You might not stop to think about it often but, even when you’re sleeping and not scurrying around the lab (seriously, if I had a fitbit…) there’s tons of stuff happening in each of the billions of cells in your body – molecules running all around in there doing stuff. And all of that running around and stuff-doing takes energy. So your body has to have ways to take energy from foods you eat, store it, transfer it, and use it where and when you need it.⠀

Your body’s main form of energy storage and transfer is a molecule called ATP – Adenosine TriPhosphate. Much more on it here, and more details below: but basically it has 3 phosphate groups (phosphorus surrounding by 4 oxygens) stuck together – and each of those phosphates is negatively charged. Since opposites charges repel, they want to get apart from each other so, like clamping together a stiff spring, it takes energy to keep them bonded together (we call this chemical potential energy)- if you let one go (split ATP into ADP + Pi) you release energy, and you can use that energy to do things (like build proteins and stuff)⠀

Your body can take all sorts of different fuel sources (sugars, fats, proteins) and – like using different currencies and denominations to purchase arcade tokens – generate ATP from them, which can be “spent” for all sorts of things in sort of “point of sale” transactions. Making ATP from food is called CATABOLISM. You might be more familiar with the term METABOLISM – and catabolism’s part of that (the breakdown part), but the term “metabolism” also encompasses ANABOLISM which is the building part. ⠀

One form of catabolism is called GLYCOLYSIS – which is a process used to break down sugars. But that’s not where the big ATP payout from sugars comes from – it’s just the starting part! Where next? Different methods of ATP-making have different “broker’s fees” – so if you start with the same fuel input you can get different ATP outputs depending on which catabolic process you take. The best bang for your buck (or I guess it’d be more like buck for your bang…) is from AEROBIC RESPIRATION. As the “aerobic” implies, this requires oxygen. In aerobic respiration, most of the energy you produce from sugar doesn’t come from glycolysis – though you still start there, and then go through the citric acid cycle where you can pick up some more – but the big payoff comes from something called “oxidative phosphorylation” (ox-phos) that occurs in “mini-cells” inside your cells called mitochondria (the citric acid cycle happens here too, but glycolysis happens in the general cell interior (cytoplasm). Much more detail below I assure you (and apologies for the formatting and some repetition, but I mismashed some parts of other posts together and am now going back to my research 🙂 )

note: The pathways are interconnected so you can take parts from catabolism & use them for building things (anabolism) and different broken-down things can enter pathways at different points – so although I’m gonna talk in terms of glucose (blood sugar) catabolism, different molecules can enter at different points in the process (i.e. most of this doesn’t just apply to glucose!)⠀

ATP’s great, but it’s only part of the story. Another main player in the energy transfer pathway is Nicotinamide Adenine Dinucleotide (NAD⁺) and it’s cousin NADP⁺ (just add phosphate) and their reduced forms NADH & NADPH. But if ATP’s cellular energy money, NADH is more like an energy IOU! NAD comes from vitamin B3 (niacin) and other places and by accepting (in the NAD⁺) form & transporting electrons (in its NADH form), NADH can take energy IOUs in the form of electrons up the electron transport chain of command to the bank boss to demand those energy “arcade tokens” (ATP molecules) in a process called oxidative phosphorylation (ox-phos).  Put these these energy-making processes together: glycolysis, citric acid cycle (TC) (or other initial breakdown process) to generate NADH and ATP followed by oxidative phosphorylation (electron transport chain + chemiosmosis) to make use of that NADH to make more ATP and you get an overall energy-making process referred to as cellular respiration. 

I just introduced a lot of jargon, so let’s step back a second… Atoms (like individual O’s and P’s and C’s and H’s) are made up of smaller things called subatomic particles which include protons (positively-charged), neutrons (neutral), and electrons (negatively-charged). Elements have a set # of protons which define them (e.g. carbon has 6, oxygen has 8, hydrogen has 1) but they can have different numbers of electrons, which whizz around the nucleus where the protons and neutrons are housed, and can be swapped & shared to form strong, covalent, bonds between atoms. ⠀

Such sharing occurs so that atoms can achieve their “ideal numbers” of electrons in terms of electron housing arrangements, but, since opposite charges repel, reaching that “ideal” can mean sticking a lot of negative charges close together, which is less than ideal. So this is how we end up with ATP being negatively charged. Each phosphate (in its free form) has 3 “extra” electrons (compared to the # of protons), so it has a negative charge of 3, (PO₄³⁻). When you hook them up the inner 2 go down to a -1 each (and the end is -2) but that still means you have a concentrated negative charge of 4. And charges want to get away from one another, so the P-P bonds require energy investment to clamp them together and thus we call the P-P bonds “high energy”

So that’s how ATP can help provide energy, but what about NAD? There it’s less obvious how it’s helpful, because we’re dealing with something called “redox,” short for “reduction” and “oxidation” which involves the giving and taking of electrons. “Giving of electrons” is called OXIDATION. When something gives up electrons, we say it gets oxidized and when something accepts electrons, we say it’s been reduced – they always go hand in hand so we call such passings REDOX reactions. You can remember which is which with the acronym OIL RIG: Oxidation Is Loss (of electrons); Reduction is Gain (of electrons). Much more on redox here:

Some molecules want electrons more than others – and if something that doesn’t want or only kinda wants an electron (has a lower reduction potential) gives an electron to something that’s happier with it than it is (has a higher reduction potential) you have a net gain in happiness. And when you make molecules happier, they release energy (I like to think of it as them not having to fidget as much to get comfy so they can relax). So you can pass electrons from things with low reduction potential (like NADH) to things with high reduction potential (like O₂), and release little bits of energy as you do, which you can put to use. A chain of such pass-offs occurs in double-membrane-bound cellular “rooms” called mitochondria. The energy released through this “Electron Transfer Chain” (ETC) is used to pump protons (H+) out of the inner sanctum of the mitochondria (the matrix) and into the intermembrane space between the 2 membranes. This creates a gradient where protons really want to flow in. But the matrix will only let them in through a specific entrance, a membrane-spanning protein called ATP synthase. 

I still remember the first time I “saw” this protein – it was in undergrad biochem class and I was immediately in awe. Because this thing is crazy beautiful and powerful, a tiny little molecular machine which uses the movement of the incoming flow of protons to literally turn its molecular cranks and catalyze (speed up) the production of ATP.

But before we get to that part, we need to go back to the beginning – the breakdown. First off, a key thing to keep in mind is how interconnected metabolic pathways are. So even though I’m going to focus on glycolysis, which is the starting point for breaking down blood sugar (glucose) for energy, different molecules which are breakdown products of other sugars, etc. can join in at various steps. Another key thing to keep in mind is that a lot of the same metabolic pathways can go either way and use the same workers. You can think of it kinda like LEGOs – you can take apart and rebuild the same thing over and over or you can use pieces you break off from one thing to make another thing, etc.⠀

Say you have 2 LEGOs – a blue one (B) and a red one (R). You can stick them together to get BR, so B + R -> BR. And you can take them apart B + R <- BR. When a step can go in either direction we call it reversible, and we draw a double arrow facing both ways, like⠀

B + R ⇆ BR⠀

In theory, *all* steps are reversible – but we call some of them irreversible because it’s so unfavorable to go the other way. Unlike some steps, which are easily reversible – kinda like loose LEGO pieces – the “irreversible” steps are more like those pieces that stick together so tightly you’re likely to take off a fingernail before you get the piece to come off!⠀

Often such steps are accompanied by the addition of ATP – you can think of it as energy money being spent as a “downpayment” to “show commitment” to a process and prevent the molecules from “backing out”⠀

A great example of this is in GLYCOLYSIS, which starts with an “investment phase” in which energy is spent followed by a “payout phase” in which energy is produced. In GLYCOLYSIS (a catabolic process) you split the sugar glucose (which has 6 carbons (6C)) into 2 molecules of pyruvate, each of which have 3 carbons (3C). This nets you 2 ATP (energy “arcade tokens”) and 2 NADH (energy “IOUs”)(more below), and those 2 pyruvate, which can be further broken down in the citric acid cycle (aka Kreb’s cycle).⠀

If you reverse glycolysis to its anabolic counterpart, so that instead of breaking glucose down, you’re making it, the process is called GLUCONEOGENESIS (literally, birth of new glucose – yes, kids, this is where glucose babies come from). As you’d expect, gluconeogenesis requires energy. But, what you might not have expected – glycolysis requires energy too – in terms of ATP, it takes 2. It then makes 4, so you have a net gain of 2, but this energy-maker starts out as an energy-taker! 

Unlike the ATP & NADH-generating steps, some of the steps of metabolic processes like glycolysis don’t seem helpful at first glance… But if you geek out and don’t just glance, but instead “think like a molecule” you can see that what’s going on is “forward planning” that not just makes biochemical sense, but is biochemically beautiful! Glycolysis has 10 main steps, and you can follow along in the figures⠀

One thing you’ll notice is that each of these steps is catalyzed (sped-up) by things ending in “-ase” – when you see “-ase” think ENZYME! Enzymes are usually proteins (sometimes protein/RNA complexes (like the case with ribosomes) or just RNA (we often call such enzymes ribozymes) and they mediate and speed up reactions by doing things holding the molecules together in the right positions for whatever they need to do and providing an optimal environment for the reaction to take place.⠀

Think back to the LEGOs. Since LEGOs have matching bumps (which are apparently officially called “studs” or “knobs”) and indents (which are called “anti-studs” or “stud receptacles” or “tubes”) if you were to put a couple LEGOs in a bag and shake them a little you might, by chance, get them to meet just the right way that studs & anti-studs alight and stick. But this is much more likely to happen if you grab them with your fingers in the right orientation. This is kinda like what enzymes do.⠀

The bricks still have to choose to stick (form new bonds) but the enzyme makes it much easier to do so. But they also make it easier to “undo so” – basically they just give the pieces more chances to choose. (i.e. if 2 molecules don’t want to join you can hold them together all you want and they won’t form a new bond – but if 2 molecules do want to join and you hold them together they’re more likely to form a new bond – and they’re more likely to form a new bond if they’re brought together with the help of enzymes than if they have to find each other on their own). So the same enzyme can help out a reaction in both directions (and so sometimes the names might seem confusing because they’re written in terms of the “opposite direction”)⠀

So, back to glycolysis (which literally means sweet (glyk) dissolution (lysis):⠀


STEP 1: enzyme: hexokinase; reaction: glucose + ATP -> glucose-6-phosphate (G6P)⠀

  • a kinase is a phosphate adder, and in this step phosphate is added to the “6th” of glucose’s 6 carbons⠀
  • this is the first of the “irreversible” steps – you start by putting down an energy-money downpayment⠀
  • it also helps with the committing because the charge makes it “impossible” for glucose to get out of the cell – what with the cell’s fatty membrane and all – so the glucose gets trapped⠀

STEP 2: enzyme: phosphoglucose isomerase; reaction: glucose-6-phosphate ⇆ fructose-6-phosphate⠀

  • isomers are different arrangements of the same atoms so, as the “isomerase” name suggests, what goes on in this step is just some rearrangement – specifically carbonyl (C=O) that glucose ends with gets shifted over, so that there’s an OH on the end. This might seem weird but just wait – it’s gonna be important for…⠀

STEP 3: enzyme: phosphofructokinase; reaction: fructose-6-phosphate + ATP -> Fructose-1,6-bisphosphate⠀

  • this is the second “irreversible step” – and it involves another phosphate adding – this time to the 1st carbon which you graciously made available in step 3⠀
  • why the second “downpayment”? if you look at the structures you’ll see that you now have a phosphate on both ends, so when you split it in half each half gets one⠀
  • the kinase involved is highly regulated to help regulate glycolysis as a whole⠀

STEP 4: enzyme: fructose bisphosphate aldolase; reaction: fructose-1,6-bisphosphate ⇆ dihydroxyacetone phosphate (DHAP) + glyceraldehyde-3-phosphate (G-3-P)⠀

  • this is the splitting I was talking about – you take a 6C thing and split it into two 3C things⠀
  • the “halves” aren’t identical in the beginning (they have carbonyls in different spots) but, that’ll get sorted out with the help of another isomerase in…⠀

STEP 5: enzyme: triose phosphate isomerase; reaction: DHAP ⇆ G-3-P⠀

  • those unidentical halves have the same atoms just arranged a little different – they’re isomers – so, with the help of an isomerase, you can shift between them⠀
  • only G-3-P can be directly used in the next step so, even though the reaction’s easily reversible, since you keep taking away the G-3-P, if you want to convert something, you’ve gotta convert the DHAP since there’s “no” G-3-P available. so eventually all the DHAP gets turned into G-3-P so you have 2 identical G-3-P’s entering the….⠀


This is where you finally start making energy money (ATP) & energy IOUs (NADH). And the important thing to remember is that you’re going in with 2 copies of the G-3-P so each of the reactions gets “multiplied by 2”⠀

STEP 6: enzyme: glyceraldehyde-3-phosphate dehydrogenase; reaction: glyceraldehyde-3-phosphate + NAD+ + Pi ⇆ 1,3-bisphosphoglycerate + NADH + H+⠀

  • this is a kinda “weird” step because it adds a phosphate but a free-floating one (so it doesn’t cost any ATP) – the phosphate gets added to the “other end” so you now have one on each end again⠀
  • the energy for the adding comes from the coupled redox reaction of NADH reduction & glyceraldehyde-3-phosphate oxidation, which is exergonic (energy-releasing) because the NAD+ wants the electrons more⠀
  • this is your first IOU payment – it reduces NAD+ to NADH which can then be “cashed in” for ATP later in oxidative phosphorylation⠀

STEP 7: enzyme: phosphoglycerate kinase; reaction: 1,3-bisphosphoglycerate + ADP ⇆ 3-phosphoglycerate + ATP⠀

  • our first real payout!⠀
  • don’t get confused by the enzyme name – before we had kinases *using* ATP to *add* phosphates, but here we’re adding a phosphate to ADP – and we’re getting that phosphate by taking off the one we just added in step 6 (the name refers to the reverse reaction)⠀

STEP 8: enzyme: phosphoglycerate mutase; reaction: 3-phosphoglycerate ⇆ 2-phosphoglycerate⠀

  • this is another isomerase, but I guess they thought “mutase” sounded cooler or something – but “all” you’re doing here is shifting the phosphate to the middle C⠀

STEP 9: enzyme: enolase; reaction: 2-phosphoglycerate ⇆ phosphoenolpyruvate (PEP) + H₂O⠀

  • making PEP is a kind of “prep” – when you kick out that water you make a double bond between 2 of the carbons – making it a really awkward situation for that middle carbon – this molecule is really unstable⠀
  • so the phosphate is now attached to a carbon that doesn’t really want it and can better survive without it – so that phosphate can more easily get removed in…⠀

STEP 10: enzyme: pyruvate kinase; reaction: PEP + pyruvate kinase + ADP -> pyruvate⠀

  • the last step & final payout of glycolysis⠀
  • involves another kinase named for the reverse reaction⠀
  • because PEP is so unstable, it’ll easily give up that phosphate to an ADP that pyruvate kinase helps it meet⠀
  • and because of how unstable PEP is, although you’re not spending energy money, the reaction’s really unlikely to go backwards.⠀

So you do all that for each copy of G-3-P you got from the investment phase. So, you end up with 2 X NADH (from step 6), 2 X ATP from step 7 and 2 x ATP from step 8. And you used 2 ATP in the investment phase. So, on net you have: 2 NADH + [(-2) + 2 + 2 = 4] ATP. And you also have those 2 pyruvates which can get further processed for more energy.⠀

reverse segue? Gluconeogenesis is the “reverse” of glycolysis but it’s not a direct reverse because it has to “reroute” around the irreversible steps using different enzymes. It reverses step 10 (the de-pep-ing) in 2 or 3 steps – it’s a bit “steppy” because that pyruvate that got made in glycolysis gets shipped into the mitochondria. In the mitochondria, carboxylase converts pyruvate to oxaloacetate (at the cost of 1 ATP) & then phosphoenol-pyruvate carboxykinase converts that oxaloacetate. That can’t go through the mitochondrial membranes, so it first gets made into malate by malate dehydrogenase, then that malate goes into the cytoplasm where another malate dehydrogenase turns it back to oxaloacetate which can then be turned into to phosphoenolpyruvate (PEP) (at the cost of 1 GTP) by PEP carboxykinase in the cytoplasm. Alternatively, the PEP can be made in the mitochondria in some animals and transported out like that. It uses fructose 1,6-bisphosphatase to reverse step 3 (remove the second phosphate that was added to go from fructose 1,6-bisphosphate to fructose 6-phosphate. And to reverse the 1st step of glycolysis (initial phosphate-adding), glucose-6-phosphatase removes the phosphate to form glucose. This happens in the lumen of the endoplasmic reticulum (another membrane-bound compartment that’s often used for protein-modifying) and shipped out into the cytoplasm by glucose transporters.⠀⠀

back to the breakdown: at this point we’ve got some NADH we want to put to use. And we can get even more if we send that pyruvate for further breakdown through the citric acid cycle (TCA) aka Kreb’s cycle. So let’s take it to the mitochondria for some ox-phos!

Unlike some of your other organelles (membrane-bound compartments inside your cells like the endoplasmic reticulum (involved in protein processing)), mitochondria have double membranes because they come from an ancient ancient ancient cell swallowing a bacteria and then adopting that bacteria to turn it into a “powerhouse” for taking those electronic “IOUs” from NADH (and FADH₂) and turning them in for ATP. And it works by passing electrons (ELECTRON TRANSPORT CHAIN (ETC)) to pump out protons (H⁺) and letting the protons rush in to make an ATP-making motor “spin” so you get ATP from ADP that went in (CHEMIOSMOSIS).  ⠀

How? Because the mitochondria has those 2 membranes it has a room of its own – an inner room called the mitochondrial matrix – that’s separated from an “outer room” called the intermembrane space by a phospholipid bilayer called the inner mitochondrial membrane (which is really “squiggly” – it has lots of folds so you have a large surface area for the stuff we’re gonna talk about below…) Because it has that internal room (mitochondrial matrix), it can control what goes in and out of it (and where the “entrances” and “exits” (in the form of membrane-spanning protein channels & pumps) are).⠀

And, they can use this to purposely keep concentrations of things imbalanced – going by the whole “absence makes the heart grow fonder” strategy, they can keep kicking protons of the matrix so the matrix “wants them more” and then they can let it back in in a rush that, like a waterfall, can move motors. But then they have to kick the protons back out to maintain that gradient. And the “kicking out” takes energy from electron passing. ⠀

The electron transport chain (ETC) occurs at the inner membrane (between matrix & inter membrane space) and it passes electrons in a series of pass-offs from a molecule that kinda wants it to one that wants it more to one that wants it even more until you finally pass it off to oxygen that wants it the most. Because you’re forming lower energy bonds with each pass-off, you release a little energy, and this energy is used to pump out protons to generate a proton gradient where you have more protons in the inter membrane space then you do in the matrix. ⠀

Since the concentration of protons is high in the intermembrane space, but low in the matrix, the protons will rush in when you let them (the CHEMIOSMOSIS part). You can think of it a bit like rain – imagine you have a certain amount of rain that’s gonna fall. If it falls as sprinkles over a wide area people might barely feel it. But if it all falls in one location, the person in the path will get drenched. The force of the water might even knock them over. Chemiosmosis is kinda like this but instead of water, it’s protons falling and instead of knocking people over it’s getting ATP synthase to make ATP. ⠀

The rain all falls in one place because the protons can’t just slip back in any ole place because the membrane is hydrophobic (water-avoiding (literally water-fearing but I think they more just find it gross)) and protons are hydrophilic (water-loving) – so the protons can only get through if there’s a channel – and the only channel available to them is the ATP synthase, which does the ADP + Pi -> ATP magic⠀

Just like water evaporating and going into clouds takes energy (heat from the sun), proton pumping takes energy in the form of those electron pass-offs, so let’s look at them a bit closer. They occur through a series of protein complexes and small molecules, starting with the original donor. NADH can pass of its electrons to Complex I, with concurrent proton pumping, but NADH isn’t the only electron-donator. FADH₂ can also contribute, but it’s not as good of a donor – it wants the electrons more than complex I does, but not as much as complex II – so it can donate to complex II but not complex I, and complex II doesn’t pump (there’s not enough energy difference between FADH₂ and complex II having the electrons), so you get less proton pumpage (and thus less ATP from FADH₂ than NADH). You get ~10 protons pumped per NADH but only 6 per FADH₂. And it takes 4 H⁺ flowing through the ATP synthase to give you 1 ATP. So you get ~2.5 ATP per NADH & only ~1.5 per FADH₂. ⠀

After complex II, the route’s the same -> I & II pass off to ubiquinone (Q) which is small and unlike the complexes that are kinda stuck in place, it’s mobile so it can travel through the membrane – to complex III, which pumps out more protons. Then another mobile carrier, cytochrome C (Cyt C) takes the electrons to complex IV that passes them (finally) to oxygen (with some more proton pumping). With the extra electrons, O₂ splits and adds protons to form water. For each O₂ you get 2 waters, but you need 4 electrons. ⠀

Oxidative phosphorylation requires oxygen, because oxygen serves as the last in a line of electron acceptors. Oxygen REALLY wants them, so, by serving as the last acceptor it “eggs on” the acceptors ahead of it, motivating the whole process. So, no oxygen, no oxidative phosphorylation and no ATP made this way (but you can still get that initial smaller gain from glycolysis)⠀

But ATP wasn’t the only thing you were getting out of ox-phos – it was also replenishing your NAD⁺, which you need for glycolysis and the citric acid cycle (plus a bunch of other things – In addition to their electron-shuttling roles, pyridine nucleotides can serve as coenzymes (helpers) for dehydrogenase enzymes, electron donors for regulating redox environment, sources of building pieces for things like poly(ADP-ribose) polymerases (PARPs) to use, etc.)⠀⠀

When it gives up its electrons in oxidative phosphorylation, NADH goes back to being NAD⁺, so you can do it all again. But if there’s no oxygen around, NADH needs to find someone else to dump those electrons off on…. and ethanol fermentation is a way that yeast & some bacteria replenish their NAD⁺ supply, giving off ethanol in the process. NADH reduces acetaldehyde to ethanol, getting oxidized back to NAD⁺ in the process. In our cells, instead of making ethanol, we regenerate NAD⁺ through lactic acid fermentation – we use NADH to reduce pyruvate (the glucose parts you get from glycolysis) to lactic acid.  No energy is generated in these fermentation parts of the cycle, they just regenerates the NAD⁺ so glycolysis, etc. can make more. ⠀

If you look in the diagrams you’ll see that the citric acid cycle (aka Kreb’s cycle) also takes place in the mitochondria. And that it doesn’t require oxygen directly. But it doesn’t occur if you don’t have oxygen because it needs that NAD⁺ and FAD also. So no oxygen, and you’re stuck with what you get from glycolysis. ⠀

So, in summary: Mitochondria are often called the “powerhouses” of the cell because they’re where this electron-to-ATP conversion occurs in a process called OXIDATIVE PHOSPHORYLATION which consists of an  ELECTRON TRANSPORT CHAIN (ETC) and CHEMIOSMOSIS. Basically it consists of molecules (NADH or FADH₂) passing electrons up & up and up the chain of command, pumping out protons as they go, until they reach ATP synthase which is a bit like a hydraulically-powered ATP-making factory – similarly to how waterfalls can be used to turn a wheel, proton “falls” can be used to turn a biochemical motor to get ADP to add a P to make ATP.

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉⠀

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