acids, sugars,proteins, nucleic acids…

  • Krebs’ cycle doesn’t exist in isolation 

    • It is linked to various other processes including amino acid biosynthesis, lactic fermentation, the Embden- Meyerhoff glycolytic pathway, the respiratory chain

• Metabolic pathways: cofactors

  • Enzymes, substrates, cofactors (both bound and soluble)

  • Bound cofactors: 

    • FMN/FMNH2, FAD/FADH2, PQQ/PQQH2, chlorins, corins, hemes

  • Soluble cofactors (lipid):

    • Q/QH2

  • Soluble cofactors (aqueous):

    •  Cytochrome c, NADH, NADPH, coenzyme A, coenzyme M…, ATP/ADP/AMP, Pi/PPi

  • Cofactors carry groups and/or electrons from place to place

    • Most can be thought of as “a [something] carrier with a handle”

      • [Something] could be electrons, alkyl groups, P-P bonds, etc

  • Most are vitamin-derived: 

    • FAD/FMN (from riboflavin/B2)

    • NAD/NADP (from niacin/B3)

  • Examples:

    • L-lactate dehydrogenase (EC 1.1.1.27)

      • L-lactate + NAD+ → pyruvate + NADH + H+

    • Split into half reactions:

      • Oxidation:

        • L-lactate → 2H+ + 2ε-

      • Reduction:

        • NAD+ + H+ + 2ε- → NADH

    • NAD+ is acting as an electron carrier – the reduced form (NADH) carries electrons to the respiratory chain and/or to enzymes that use it

• FAD/FADH2 and NAD(P)/NAD(P)H

  • FAD/FADH2

    • FAD

      • Oxidized form

    • FADH2

      • Reduced form

  • NAD(P)/NAD(P)H

    • NAD(P)

      • Oxidized form

    • NAD(P)H

      • Reduced form

• Metabolic pathways: general rules!!

  • The pathway must be irreversible  

    • Pathway will be highly exergonic in one direction and highly endergonic in the other.

      • Reverse will be possible via a different suite of reactions 

        • Cf. Embden-Meyerhoff glycolytic pathway versus gluconeogenesis; cf. Krebs’ cycle versus the Arnon-Buchanan cycle

  • The first step is always committed – this avoids equilibria forming

  • First step is usually regulated

    • The rest of the pathway will just equilibrate on its own

  • In the Eukarya (and a minority of the Bacteria), organelles physically separate some pathways

  • In some other Bacteria, microcompartments physically separate pathway components (cf. Photolithoautotrophy lecture)

• Comparing pathways

  • Embden-Meyerhoff glycolytic pathway VERSUS gluconeogenesis

• Compartmentalisation

  • Organelles in the Eukarya and one phylum of the Bacteria compartmentalise processes

    • This permits high concentrations when required and groups linked reactions together physically

    • Assists in containing/controlling toxic intermediates

    • This requires transport across membranes – often at the expense of ATP

      • If a concentration gradient must be overcome – it is not without cost, especially if something has to be trafficked over two membranes (from one organelle to another)

  • Also related to this is substrate channelling from the active site of one enzyme into the active site of another, directly

• The anammoxosome

  • Found in some Bacteria of the Planctomycetota that perform the anammox reaction

    • They are chemolithoautotrophs

  • Ammonium ions are the electron donor (ours are sugars)

  • Nitrite ions are the terminal electron acceptor (ours is O2)

  • Molecular nitrogen is produced 

    • About 50% of the N2 produced by the oceans is made this way.

  • Ntrite is reduced to hydroxylamine (NH2OH) in the cytoplasm then in the anammoxosome reacts with ammonium to yield hydrazine (N2H4) which is a potent oxidising agent and extremely toxic!

  • Anammoxosome membranes are made of ladderane lipids and have abundant cytochromes to destroy ROS