B9: Heme Proteins - Biology

B9: Heme Proteins - Biology

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So far, we have examined three different kinds of heme proteins..

  • The first, hemoglobin (and myoglobin) serve as carriers of dioxygen. Even though they bind one of the best oxidizing agents around (dioxygen), the heme Fe2+ does not get oxidized to Fe3+. If it does, as in the case of met-Hb, the protein looses it ability to carry oxygen.
  • Cytochrome C, on the other hand, does not bind dioxygen but rather serves as a carrier of electrons which get passed to dioxygen in Cytochrome C oxidase. Its Fe ion readily cycles between the 2+ and 3+ states as it serves as an electron carrier.
  • Finally, the Fe2+ in the heme of the cytochrome P450s (so named since they have an absorbance maximum at 450 nm when they bind CO) does both. It binds dioxygen and cycles between the 2+ and 3+ states as it activates dixoxygen for hydroxylation reactions.

How could heme serve such diverse functions? We can explain this by referring to one of the main themes of the course - structure mediates function. The environment of each heme must be different. Clearly the protein ligands coordinating the Fe ions are different. The 5th ligand is the proximal His in hemoglobin while dioxygen binds to the 6th site. In cytochrome C, the 5th and 6th ligands are His and Met, respectively. In cytochrome P450, the 5th site is occupied by Cys, and the 6th by dioxygen. Presumably the environments surrounding the hemes are different as well. Once again, we have seen analogous example in which chemical properties are influenced by the microenvironment. The pKa of a given amino acid side chain can vary considerably depending on the polarity of the local environment. Likewise, the standard reduction potential of tightly bound FAD/FADH2 depends on the microenvironment.

As we have seen (from the study of heme proteins and the oxidative enzymes of cells), transition metals such as Fe, Zn, and Cu have vital biological roles as binding sites and cofactors in many reactions. Yet they also pose problems since they can lead to oxidative damage in cells. As we saw with cytoplasmic metallothioniens, which bind to heavy metals and protect the cell from such damage, many proteins are involved in binding and regulation of transition metals in the cell. Integral membrane proteins are required to bind and transport these cations into the cytoplasm. Other proteins act as sensors of transition ion concentration (such as latent transcription factors which bind heavy metals and become active transcription factors for metallothioniens. Others act as chaperone proteins which bind metal ions and transfers them to apometalloproteins. Recent work has suggested transporters and chaperones involved in metal ion biology bind these ions with unusual coordination geometry, which presumably facilitates transfer of the ion to the apo-target protein.

The transition metals Zn and Fe are often found in E. Coli at a concentration of 0.1 mM, compared to Cu and Mn which are present at concentrations from 10 to 100 μM. Also, about one third of all proteins demonstrate specific binding of metal ions and can be classified as metalloproteins. Mass balance suggests that metal ions would be distributed in proteins with low, intermediate, and high metal binding affinity as well as in free pools, which which potentially be toxic to cells. Metalloproteins, depending on their Kd for metal ion binding, would hence be in various state of ligation. The free concentration of some ions (Cu and Zn) is so low that newly synthesized apoproteins which bind these ions would not obtain the ion from the free pool. In such cases, metal chaperones would be required.

Endothelial NOS

Endothelial NOS (eNOS), also known as nitric oxide synthase 3 (NOS3) or constitutive NOS (cNOS), is an enzyme that in humans is encoded by the NOS3 gene located in the 7q35-7q36 region of chromosome 7. [5] This enzyme is one of three isoforms that synthesize nitric oxide (NO), a small gaseous and lipophilic molecule that participates in several biological processes. [6] [7] The other isoforms include neuronal nitric oxide synthase (nNOS), which is constitutively expressed in specific neurons of the brain [8] and inducible nitric oxide synthase (iNOS), whose expression is typically induced in inflammatory diseases. [9] eNOS is primarily responsible for the generation of NO in the vascular endothelium, [10] a monolayer of flat cells lining the interior surface of blood vessels, at the interface between circulating blood in the lumen and the remainder of the vessel wall. [11] NO produced by eNOS in the vascular endothelium plays crucial roles in regulating vascular tone, cellular proliferation, leukocyte adhesion, and platelet aggregation. [12] Therefore, a functional eNOS is essential for a healthy cardiovascular system.

Biological significance and applications of heme c proteins and peptides

Hemes are ubiquitous in biology and carry out a wide range of functions. The heme group is largely invariant across proteins with different functions, although there are a few variations seen in nature. The most common variant is heme c, which is formed by a post-translational modification in which heme is covalently linked to two Cys residues on the polypeptide via thioether bonds. In this Account, the influence of this covalent attachment on heme c properties and function is discussed, and examples of how covalent attachment has been used in selected applications are presented. Proteins that bind heme c are among the most well-characterized proteins in biochemistry. Most of these proteins are cytochromes c (cyts c) that serve as electron carriers in photosynthesis and respiration. Despite the intense study of cyts c, the functional significance of heme covalent attachment has remained elusive. One observation is that heme c reaches a lower reduction potential in nature than its noncovalently linked counterpart, heme b, when comparing proteins with the same axial ligands. Furthermore, covalent attachment is known to enhance protein stability and allow the heme to be relatively solvent exposed. However, an inorganic chemistry perspective on the effects of covalent attachment has been lacking. Spectroscopic measurements and computations on cyts c and model systems reveal a number of effects of covalent attachment on heme electronic structure and reactivity. One is that the predominant nonplanar ruffling distortion seen in heme c lowers heme reduction potential. Another is that covalent attachment influences the interaction of the heme iron with the proximal His ligand. Heme ruffling also has been shown to influence electronic coupling to redox partners and, therefore, electron transfer rates by altering the distribution of the orbital hole on the porphyrin in oxidized cyt c. Another consequence of heme covalent attachment is the strong vibrational coupling seen between the iron and the protein surface as revealed by nuclear resonance vibrational spectroscopy studies. Finally, heme covalent attachment is proposed to be an important feature supporting multiple roles of cyt c in programmed cell death (apoptosis). Heme covalent attachment is not only vital for the biological functions of cyt c but also provides a useful handle in a number of applications. For one, the engineering of heme c onto an exposed portion of a protein of interest has been shown to provide a visible affinity purification tag. In addition, peptides with covalently attached heme, known as microperoxidases, have been studied as model compounds and oxidation catalysts and, more recently, in applications for energy conversion and storage. The wealth of insight gained about heme c through fundamental studies of cyts c forms a basis for future efforts toward engineering natural and artificial cytochromes for a variety of applications.

Disturbances in Energy Supply

4.3.3 Neuroglobin and Cytoglobin

Dioxygen-binding heme proteins like neuroglobin and cytoglobin have been also described in other tissues. In comparison with hemoglobin and myoglobin, this knowledge is fragmentary as these proteins are only recently discovered on the basis of proteomic analysis [70–73] .

Neuroglobin is expressed in the cells of the central and peripheral nervous system, cerebrospinal fluid, retina, and endocrine cells [70,73,74] . This monomeric heme protein reversibly binds dioxygen with an affinity higher than hemoglobin, but lower than myoglobin. Unlike hemoglobin and myoglobin, the deoxygenated neuroglobin has a hexacoordinated heme iron in the reduced and oxidized form. On binding, dioxygen displaces the sixth endogenous ligand.

Neuroglobin facilitates like myoglobin the diffusion of dioxygen to the mitochondria. It helps to provide reserves of dioxygen to neuronal cells under hypoxic-ischemic conditions [70,75,76] . An action of neuroglobin as NADH oxidase under conditions of limited dioxygen supply has been proposed [73] . The involvement of neuroglobin in NO metabolism is another hypothetic function [77] .

The forth human heme protein involved in binding and storage of dioxygen is cytoglobin that is expressed in a wide range of tissues [71] . Like neuroglobin, this monomeric protein is characterized by hexacoordinated heme iron in the deoxygenated form.

Similar functional activities as described for neuroglobin are also under discussion for cytoglobin. This heme protein also exhibits a catalase activity [78] .

Anatomy of an Impossible Burger

Burger King is going to sell the Impossible Burger and McDonald&rsquos is soon to follow with its own meatless patty. So I thought I&rsquod check out the brilliantly-branded burger, both in the patent and on my plate.

A Variation on the Heme Theme

My first encounter with the Impossible Burger was pinching a piece off my dinner companion&rsquos plate in February. It looked and seemed to bleed like a real burger. As I chewed, I googled the product on my phone, stopping at the word &ldquoheme.&rdquo

I stopped chewing. Once I got past the image of a bovine muscle pulsating on the plate, I envisioned the iron atom within its porphyrin ring, both lying within a surrounding globular protein, a little like a tootsie roll pop.

Heme in various guises is found in all species, from bacteria to beans to buffalos. It&rsquos at the heart of the myoglobin in our muscles and the hemoglobin in our blood, packed most densely into the muscle cells of beef cattle.

Proteins that are the same or similar among diverse species are called &ldquohighly conserved.&rdquo They&rsquove not changed much through evolution because they work. Natural selection weeds out mutations that stifle the ability to bind oxygen, which is what the iron atom at the center of the action does. All hemes have iron, but the protein globin parts vary, ever so slightly, among species.

The trick in creating a meatless burger that tastes and feels meaty is in finding an organism whose heme-plus-protein imparts what&rsquos described as savory, bloody, or just beefy.

I&rsquove been trying different brands of veggie burgers since ditching beef 18 months ago in the wake of a near-simultaneous cancer diagnosis/trip to Costa Rica, against a backdrop of our daughter urging us to do so for more than a decade (see How Genetic Testing Guided my Breast Cancer Journey to Eschewing Beef). The images on the packages depict enticing chunks of sweet potato, black beans, peas, and carrots peeking from patties consisting of the ubiquitous soy protein. These products provide palatable alternatives to burgers, but they&rsquore not quite the real deal. The Impossible Burger comes closest.

The 52-page patent, awarded in 2017 after many years of work to Impossible Foods of Redwood City, CA for &ldquomagic mix,&rdquo opens with two-columned pages of patent and article citations.

The meat of the patent begins with a list that might ring a bell for biology or chemistry majors: the amino acid sequences of the heme proteins from 25 species, including the top candidates for the Impossible Burger vertebrate flesh equivalent. The 25 contenders include peas, bacteria, algae, soil fungi, horse, cattle, tobacco, wild boar, and a paramecium.

The winner is at the end of the first patent claim: &ldquoA ground beef-like food product comprising 0.1%-5% by weight of a heme-containing protein comprising an amino acid sequence having at least 80% sequence identity to the polypeptide set forth in SEQ ID NO:4.&rdquo

SEQ ID No. 4 is Glycine max, aka soybeans.

The heme protein, leghemoglobin (legHB), reddens root nodules of soybean plants. It provides oxygen to its symbiotic bacteria, similar to hemoglobin transporting oxygen in our blood and myoglobin in our muscles. But even the millions of acres covered in soybeans in the US aren&rsquot enough to meet the projected demand for legHB in burgers.

Recombinant DNA Technology to the Rescue

The obvious way to scale up production of a specific protein is to use recombinant DNA technology: make the soybean protein in cells of another, easier-to-harness, species.

When recombinant DNA technology was invented in the 1970s, the tag GMO was still years away. Early on a pattern emerged of some people objecting to agricultural experiments and even destroying experimental fields and one notable strawberry patch, while people with diabetes began to use insulin made in bacteria like E. coli, as they still do. It&rsquos always been a fractured field, but recombinant DNA technology is here to stay. The pharmacopeia courtesy of the technology today includes clotting factors, enzyme replacements, heart drugs, cytokines, surfactant, hormones and growth factors, and lots more.

The researchers at Impossible Foods stitched the soybean gene that encodes legHB protein into the genome of a different organism that can pump it out more efficiently (cheaply): the yeast Picchia pastoris. A yeast is a single-celled fungus, but it&rsquos a complex cell, unlike a bacterium.

A single sentence in the 52-page patent spells this out: &ldquoHeme-containing proteins also can be recombinantly produced using polypeptide expression techniques&rdquo and can be grown in the cells of bacteria, insects, fungi, plants, or mammals. &ldquoAlso&rdquo refers to extraction from natural sources or synthesis in a lab.

Does an Impossible Burger contain a GMO? Well, yes and no. Yes because a soybean gene wouldn&rsquot naturally be in a yeast cell. But no because the legHB that the yeast cells crank out is identical, amino-acid-by-amino-acid, to the protein from soybean root nodules. So the yeast is genetically modified, the product, not.

It&rsquos a distinction with a precedent, and Impossible Foods&rsquo Chief Science Officer David Lipman grabs it in this 2018 interview for Food Dive. He cites the 1990 FDA approval of the first recombinant DNA food product, rennin (aka chymosin), after being deemed &ldquogenetically recognized as safe,&rdquo or GRAS.

Rennin, used to curdle milk in cheese-making, is part of a mixture of digestive enzymes collected from calf intestines. Making it in E. coli given the cow gene for the needed enzyme is much cheaper. Regulations can be confusing. In the US, recombinant chymosin need not bear the GMO label because the protein that makes it into the product is identical to the protein straight from the natural source organism.

In the interview, Lipman uses the word &ldquofermentation&rdquo repeatedly, which conjures folksy images of vats of sweet wine and aging pungent cheeses. &ldquoRecombinant DNA&rdquo and &ldquogenetically modified&rdquo elicit different responses, such as in this post calling the Impossible Burger out on its GMO ingredient. But the FDA deemed magic mix GRAS in January 2019 and so the burger doesn&rsquot require GMO labeling.

The heme protein is only one ingredient of magic mix.

To fashion the burger, &ldquoflavor precursor molecules&rdquo are added. These include coconut and other plant oils, potato and texturized wheat protein, sugars, amino acids (like monosodium glutamate), one vitamin, and familiar compounds like lactic acid and creatine.

Here&rsquos the patent lingo: &ldquoa compound selected from glucose, ribose, fructose, lactose, xylose, arabinose, glucose-6-phosphate, maltose, and galactose .. and &hellip cysteine, cystine, selenocysteine, thiamine, methionine, and mixtures of two or more thereof.&rdquo But all foods are, ultimately, chemicals. Everything is a chemical.

Creating the Impossible Burger also analyzed the volatiles the concoction emits upon cooking. &ldquoTrained human panelists&rdquo and other humans who deployed gas chromatography&ndashmass spectrometry, a standard analytical chemistry test, analyzed the release of the meaty aromas upon cooking, producing &ldquoolfactory maps.&rdquo The goal was to optimize the flavor, taste, smell, texture, and the all-important &ldquomouthfeel&rdquo of the product. Like software, new versions would come out periodically.

The meaty mash is also malleable, molded into faux body parts like wings and steaks, extruded as sausages, crumbled up delicately in soup and stew bases, and easily converted into a smorgasbord of snacks, cubes, and powders.

I&rsquod give the Impossible Burger a grade of &ldquoA&rdquo for mouthfeel and texture, which may be the same thing. But I&rsquod give only a B-plus for flavor, taste, and smell, because it didn&rsquot have them. But add cheese, fried onions, a pickle, and a dollop of Sweet Baby Ray&rsquos, and it can indeed pass for a real patty of Bos taurus flesh. At least the one I had lacked that deep flavor of 85%-fat burger coming straight off the grill.

On a scale of 0 to 10, with 10 being a beef burger and 0 the worst veggie burger imaginable, most products I&rsquod rate in the 4 to 6 range, with an Impossible Burger a robust 9.

A data-packed paper in PLOS One reaches a similar conclusion: &ldquoBy fulfilling the same gustatory, culinary and nutritional functions as traditional beef, the PBB (&ldquoplant-based burger&rdquo) aims to lower the adoption barrier associated with the consumption of vegetal proteins in lieu of animal products.&rdquo Two of the four authors work at Impossible Foods, but still, I think they&rsquore right. This product can replace burgers for omnivores, and perhaps win over some vegetarians.

Beyond personal preferences and tastes, the Impossible Burger achieves its stated goal: producing it doesn&rsquot kill any animals. It also earns high marks for environmental friendliness. According to a post at Fast Company, the carbon footprint of an Impossible Burger is 89% smaller than that for a cowburger and uses 87% less water, 96% less land, and cuts water contamination by 92%. The PLOS One article analyzes the environmental impact too.

I&rsquom looking forward to Impossible Burger, in whatever form, hitting supermarket shelves.

Hemoglobins: Structure and Properties | Biochemistry

In this article we will discuss about the structure and properties of hemoglobins.

Structure of Hemoglobins:

As indicated by their name, hemoglobins consist of a prosthetic group the heme (4%) and a protein part: the globin (96%).

A. Heme:

This is the prosthetic group common to various hemoglobins (while globin varies in different hemoglobins). It contains one molecule of protoporphyrin and one iron atom.

Four pyrrole rings, linked by methenyl bridges = CH — between their α and α’ carbon atoms form the porphin (see fig. 1-27).

We must note the alternation of the double bonds which are all conjugated.

Porphyrins are simply derivatives of porphin where the 8 β and β’ carbon atoms are carriers of different substituents. It is clear that there are pos­sibilities of isomerism we will study these, taking the example of uroporphyrins where the 8β and β’ carbon atoms carry 4 acetic radicals (A) and 4 propionic radicals (P) figure 1-28 shows that with 2 different substituents there are 4 possible arrangements among natural pigments, mostly type III and some­times type I, are found.

Protoporphyrin of hemoglobins is of type III as maybe seen in figure 1-29, it derives from uroporphyrin III by decarboxylation of the 4 acetic radicals (into methyl groups) and decarboxylation and then dehydrogenation of two of the four propionic radicals (which are thus transformed into vinyl groups).

This is denoted protoporphyrin IX by certain authors, because with 3 dif­ferent substituents there are actually 15 possible isomers (and not 4) and the comparison with different porphyrins obtained by synthesis has shown the identity of natural protoporphyrin with the type IX of synthetic isomers. Protoporphyrin therefore contains 4 methyl groups in 1, 3, 5 and 8 2 propionic radicals in 6 and 7 and 2 vinyl groups in 2 and 4.

b) Linkage of Iron and Protoporphyrin:

Porphyrins have the property of binding, by their pyrrole nitrogen atoms, metals like Fe, Mn, Ni, Co, Mg metalloporphyrins containing ferrous iron (Fe ++ ) are called hemes in the particular case of protoporphyrin, the ferroporphyrin is called protoheme (the term “heme” is used by abbreviation). Metalloporphyrins containing ferric iron (Fe + ++ ) are called hematins (the one corresponding to protoporphyrin is protohematin).

In the protoheme, the iron atom replaces the 2 hydrogen atoms carried by two of the four nitrogen atoms but it is linked with the 4 nitrogen atoms by coordination. Besides, Fe ++ can still exchange 2 coordination linkages with nitrogen bases for example, forming a hexacoordinated complex (as in ferrocyanide). These two linkages are perpendicular to the plane of the heme.

c) Important Properties of the Heme:

We have seen that the heme is a ferrous compound under the action of oxidants (like alkaline ferricyanides, for example), it can be oxidised into hematin (where iron is ferric) which can be again reduced into heme (by sodium hydrosulphite, for example).

This reversible reaction forms the basis of the participation of ferropor- phyrins, like the cytochromes, in the oxidation — reduction processes (but it does not take place during the conversion hemoglobin ⇋ oxyhemoglobin).

As will be seen below, the heme can link with globin. It can also link with other nitrogen bases (pyridine, nicotins) or with the nitrogen groups of proteins, to form homochromogens.

Similarly, hematin can link with different proteins to form parahematins, often having catalase or peroxidase properties.

B. Globin:

This is the protein part of the chromoprotein it is easily obtained by treating hemoglobin with acetone containing 5% HCl (globin precipitates). Globin is the specific part of hemoglobin which varies according to age, species, and in certain diseases.

In myoglobins there is a single polypeptide chain (combined with one heme) whose primary, secondary and tertiary structure is known, at least in certain species (see figure 1-19). Molecular weight is about 17 000. Histidine content is comparatively high (6 to 10%).

Blood hemoglobins are tetramers they contain 4 polypeptide chains (each combined with one heme) and their molecular weight is about 68 000.

The 4 chains are united by linkages which are easy to break in the case of adult human hemoglobin (HbA), 2 types of chains (α and β) are obtained these are combined in pairs in the original molecule this is expressed by writing HbA = α2 A β2 A (A means “Adult”). The α chain contains 141 amino acids and the β chain 146 the sequences are fully known.

The foetal hemoglobin also contains 4 chains, 2 α chains and 2 γ chains (the latter have about ten amino acids different from those present in β chains). We can write HbF = α2 A γ2 F .

The pathological hemoglobins differ from HbA, either by anomalies in the distribution of chains (there are hemoglobins having 4 β chains or 4 γ chains), or by anomalies in the sequence of the α chain or β chain (for example, in sickle-cell anaemia, glutamic acid normally present in position 6 of the β chain is replaced by a valine, and just this difference is sufficient to alter the physiological properties of hemoglobin).

C. Union of Heme and Globin:

In the myoglobin of the sperm whale, which was thoroughly studied from the structural point of view, the heme is lodged in a fold of the tertiary structure of globin (see fig. 1-19).

In all these ferroporphyrin — protein complexes, the iron atom has an oc­tahedral structure of chemical type d 2 sp 3 (double pyramid with square base formed by the 4 pyrrolic nitrogen atoms).

The 5th coordination position of the iron is occupied by the imidazole nitrogen N3 of the histidyl residue in position 93 (F8 or 8th residue of the helix F in the Perutz nomenclature). The 6th coordination position is free in the non-oxygenated or Deoxy state, but it is occupied by oxygen in the oxygenated or Oxy state (or by the carbon of carbon monoxide, competitive inhibitor of oxygen).

In the case of the α or β chains of hemoglobin, we have exactly the same Deoxy and Oxy structures (fig. 1-30): iron is linked with the nitrogen N3 of histidine F8 called proximal (position 87 for α and 92 for β).

If the iron atom is oxidized to the ferric state, its 6th coordination position cannot any more be occupied by an oxygen molecule, it is occupied by the oxygen atom of a water molecule, one proton of which is attached by hydrogen bond to the nitrogen N3 of another histidine called distal and which occupies the position E7 in the Perutz nomenclature (7th residue of the helix E, respec­tive positions 64, 63 and 58 in myoglobin, chain β and chain α).

This form called metmyoglobin or methemoglobin is a stable Deoxy state in which hexacoordinated ferric iron can no longer play the role of oxygen fixing agent, contrary to the previous case.

It is observed that in the Deoxy state, the external electron layer of the ferrous iron atom contains 16 electrons of which 2 are unpaired. The ferroporphyrin-protein complex is then paramagnetic. In the Oxy state, this external layer is saturated with 18 electrons all paired, and the complex is therefore diamagnetic.

In the metmyoglobin or methemoglobin form, the external layer has 17 electrons of which 1 is unpaired, and the complex is paramagnetic. This form can be stabilized by a CN – cyanide ion which brings 3 electrons to the ferric ion, the external layer of which is saturated with 18 electrons. We then obtain a diamagnetic stable Oxy form where oxygen cannot displace the CN – ion.

Beside this primary linkage between Fe and N 3 of the proximal histidine, two saline linkages (between 2 propionyl radicals of heme and 2 basic groups of lysine or arginine) and Van der Waals type linkages (between hydrophobic groups of heme and globin) maintain the stability of the structure.

It must be noted that if we have a non-denatured globin, it is possible to effect in vitro the combination heme + globin we obtain a hemoglobin having the properties of the chromoprotein which was used for the isolation of globin (whatever the origin of the heme). Inversely, the combination of a heme with diverse globins produces chromoproteins which differ according to the origin of the globin used.

Properties of Hemoglobins:

When we centrifuge fresh blood or blood made incoagulable, the red cells sediment. Bringing them back in suspension in a hypotonic medium (for example distilled water) is sufficient to make them burst (hemolysis) and, after elimination of the cellular debris by centrifugation, we obtain a red coloured solution.

The solution is dark red for hemoglobin and bright red for oxyhemoglobin (due to differences in the absorption spectra) there is there­fore an oxidized form of hemoglobin whose physiological role — as mentioned in the introduction – is to transport oxygen after forming with this gas an easily dissociable combination. We will now examine the most important property of hemoglobins namely their capacity to reversibly bind certain gases particularly oxygen.

A. Combinations of Hemoglobins with Gases:

a) Combination with Oxygen:

For all hemoglobins, the maximum quan­tity of oxygen which can be bound is a function of the quantity of iron: one atom-gram of iron combines with one molecule-gram of oxygen (or one molecule gram of carbon monoxide).

Since there are 4 hemes (i.e. 4 iron atoms) per molecule of hemoglobin, we can write:

(but very often, oxyhemoglobin is represented by HbO2). This is an oxygenation and not an oxidation because iron remains in the ferrous state, 17 000 g Hb (corresponding to the monomer which contains 1 iron atom) can therefore combine with 32 g of O2 and since 1 mole of oxygen occupies 22.4 1, it may be derived that 1 g Hb can bind 1.34 ml O2. But, 100 ml of blood contains in average 15gHb, so that it can bind 1.34 x 15, i.e. about 20ml O2 this represents complete saturation in O2.

The equation of oxygenation reaction shows that oxygen pressure is an important factor controlling this equilibrium. In the air that we breathe where mean pressure is 760 mm Hg and oxygen concentration about 20%, the partial pressure of oxygen is 760/5, i.e. about 150 mm.

In arterial blood, oxygen pressure is about 80 mm, so that (see fig. 1-31) 19 ml of O2 can be bound for 100 ml blood (95% saturation). In the capillaries, partial pressure of oxygen falls to 40 mm or even 20 mm, therefore O2 fixation can be only 77% and 40% of saturation respectively.

Figure 1-31 shows that this stage corresponds to the portion of the curve where a comparatively small decrease of oxygen pressure causes a comparatively major liberation of oxygen when pressure falls from 40 to 20 mm, the quantity of oxygen which can be present in blood falls from 20 x 77/100 i.e. 15.4 ml O2/100 ml to 20 X 40/100 i.e. 8 ml O2/100 ml in other words there is liberation of 7.4 ml O2/100 ml of blood in favour of the tissues.

It is seen in figure 1-31 that the saturation curve of myoglobin is a branch of hyperbola while that of hemoglobin is a sigmoid (S-shaped). This difference is due to the fact that hemoglobin is a tetramer whose 4 hemes are not in­dependent, because the oxygenation of one of them favours that of others (which thus requires less energy).

This interaction does not take place directly because the hemes are too distant from one another (about 30 Å) it takes place through the quaternary structure of hemoglobin to obtain this cooperative effect between hemes, the quaternary structure must be intact, and a pathological hemoglobin (by anomaly of the distribution of chains) or a denatured hemoglobin behaves like the myoglobin furthermore, X-ray studies have actually shown – during the transformation [Hb]4 → [HbO2]4 or inversely — a change of the conformation of the molecule, called allosteric transition, which takes place only with a hemoglobin whose quaternary structure is native.

This is an example of relationship be­tween quaternary structure and biological activity we will come across other such cases while studying enzymes and we will see that the presence of al­losteric enzymes ensures a regulation of cellular metabolism.

Lastly, it should be noted that oxyhemoglobin, because of changes of confor­mation, has a larger number of dissociated acid groups.

Therefore, H + ion concentration also has an effect on the equilibrium which may be written:

In the tissues, there is production of CO2 which will lead to an increase of H + concentration and favour liberation of oxygen. In the capillaries, this liberation is therefore caused, mostly by the decrease of O2 pressure, but also by the increase of CO2 pressure-, figure 1-31 shows that for a given O2 pressure, the quantity of oxygen which may be combined is smaller when CO2 pressure is higher (curve no. 3).

On the contrary, in the lungs, the O2 pressure is high and CO2 pressure falls (because CO2 is eliminated) and this decreases acidity these two factors — particularly, the former — favour the formation of oxyhemoglobin.

b) Combination with Carbon Monoxide:

This is very similar to the combination with oxygen there is binding of one molecule-gram CO per atom-gram iron, but the stability of the combination (Hb-CO)4 called carbonyl- hemoglobin (or carboxyhemoglobin) is about 200 times greater the dissocia­tion is very difficult this explains the toxicity of the gas, and the fact that high concentrations of oxygen (oxygenotherapy) are required in the treatment of intoxication by carbon monoxide.

c) Combination with Other Gases:

Carbon dioxide can combine with hemoglobin to give carbhemoglobin, but the heme plays no part, because CO2 binds to the basic groups of globin it also binds to those of other proteins (but it should be remembered that hemoglobin represents about 75% of the total blood proteins).

About one fourth of the CO2 carried by blood travels in the form of carbaminoproteins:

It should be of interest to examine here, how, in general, CO2 is transported from the tissues where it is formed to the lungs where it is eliminated. This transport must prevent, on the one hand, a progressive acidification of blood as it flows through the tissues liberating CO2, and on the other hand, the formation of CO2 bubbles, because the solubility of this gas is comparatively limited.

These difficulties are overcome by the buffer capacity of blood proteins which can bind H + ions as mentioned earlier and so, more H2CO3 can be ionized and therefore more CO2 can be dissolved (see fig. 1-32). On the contrary, when blood reaches the lungs, CO2 is liberated and the reactions of figure 1-32 take place in the opposite direction the proteins are again ready to bind H + ions.

It must be noted that the system H2 CO3 ↔ HCO3 – is a buffer system and that it is largely responsible (together with the system H2PO4 – ↔ HPO4 = ) for protecting blood from excesses of acid or base.

B. Reversible Oxidation of Hemoglobin and Oxyhemoglobin:

Hemoglobin treated by a mild oxidising agent like potassium ferricyanide is oxidized into methemoglobin. Iron changes from Fe ++ state to Fe + + + state, so that the heme is oxidized into hematin this oxidation is accompanied by a modification of the absorption spectrum and the loss of the property of the prosthetic group to combine with O2 or CO.

If oxyhemoglobin or carboxyhemoglobin is subjected to this oxidation, we also observe the formation of methemoglobin accompanied by the liberation of the gas combined, which incidentally permits the titration of the gas combined with hemoglobin.

The formation of met hemoglobin in vivo, either as a result of a serious intoxication or after a metabolic disease, has grave consequences because oxygen transport is made impossible. However, methemoglobin can be again reduced into hemoglobin in vitro as well as in vivo in normal red blood cells, by the action of reducing agents. These inter-conversions may be summarized in a diagram (see fig. 1-33).

Globins and Other Nitric Oxide-Reactive Proteins, Part A

Alessandra Pesce , . Martino Bolognesi , in Methods in Enzymology , 2008


Crystal structures of group I, group II, and group III 2/2Hbs show that their fold is based on a subset of the classical globin fold (the so‐called 3‐on‐3 α‐helical sandwich), typical of sperm whale myoglobin (Mb) ( Holm and Sander, 1993 Perutz, 1979 ). Indeed, 2/2Hbs host the heme in a 2‐on‐2 α‐helical sandwich (2/2 fold) based on four α‐helices, corresponding to the B‐, E‐, G‐, and H‐helices of the classical globin fold ( Milani et al., 2003 Nardini et al., 2007 Pesce et al., 2000 Vinogradov et al., 2006 ). The antiparallel helix pairs (B/E and G/H) are arranged in a sort of α‐helical bundle, which surrounds and protects the heme group from the solvent phase ( Fig. 17.1A ). Residue deletions, insertions, and replacements relative to the classical vertebrate globin sequences have been shown to be distributed throughout the whole 2/2Hb chain. The most noticeable differences between the 2/2Hb and the full‐length globin folds are the drastically shortened (or absent) A‐helix, the absence of the D‐helix, and a long polypeptide segment (pre‐F) in extended conformation followed by a very short F‐helix that supports the heme proximal HisF8 residue, enabling heme iron coordination. Local structural variations in the 2/2Hb fold are evident in each of the three evolutionary groups ( Milani et al., 2003 Nardini et al., 2006, 2007 Pesce et al., 2000 ).

Figure 17.1 . Panel A: Stereo view of the 2/2Hb fold, displaying the four main helices building up the protein scaffold and the heme group. Labels identify the B, E, G, and H helices and the protein termini. Panel B: Stereo view of Ce‐2/2HbN displaying the protein matrix tunnel, shown as the gap‐density grid provided by SURFNET. In addition, four Xe atoms identified through the crystallographic analyses described are shown as spheres, with a radius proportional to their occupancy in the crystal structure ( Milani et al., 2004 ). Figure drawn with MOLSCRIPT program ( Kraulis, 1991 ).

Results and Discussion

Non-redundant dataset of heme binding proteins

There are 1998 and 113 PDB entries containing ligand HEM (heme type-b) and HEC (heme type-c) respectively with resolutions of 3Å or better as of November 24, 2009 [18]. Among these entries, 10 (1BE3, 1BGY, 1FGJ, 1GWS, 1PP9, 1PPJ, 1S56, 1S61, 2A06, and 3H1J) contain both heme type b and c. In toto 4272 protein chains were identified as heme interacting protein chains as described in Methods. A non-redundant dataset of 125 protein chains (114 heme-b and 11 heme-c, Additional file 1, Table S1) were generated using PISCES with a sequence identity cutoff of 25%[43]. Eighty-two percent of these protein chains contain only one heme molecule while the number of heme molecules in the remaining protein chains ranges from 2 to 8 (Additional file 1, Table S1). Two examples of multi-heme protein chains, 1FS7A with 5 type b and 3F29A with 8 type c heme molecules, are shown in (Figure 2A & 2B).

Examples of three-dimensional structure of multi-heme proteins and identification of heme-binding environment. (A) Cytochrome c nitrite reductase of Wolinella succinogenes (PDB chain: 1FS7A) with 5 heme b molecules (B) Thioalkalivibrio nitratireducens cytochrome c nitrite reductase (PDB chain: 3F29A) with 8 heme c molecules (C) globin domain of globin-coupled sensor in Geobacter sulfurreducens (PDB chain: 2W31A). The red sticks are axial ligands of the heme iron and the blue sticks represent other heme interacting residues. For better visualization, the neighboring heme residues in A and B are colored yellow and green respectively. The heme molecules are shown as spacefill. The images were generated using Pymol

The dataset of heme binding proteins includes a wide variety of protein folds. A total of 86 protein chains (

69% of the dataset) have SCOP annotations (based on release 1.75 and Pre-SCOP) and belong to 31 distinct structural folds in all four major classes (Table 1) [42]. The dataset is dominated by proteins in the all-α class, making up 64% (55 of 86) of the total. The top 4 folds, Globin-like (a.1), Cytochrome P450 (a.104), Cytochrome c (a.3), and Multi-heme cytochromes (a.138) represent the well-known heme binding proteins that have been investigated extensively (Table 1).

Structural environment of the heme binding pockets

To investigate the structural environment of heme binding pockets, we identified both residues that make coordinate bonds with the heme iron and the ones that interact with the heme porphyrin structure (Figure 2C and Methods). Out of the 125 heme binding protein chains, only 2PBJA and 3HCNA do not have residues identified as axial ligands to heme iron though both have extensive interactions with heme instead other small molecules, such as glutathione (GSH) in 2PBJA (microsomal prostaglandin E synthase)[57] and imidazole (IMD) in 3HCNA (human ferrochelatase) [58] form coordinate bonds with heme iron. Five different amino acids (H, M, C, Y, K) are found to serve as axial ligands to the heme iron with histidine as the dominant residue (

80%) in both heme b and heme c types (Figure 3). Heme b utilizes more cysteine residues while heme c has slightly more methionine residues as axial ligands. It should be pointed out that there are only 41 residues as heme c ligands. Therefore the percentages of non-histidine ligands may have a relatively large change with a slight increase or decrease of ligand numbers due to the small dataset.

Distribution of the axial ligands for heme b (HEM) and heme c (HEC).

The conserved interactions between protein residues and heme were previously studied by calculating either the frequencies of residues that are in van der Waals contact with heme for each fold class of b-type heme proteins [27] or by calculating the mean number per binding site [41]. Smith et al also applied normalized amino acid profiles to assess the composition and conservation of heme binding sites [41]. Here we explored the residue preferences in the heme binding pockets through calculating the relative frequencies of heme binding residues in our non-redundant dataset. The relative frequency of each amino acid is normalized to its background frequency.

Normally, the background frequencies used for comparisons are calculated from a non-redundant protein dataset. However, due to the dominant presence of all-α folds, it is not clear whether the residue distribution in heme proteins is different from that in other proteins. Therefore we first compared the residue distributions between non-redundant heme proteins and non-redundant all proteins. To avoid issues with missing residues and cloning artifacts (His-tags etc.) associated with PDB sequences, we used native full-length protein sequences to calculate residue compositions by mapping the PDB chains to Uniprot entries with PDBSWS [46]. The relative residue frequencies between heme proteins and all proteins show that heme proteins tend to contain more alanine, phenylalanine, histidine, methionine, and tryptophan residues and fewer cysteine, aspartic acid, isoleucine, lysine, asparagine, and serine residues (Additional file 2, Figure S1). Statistical analysis (χ 2 ) revealed a significant difference between these two frequency profiles (data not shown). In order to have a meaningful description of the enrichment or deficiency of residues in the heme interacting environment, we used the background frequencies from the non-redundant set of heme proteins as references.

The top five residues with high relative frequencies are cysteine (C), histidine (H), phenylalanine (F), methionine (M), and tyrosine (Y) (Figure 4A). Because four of the top five (C, H, M, and Y) can serve as axial ligands to heme iron (Figure 3), we removed axial ligands from the dataset and recalculated the relative frequencies. Figure 4B shows that the level of histidine decreases to the background level, suggesting the enrichment of histidine is essentially due to the large number of heme histidine ligands. The other four residues, on the other hand, have almost the same relative frequencies with or without ligand residues (Figure 4B). In heme c proteins, the occurrence of cysteine residues is extremely high with an eight fold enrichment compared to the background distribution. This is not surprising as the classic CXXCH binding motif, in which the histidine serve as ligand and the cysteine residues form covalent thioether bonds with the heme vinyl groups, has dominant presence in heme c proteins[28].

Relative frequency of the heme interacting amino acids. (A) Relative frequency of residues in heme b (HEM), heme c (HEC), and heme b and c (ALL) (B) the relative frequency of the 5 residues with or without them as axial heme ligands.

Consistent with earlier reports, the aromatic residues (phenylalanine, tyrosine, and tryptophan) play important roles in protein-heme interactions through stacking interactions with the porphyrin[27, 41]. One exception is tryptophan in heme c proteins, which showed a similar level of occurrences compared to the background (Figure 4A). Leucine, isoleucine, and valine, which make hydrophobic interactions with the heme ring structure, are slightly increased over the background frequencies. The residues with the fewest occurrences, aspartic acid, glutamic acid, and lysine are charged residues, suggesting the heme binding pocket is mainly a hydrophobic environment. In contrast, arginine, a positively charged residue that has been considered a major player in anchoring the heme propionates, has a much higher occurrence than other charged amino acids and shows a similar (HEM) or slightly higher (HEC) level of frequency to the background (Figure 4A) [27].

The secondary structure types for heme interacting residues are shown in Figure 5. There are more helical and less coil types in proteins with heme b no matter what dataset (heme proteins or all proteins) is used as a reference. Therefore the difference is not due to the large number of all-α proteins in the dataset. As for heme interacting residues in heme c, they have similar distribution to the background (Figure 5). Based on our 3-category classification of relative solvent accessibility [49], the heme interacting residues are less likely to be exposed. The buried residues are comparable to the background distribution. About 20% increase is observed in the intermediate category (Additional file 2, Figure S2).

Frequencies of secondary structure types for heme interacting residues.

Heme binding sequence motifs

To investigate possible sequence motifs involved in heme binding, the flanking sequences with four residues on each side of heme axial ligands were collected and aligned. The non-redundant dataset has 34 heme c ligands, 32 of which have histidine as axial ligands. The alignment of these sequences shows the classic CXXCH heme c binding motif [4, 28] (Figure 6A).

Sequence motifs surrounding the axial ligands. (A) Sequence logo from 32 heme c proteins with histidine as axial ligand shows the classic CXXCH heme c binding motif (B) Sequence logo from 18 heme b proteins with cysteine as axial ligand. The sequence logos were created with WebLogo [74] (C) Arginine-334 (red sticks) of 1N97A interacts with heme propionates (red spheres) and (D) Interactions between histidine-353 (red sticks) of 1GWIA and heme propionate groups (red spheres).

Another motif worthy of note, G X[HR]XC[PLAV]G, comes from the heme b proteins with cysteine as axial ligands (Figure 6B). The motif represents the classic CYP signature heme binding motif FXXGXXCXG in bacteria, plant, and mammalian cytochrome P450 s [59–61]. At the -4 and +2 positions (with ligand cysteine as reference position) are small amino acids (glycine) while the -2 position prefers a positively charged amino acid such as histidine or arginine. These positively charged residues interact electronically with the negatively charged heme propionates (Figure 6C and 6D). The small glycine residue at the -4 position may provide the flexibility needed for positioning the positively charged residues close to heme propionate groups. The +1 position is dominated by proline and hydrophobic amino acids, leucine, alanine, valine and isoleucine. Six of the eighteen cases have proline right after the axial ligand cysteine, reminiscent of the dipeptide CP motif being implicated in heme sensing and regulation [31–35, 62]. While the importance of CP motif has been studied through deletion or site-directed mutation experiments in several important proteins, including transcription repressor Bach1[63], iron regulatory protein 2 (IRP2) [31], circadian factor period 2 (Per2) [34] and δ-aminolevulinic acid synthase (ALAS) [33], the possible role of the CP motif in heme interaction from a structural point of view remains unclear as the structures for most of these proteins with such CP motifs are unknown.

All the six CP dipeptides that have direct physical interactions with heme exhibit similar structural roles with the cysteines serving as ligands to the heme iron and the proline residue introducing a bend for the downstream structures, mainly α-helices, to steer them away from the heme face (Figure 7B and 7C). A seventh protein chain, 2PBJA, contains a CP where the proline shows highly similar structural implication, whereas the cysteine residue interacts with heme but not as a ligand. Instead, the presence of a glutathione molecule (GSH), which forms a coordination bond with the heme iron, seems to push the cysteine slightly away from the axial ligand position (5.25 Å from heme iron) [57]. Considering the conformation in the proline-bend structure and the small distance between cysteine and heme iron, it is likely that the cysteine could serve as a heme ligand if GSH is not present in the structure. Interestingly, a closer examination of the structural conformation downstream of the proline residue in 2CIWA (cloroperoxidase), 3CQVA (Rev-erb), and 2PBJA (microsomal prostaglandin E synthase), which have the CP heme motifs with conserved proline, indicates nearly perpendicular orientation to the heme plane (Figure 7A, 7B and 7D). In contrast, in the P450 family where the proline residue is less conserved, with leucine, isoleucine, and methionine also found at the position of proline as shown in the motif logo (Figure 6B), the α-helices following the proline residue are in parallel with the heme plane (Figure 7C). The difference suggests a different structural role for the proline in conserved CP dipeptides from that in the less-conserved CP dipeptides, more specifically at the proline position.

Three-dimensional structures of heme proteins with "CP" motifs. (A) 3CQVA (B) 2CIWA (C) 1GIWA and (D) 2PBJA. The CP dipeptides are shown as red sticks. The immediate downstream structures of the CP dipeptides are shown in blue.

CP dipeptides have also been implicated in indirect interaction with heme. Ragsdale and colleagues reported a novel role for CP motifs in heme oxygenase 2 (HMOX-2) as a thiol/disulfide redox switch that localizes outside the heme-binding pocket [62, 64, 65], therefore regulating heme-protein interaction via sensing redox status in the environment. There are a total of twenty-nine CP dipeptides in our dataset. Less than a quarter of them (in 7 protein chains including 2PBJA) show physical interactions with heme molecules. It would be impractical at this point to predict the functional role of the remaining CP dipeptides in heme-protein interaction, mainly due to the limited sample size and the lack of structural details on heme pocket-CP interaction. Here we made use of statistical analysis to indirectly assess the functional relevance of CP dipeptides in heme interaction. The rationale behind the assay is that, if CP dipeptides are important heme signatures for heme interaction, the expected occurrences of CP dipeptides in hemoproteins should be higher compared to control population. We found no statistically significant difference between the presence of CP dipeptides in heme proteins and non-heme proteins (data not shown), suggesting other yet to be identified factors may exist to help determine the role CP dipeptides play in heme binding [31]. It should be noted that we do not exclude the possibility that in the control sample there exist unknown hemoproteins however for them to significantly affect the frequency of CP signals there would have to be a considerably large fraction of the control proteins being analyzed to be heme-interacting, which we anticipate as less likely.

Structure comparison between apo and holo heme proteins

An interesting question related to structure-based heme binding protein design and prediction is the degree of global conformational transition and the local changes of the heme-binding pocket upon heme binding. We collected 446 heme protein chains (after removing heme protein chains with at least 90% sequence identity) and compared their sequences with the protein chains without heme or heme-like ligands (Additional file 1, Table S2). One hundred seventy-nine heme protein chains are found to have apo structures with high sequence similarity and coverage. After removing redundant apo/holo pairs with a 25% sequence identity cutoff and proteins with non-heme or non-heme-like ligands occupying the heme binding pocket, the final dataset consists of 10 apo-holo protein pairs. Table 2 shows that 9 out of 10 proteins undergo very small global conformational changes after heme binding with RMSDs of 1.03Å or less. For example the 2ZDOA-1XBWD pair (iron-regulated surface determinant IsdG from Staphylococcus aureus) has an RMSD of 0.59 Å. In the absence of heme, the protein assumes the same conformation as the holo protein with heme (Figure 8A, B). Even the side chain positions of the histidine ligand are similar. The one with relatively large conformational changes is Rev-erb (3CQVA-2V7CA). Without heme the C-terminal helix (residues 568-576) moves towards the heme pocket with His568 (heme-binding ligand) facing away from the binding pocket (Figure 8C, D) [66].

Structural comparison of apo-holo heme protein pairs. (A,B) 2ZDOA-1XBWD (C,D) 3CQVA-2V7CA.

Three of the ten heme proteins in Table 2 have multiple known apo structures. 1KBIA (flavin-binding domain of Baker's yeast flavocytochrome b2), 1N45A (human heme oxygenase-1), and 1N5UA (human serum albumin) have 9, 3, and 28 apo structures respectively (with at least 99% sequence identity, Additional file 1, Table S3). Because proteins are inherently dynamic and conformational selection has been considered as a major mechanism for biomolecular recognition [67–69], we checked the conformational differences between each of the apo structures and the holo structures. Figure 9A shows the RMSD (Cα atoms of aligned residues) values of the apo-holo structural differences. The RMSDs are generally less than 1Å for 1KBIA and 1N45A. On the contrary, apo structures of 1N5UA form two clusters. Members of one cluster with 12 apo structures have RMSDs around 0.8Å while the other contains 15 apo structures with RMSDs ranging from 4 to 5Å. Through manual inspection, we found that the differences are caused by the numbers of non-heme ligands in structures. In addition to heme, 1N5UA also has 5 myristic acid (MYR) molecules (Figure 9B). The apo structures with higher RMSDs either do not have ligands (Figure 9C) or have only one or two non-MYR ligands. For example, 1E7AA and 2BX8B have 2 PFL and 1 AZQ respectively. On the other hand, apo structures with MYR ligands in similar positions as those in 1N5UA generally have smaller RMSDs (Figure 9D). Therefore, under similar environment, there are relatively small structural differences between holo and apo heme protein structures.

Examples of heme proteins with multiple apo structures. (A) RMSD distribution of apo structure of three heme protein chains, 1KBIA, 1N45A, and 1N5UA. The 28 apo structures of 1N5UA form two clusters (red ovals). (B) Structure of 1N5UA with one heme and five myristic acid molecules (MYR, red spacefill). (C) Structure of 1AO6A with no ligands. (D) Structure of 3CX9A with five myristic acid molecules (MYR, red spacefill) and one LPX ligand (orange spacefill).

It should be noted that the above comparisons are based on heme proteins that have stable apo structures solved through X-ray crystallography. For some proteins, as in the case of hemoglobin, the absence of ligand(s) can increase the flexibility and cause partial unfolding of the protein structure, making it difficult for structure determination [70, 71]. Furthermore, intrinsically disordered or unstructured regions are considered to be responsible for many important cellular functions such as ligand binding [72, 73]. However the existence of such flexible apo structures would not interfere with our goal in structure-based heme protein prediction as we aim to take the existing apo structures in PDB as inputs [18].

Other features useful for comparing apo-holo heme proteins are the pocket size and shape. Due to different heme binding modes (partially exposed or fully embedded, Additional file 2 Figure S3) and the difficulty in identifying the exact heme binding pocket from existing automatic programs, the sizes of heme binding pockets vary from small (

400 Å 3 ) to very large (over 2000 Å 3 ) (Table 2). In addition, the changes in absolute pocket volumes after heme binding are variable. Small changes are seen in 2ITFA-2ITEB, 2R7AA-2RG7 D, and 2ZDOA-1XBWD. Other pairs exhibited significant changes in volume despite the minimal conformational change (Table 2). To take the shape into consideration we calculated the Rvs value (the ratio of pocket volume over the pocket surface area) of each pocket. Most of the apo or holo proteins have Rvs values around 1.4. To further investigate whether the binding pocket can be used as one of the characteristics for heme protein prediction, we compared the Rvs distributions between heme binding pockets and pockets in non-heme proteins (proteins that don't have heme ligand(s) and are not homologous to heme proteins) with similar sizes ranging from 350 to 2000Å 3 . The Rvs of heme binding pockets has a narrow distribution whereas the Rvs from similar pocket sizes of non-heme proteins has a wide spread with a long right tail (Additional file 2, Figure S4-A). We also investigated the distribution of Rvs normalized to a sphere shape as introduced by Sonavane and Chakrabarti [56]. A similar trend was found (Additional file 2, Figure S4-B). It should be pointed out that, even though unknown heme proteins may be included in the non-heme dataset, many non-heme proteins share similar pocket characteristics.


Cytochromes were initially described in 1884 by MacMunn as respiratory pigments (myohematin or histohematin). [4] In the 1920s, Keilin rediscovered these respiratory pigments and named them the cytochromes, or “cellular pigments”. [5] He classified these heme proteins on the basis of the position of their lowest energy absorption band in their reduced state, as cytochromes a (605 nm), b (≈565 nm), and c (550 nm). The ultra-violet (UV) to visible spectroscopic signatures of hemes are still used to identify heme type from the reduced bis-pyridine-ligated state, i.e., the pyridine hemochrome method. Within each class, cytochrome a, b, or c, early cytochromes are numbered consecutively, e.g. cyt c, cyt c1, and cyt c2, with more recent examples designated by their reduced state R-band maximum, e.g. cyt c559. [6]

The heme group is a highly conjugated ring system (which allows its electrons to be very mobile) surrounding an iron ion. The iron in cytochromes usually exists in a ferrous (Fe 2+ ) and a ferric (Fe 3+ ) state with a ferroxo (Fe 4+ ) state found in catalytic intermediates. [1] Cytochromes are, thus, capable of performing electron transfer reactions and catalysis by reduction or oxidation of their heme iron. The cellular location of cytochromes depends on their function. They can be found as globular proteins and membrane proteins.

In the process of oxidative phosphorylation, a globular cytochrome cc protein is involved in the electron transfer from the membrane-bound complex III to complex IV. Complex III itself is composed of several subunits, one of which is a b-type cytochrome while another one is a c-type cytochrome. Both domains are involved in electron transfer within the complex. Complex IV contains a cytochrome a/a3-domain that transfers electrons and catalyzes the reaction of oxygen to water. Photosystem II, the first protein complex in the light-dependent reactions of oxygenic photosynthesis, contains a cytochrome b subunit. Cyclooxygenase 2, an enzyme involved in inflammation, is a cytochrome b protein.

In the early 1960s, a linear evolution of cytochromes was suggested by Emanuel Margoliash [7] that led to the molecular clock hypothesis. The apparently constant evolution rate of cytochromes can be a helpful tool in trying to determine when various organisms may have diverged from a common ancestor. [8]

Several kinds of cytochrome exist and can be distinguished by spectroscopy, exact structure of the heme group, inhibitor sensitivity, and reduction potential. [9]

Four types of cytochromes are distinguished by their prosthetic groups:

Type Prosthetic group
Cytochrome a heme A
Cytochrome b heme B
Cytochrome c heme C (covalently bound heme b) [10]
Cytochrome d heme D (Heme B with γ-spirolactone) [11]

There is no "cytochrome e," but cytochrome f, found in the cytochrome b6f complex of plants is a c-type cytochrome. [12]

In mitochondria and chloroplasts, these cytochromes are often combined in electron transport and related metabolic pathways: [13]

Cytochromes Combination
a and a3 Cytochrome c oxidase ("Complex IV") with electrons delivered to complex by soluble cytochrome c (hence the name)
b and c1 Coenzyme Q - cytochrome c reductase ("Complex III")
b6 and f Plastoquinol—plastocyanin reductase

A distinct family of cytochromes is the cytochrome P450 family, so named for the characteristic Soret peak formed by absorbance of light at wavelengths near 450 nm when the heme iron is reduced (with sodium dithionite) and complexed to carbon monoxide. These enzymes are primarily involved in steroidogenesis and detoxification. [14] [9]

Structure𠄿unction Relationships in Heme-Proteins

Biological systems rely on heme-proteins to carry out a number of basic functions essential for their survival. Hemes, or iron–porphyrin complexes, are the versatile and ubiquitous active centers of these proteins. In the past decade, discovery of new heme-proteins, together with functional and structural research, provided a wealth of information on these diverse and biologically important molecules. Structure determination work has shown that nature has used a variety of different scaffolds and architectures to bind heme and modulate functions such as redox properties. Structural data have also provided insights into the heme-linked protein conformational changes required in many regulatory heme-proteins. Remarkable efforts have been made towards the understanding of factors governing redox potentials. Site-directed mutagenesis studies and theoretical calculations on heme environments investigated the roles of hydrophobic and electrostatic residues, and analyzed the effect of heme solvent accessibility. This review focuses on the structure–function relationships underlying the association of heme in signaling and iron metabolism proteins. In addition, an account is given about molecular features affecting heme's redox properties this briefly revisits previous conclusions in the light of some more recent reports.


The deazaflavin cofactor F420 enhances the persistence of mycobacteria during hypoxia, oxidative stress, and antibiotic treatment. However, the identities and functions of the mycobacterial enzymes that utilize F420 under these conditions have yet to be resolved. In this work, we used sequence similarity networks to analyze the distribution of the largest F420-dependent protein family in mycobacteria. We show that these enzymes are part of a larger split β-barrel enzyme superfamily (flavin/deazaflavin oxidoreductases, FDORs) that include previously characterized pyridoxamine/pyridoxine-5′-phosphate oxidases and heme oxygenases. We show that these proteins variously utilize F420, flavin mononucleotide, flavin adenine dinucleotide, and heme cofactors. Functional annotation using phylogenetic, structural, and spectroscopic methods revealed their involvement in heme degradation, biliverdin reduction, fatty acid modification, and quinone reduction. Four novel crystal structures show that plasticity in substrate binding pockets and modifications to cofactor binding motifs enabled FDORs to carry out a variety of functions. This systematic classification and analysis provides a framework for further functional analysis of the roles of FDORs in mycobacterial pathogenesis and persistence.

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