Yesterday, in honor of Passover, I told you about how yeast use anaerobic respiration to make energy from sugar – and then have to go through fermentation to be able to keep going, generating bread-leavening gas and wine wooziness producing ethanol along the way in order to regenerate NAD⁺. Today (as I continue working on a new post and catching up on energy production…) I thought I’d tell you more about NAD⁺, how it’s crucial for energy-making. And how aerobic respiration allows us to regenerate it without making alcohol!

I’ve learned a lot about ATP, but I realized I know next to nada about its metabolic colleague NAD! Nicotinamide Adenine Dinucleotide (NAD⁺) and it’s cousin NADP⁺ (just add phosphate) play important energy-storage & transfer roles in cells too – but if ATP’s cellular energy money, NAD⁺ is more like an energy IOU! And before you *wine* about not getting paid right away – you can thank this molecule for alcoholic beverages like Chardonnay (you can tell I don’t drink because I initially spelled this chardoney and then chardonet which my autocorrect still couldn’t figure out…)  

NAD comes from vitamin B3 (niacin) and other places and by accepting (in the NAD⁺) form & transporting electrons (in its NADH form), NAD⁺ can take energy IOUs in the form of electrons up the electron transport chain of command to the bank boss to demand energy “arcade tokens” in the form of ATP. We looked a lot at ATP yesterday  but here’s a quick review. 

ATP stands for Adenosine TriPhosphate and it’s an RNA letter that can also serve as a form of cellular “energy money” – it has a ribose sugar linked to an “adenine” nucleobase and 3 phosphate groups.  A phosphate is a central phosphorus (P) linked up to 4 oxygen atoms – so (PO₄³⁻). A negative charge of 3! that means it has 3 “extra electrons” – and by extra I just mean that it has 3 more electrons than protons (the +-charged counterparts) – atoms 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 are negatively-charged, whizz around the nucleus where the protons are housed, and can be swapped & shared to form 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. Like in the case of ATP – it has 3 of those negatively-charged phosphate groups linked together – and they want to get away from one another. So the bonds holding them together have quite the “clamping” task to do, like compressing a stiff spring. It takes energy to keep them from “springing apart” so we call the P-P bonds “high energy” – this just means that they have high chemical potential energy – the biochem equivalent of a roller coaster car at the top of a hill – if you let go of one of the phosphates, you release the energy that you were using for the clamping to be used to do other things, like build proteins.

But it’s really important this process is controlled so that you don’t just have molecular fireworks going off randomly in your cells. So you need a way to convert the energy you can get from burning food fuel to a form you can spend when you’re ready, at biochemical “point of sale” transactions. And ATP is only part of the story.

Instead of money, which can come in all forms of different denominations (e.g. quarters, nickels, pennies) and currencies (is that a US dollar or a Canadian one?), ATP is a more like an arcade token – you can “trade in” all different forms of money (sugars, fats, proteins) for a universal thing you can spend (a molecule of ATP). 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. 

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!)

One form of catabolism 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! 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). 

For every molecule of glucose (blood sugar) you break down you can get 30-32 molecules of ATP. But you only get 2 of those from glycolysis! From glycolysis you also get 2 molecules of NADH (from NAD⁺). It might look like the big difference between NAD⁺ and NADH is a hydrogen (H), but the real difference is “unspoken” – it’s the electrons! When NAD⁺ picks up that H, it picks it up as something called a HYDRIDE, which is an H with 2 electrons instead of its usual 1. Hydrogen atoms only have 1 proton & 1 electron & they often leave that electron behind when they go, so all they’re left with is a H⁺, so we often call H⁺ a proton but when H’s have 2 electrons we call it a hydride. So, for example, you can split H₂ unevenly to get a hydride and a proton, and we’ll see both of these involved in this ox-phos stuff.

Unlike your other organelles (membrane-bound compartments inside your cells like the nucleus (holds DNA) and 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. 

“Passing 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.

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. 

At the molecular level, pyridine nucleotides (NAD⁺ and NADP⁺ (oxidized forms) and NADH and NADPH (reduced forms) are made up of 2 mono nucleotides – adenosine monophosphate (AMP) & nicotinamide mono nucleotide (NMN) joined together through their phosphates. You can get it “pre-made” in your food, or make it yourself – either “from scratch” (de novo) or by breaking it off from other things (salvage pathway). 

In the de novo pathway, NAD⁺ is synthesized from the amino acid (protein letter) tryptophan – tryptophan is an essential amino acid meaning our cells can’t make it. We can make NAD⁺ from tryptophan but we can’t make tryptophan from anything, so we need to get it from our food. In the salvage pathway, NAD⁺ is made from nicotinic acid (Na), nicotinamide (Nam) or nicotinamide ribose (NR) from dietary sources or NAD⁺ metabolites

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.

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