Globin and heme make a pretty great team. But making that heme is harder than it may seem! Especially if there is lead on the scene. When it comes to lead poisoning, you mostly hear about lead’s effects on the head, but lead can also cause severe problems with the blood because it interferes with production of heme, the oxygen-binding molecule that, when bound globin proteins to form hemoglobin, allows our blood cells to transport oxygen throughout the body (and, when bound to other proteins like cytochromes, allows cells to do things like detoxify drugs and extract and store energy through oxidative phosphorylation).
Yesterday I told you a basic overview of lead poisoning and how it disproportionately affects people of color. https://bit.ly/leadpoisoningscience Today I want to tell you more of the nerdy details. Which involves a discussion of heme synthesis. So let’s go…
Lead is a trickster – when it comes to metals, it’s less picky than some others about where it likes to hang out. And this can cause ALAD-a problems… Not all of these problems are related to heme, but I’m going to focus mostly on the heme-y ones in this post. Heme is a multi-ringed molecule classified as a “porphyrin” – more on what this means chemically later, but for now just know that it can get held by proteins to serve as a “cofactor” or “prosthetic group” – and (some) proteins want to hold it because heme can also bind to iron – and iron can bind to oxygen. So heme provides a way for a protein to hold and transfer oxygen, for transport and/or to facilitate oxygen-requiring reactions.
“Porphyria” is an umbrella term for a variety of disorders involving problems with heme synthesis – in addition to its oxygen storage & carrier roles in hemoglobin (in blood) and myoglobin (in muscles), heme is a crucial component of proteins called “cytochromes” that are involved in the energy-producing process of oxidative phosphorylation, and “cytochrome P450s” (CYPs), which the liver makes and uses as an early step in the detoxification of foreign molecules like pharmaceutical drugs. Thus, heme’s 2 main sites of production and usage are red blood cells (erythrocytes) and liver cells (hepatocytes) and porphyrias can therefore affect either and/or both.
The name porphyria comes from the Greek word for purple due to the color of some of the heme intermediates that build up when there are “traffic jams” along the heme-synthesis pathway. This pathway is complex & multi stepped – it involves 8 different protein enzymes (proteins that mediate & speed up (catalyze) biochemical reactions by holding reagents together in the right orientation to react, etc.). It’s normally negatively regulated by the end product, heme such that, when plenty of heme is made, the heme stops the production of the first enzyme in the pathway, ALAS, to slow down production until more is needed. Without ALAS, the synthesis pathway can’t start.
But problems with any of those enzymes can lead to hold-ups. And, instead of stopping traffic altogether and “closing off the road” by not letting any more synthesis start, there’s a communication gap, so the enzymes at the beginning of the pathway don’t get word that there’s any sort of problem and – since they don’t get negative feedback telling them that there’s plenty of the end product, heme, they think they need to keep going and even ramp up their work. So they keep making the starter products and then those get stuck along the way, so pre-jam molecules build up and, unable to continue on their usual path, they can cause problems. For example, the first product, ALA, is “neurotoxic” meaning it can damage cells of the nervous system if the next enzyme, ALAD, doesn’t convert it into the next product, porphobilinogen (PBG) – which you also don’t want building up…
There are many *genetic* causes of porphyria – including the most common form of Acute Hepatic Porphyria (AHP), Acute Intermittent Porphyria (AIP), which is caused by mutations in the third enzyme in the pathway, porphobilinogen deaminase (PBGD) (aka hydroxymethylbilane synthase). http://bit.ly/39kvDgC
That is just one example – there’s basically a type for every enzyme in the pathway – and those enzymes can be messed up by any number of mutations. In addition to those hereditary forms of porphyria. There are also non-genetic, “acquired porphyrias” including a form of porphyria caused by heavy metal poisoning, commonly lead poisoning (aka plumbism)
As the “hepatic” in acute hepatic porphyria hints at, AHP mainly affects the liver, especially as it tries to ramp up heme production to make more of the heme-containing detoxifiers, CYPs. It’s characterized by attacks of stomach and nerve pain, racing heart rate, and numerous other potential symptoms. Lead poisoning, on the other hand, shows its heme-related effects mainly in blood cells and can cause a form of anemia (a condition where blood is unable to carry oxygen sufficiently – usually either because there are too few red blood cells and/or not enough functioning hemoglobin).
To understand what’s going on, here’s a brief overview of the heme synthesis pathway. It starts with the enzyme ALAS (δ-aminolevulinic acid synthase), which takes the amino acid glycine & succinyl-CoA (an intermediate of the energy-producing tricarboxylic acid (TCA) cycle), sticks them together with the removal of carbon dioxide, and gives you aminolevulinic acid (ALA). Two ALA hook up with the help of ALA Dehydrogenase (ALAD) to give you porhobilinogen (PBG). Then 4 of those PBGs chain up with the help of porphobilinogen deaminase (aka hydroxymethylbilane synthase aka the one that’s messed up in AIP) to give you porhobilinogen (PBG).
Then 4 of those PBGs chain up with the help of PBG deaminase (aka hydoxymethylbilane synthase) to form hydroxymethylibiane (HMB) & that chain of rings ringifys to a ring of rings called uroporphyrinogen III with the help of uroporphyrinogen III synthase (UROS). That ring of rings then gets further processed, by uroporphyrinogen decarboxylase (UROD), which removes carboxyl groups to give coproporphyrinogen-III. Then coproporphyrinogen-III oxidase (CPOX) removes a couple more to give you protoporphyrinogen IX, and then proptoporphyrinogen IX oxidase (PPOX) oxidizes (removes electrons from) protoporphyrinogen IX to give you protoporphyrin IX. I told you there were a lot of opportunities for things to go wrong!
Now, all that’s left is adding an atom of the element iron – so let’s iron out some quick background about why iron and protoporphyrin IX make a good match…
The basic units of elements are called atoms – they’re made up of smaller subatomic particles called protons (positively-charged), neutrons (neutral), & electrons (negatively-charged). Unlike the protons & neutrons, which are tightly packed into a dense central nucleus, the electrons whizz about around them in a diffuse “electron cloud.” Also unlike protons, for which each element has a fixed number, the # of electrons can vary because atoms can give, take, & share electrons, so atoms can have various charges. Metals like lead are really good at electron give & taking so they’re great for catalyzing redox reactions – reactions involving the giving of electrons (oxidation) and the taking of electrons (reduction). This is one reason some proteins like to hold heme.
In addition to giving & taking electrons, atoms can share pairs of them – and this is how you get the strong covalent bonds that hold atoms together in molecules. You need 2 electrons for a single bond & 4 for a double. Usually one bonding partner each provides an electron, but, if something has a lone pair (often oxygen or nitrogen), they can share that pair with a metal to form a coordinate covalent bond. And metals can form multiple such bonds, so they can bind multiple things all without having to give up a single electron. When a metal is bound in multiple places we say it’s “chelated” and protoporphyrin IX has 4 Nitrogen atoms ready to “bite down” – so it’s a “tetradentate chelator.” Coordinate covalent bonds still involve 2 electrons – but they’re different from usual covalent bonds because both of those electrons are coming from the same atom.
But what if you have “extra” electrons – if others around you have extras also you can get something called “resonance” or electron delocalization – a sort of electronic commune where they share extra electrons among all of them. more here: http://bit.ly/2qzMRFi
Protoporphyrin IX forms such a conjugated ring system – it has heterocyclic organic rings (rings that are mostly carbon but have atoms of other (hetero) elements at some corners) in which all of the atoms in the ring have “opted in.” This provides “resonance stabilization” for those ring atoms, which makes them happy, and it makes a great electronic “nest” for metals because all those electrons are oh-so-attractive to positively-charged (cationic) metal ions.
At this point in the heme pathway we have our metal holder (protoporphyrin IX) – now you just need the (right) metal. A ferrous iron atom (Fe²⁺) gets plopped into the nice little nest you just formed with the help of the enzyme ferrochelatase (Fe-Ch) to give you heme b (aka protoporphyrin IX).
But what if Fe-Ch isn’t working (like if it’s inhibited by lead)? And/or you don’t have enough iron (there’s an iron deficiency)?! You still have a nice inviting potential metal nest. So other metals can see it and plop themselves in – zinc (Zn²⁺) can attach itself to form zinc-protoporphyrin (ZPP) – either with the help of Fe-Ch if that’s working fine there’s just not enough iron, or on its own (non-enzymatically) if Fe-Ch’s down.
A non-cool thing about ZPP is that it doesn’t work for carrying oxygen. But a cool thing about ZPP is that, like Rudolph’s nose, you could even say it glows – if you shine the right wavelength of light at it, it will absorb that light and then spit out a different wavelength of light (i.e. it fluoresces) – so ZPP levels can be measured through fluorescence checking with a haematofluorometer – and this can be used to screen for lead poisoning and iron deficiency.
It’s easier to see why it can be useful for determining iron deficiency – without iron to stick in there, the Zn²⁺ takes its place. But with lead poisoning, you still have iron, so what’s the problem? The problem is that lead inhibits Fe-Ch – ironically (no pun intended – honestly!) because it displaces Zn²⁺ in the Fe-Ch protein.
Turns out that it’s not just Zn²⁺ that can house-steal! Zn²⁺ itself can get kicked out of various places by lead, Pb²⁺. Thanks to the whole big ole metal that can grab onto multiple things at once – thing, Zinc often plays structural and catalytic roles in enzymes and other proteins, so getting kicked out can cause Zn²⁺-requiring proteins to stop working, including a few such proteins in the heme synthesis pathway – in particular, lead can inhibit ALAD & Fe-Ch.
So, even though you have iron, you can’t stick it in there without Fe-Ch but zinc can get in without it. So, with lead poisoning, protoporphyrin & ZPP can build up in the blood & ultimately in the urine.
But that’s not the only heme-related buildup product. Similarly to what I explained in the post on that genetic porphyria, AHP, you get increased ALA synthesis because of lost negative feedback since you don’t have real heme. And not all this ALA makes it all the way (even all the way to ZPP) because iron also inhibits ALAD (the enzyme that sticks together ALAs to give you porphobilinogen. So you also get a build up of the neurotoxic ALA (but, unlike with AIP, you don’t get PBG buildup).
ALA is neurotoxic, but that’s not the only way lead causes nervous system problems. Because, in addition to displacing Zn²⁺, lead can displace calcium, Ca²⁺. A lot of the complex signaling that occurs in our brains relies on calcium. Lead can “pretend” to be calcium, sneaking past the blood-brain-barrier & confusing brain cells and making them less sensitive to legit calcium. This can cause severe neurological and psychological problems – especially in children, whose brains are rapidly developing and are more vulnerable. And kids aren’t just building their brains – they’re also building bones – and bones store calcium – and if bones think lead’s calcium, they can store lead instead – so lead can build up in their bodies and get released over time, leading to long-term consequences even years after initial exposure.
Lead leads to lots of problems – and it’s blood-related problems don’t just come for heme problems. The anemia associated with lead poisoning doesn’t just come from reduced heme synthesis – it also comes from the red blood cells that get made not lasting very long before getting destroyed by “hemolysis.” This occurs in part because lead inhibits an enzyme called pyrimidine 5′-nucleotidase (P5NT), which normally helps catabolize (break down) RNA letters. So those letters build up and cause problems. Additionally, Lead likes to bind to the sulfhydryl (-SH) groups of the amino acid (protein letter) cysteine, which antioxidant proteins usually use to regulate redox levels and protect other molecules from getting damaged by highly-energetic “reactive oxygen species (ROS).” When lead interferes with these proteins, redox is thrown out of wack and all sorts of problems can arise.
more on lead poisoning: https://bit.ly/leadpoisoningscience
more on redox: http://bit.ly/ridiculousredox
more on cysteine: http://bit.ly/cysteinecrosslinks
more on metals & coordinate covalent bonds: http://bit.ly/2XDSLjd
more on metalloenzymes: http://bit.ly/2pRukAj