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Subject Chemistry

Paper No and Title 15: Bioinorganic Chemistry

Module No and Title 20: Transport and storage of dioxygen

Module Tag CHE_P15_M20_e-Text

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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TABLE OF CONTENTS

1. Learning outcomes 2. Transport and storage of dioxygen 3. Myoglobin 3.1 Structure of myoglobin 3.2 Mechanism of oxygen binding in myoglobin 4. Hemocyanin 4.1 Structure of hemocyanin 4.2 Function of hemocyanin 5. Hemerythrin 5.1 Structure of hemerythrin 5.1 Mechanism of oxygen binding 6. Summary

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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1. Learning Outcomes

After studying this module, you shall be able to understand:

. Importance of biological oxygen carriers in living organisms. . Different respiratory metalloproteins involved in transport and storage of dioxygen. . Role of myoglobin in storage of dioxygen. . Need of hemocyanin for oxygen transportation in molluscs and arthropods living in lower oxygen environments. . Function of hemerythrin as oxygen carrying proteins in marine invertrebrates.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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2. Transport and storage of oxygen

Most of the living species perform aerobic respiration process in the presence of oxygen to breakdown organic compounds into energy storing molecule (ATP) that can be readily used by cells (Figure 1).

Figure 1: Schematic representation of aerobic respiration to harness energy

However, oxygen is sparingly soluble in water and hence only a small fraction of total oxygen content is dissolved in the circulatory fluids of organisms which would not be sufficient to sustain life. Therefore, an oxygen carrier that can reversibility form adduct with dioxygen molecule is required in order to increase the oxygen carrying capacity of the circulatory fluids. There are certain respiratory metal-containing proteins known, which can efficiently act as biological oxygen carriers for transport and storage of molecular oxygen: . Hemoglobin/Myoglobin: Vertebrates, echinoderms, insect larvae . Hemerythrin: Marine invertebrates, annelids . Hemocyanin: Molluscs and arthropods Comparative study of properties of the above mentioned heme as well as non-heme respiratory metalloprotiens has been provided below:

Properties Hemoglobin Myoglobin Hemerythrin Hemocyanin Oxidation State in deoxy form (II) (II) (II) (I)

Metaln:O2 Fe: O2 Fe: O2 Fe2: O2 Cu2: O2 Colour deoxy form Red-Purple Dull-Purple Colourless Colourless Colour of oxy form Red Bright red Violet pink Blue Metal Coordination Porphyrin ring Porphyrin Prot. side chain Prot. side ring (Glu, His) chain (His) Molecular Weight (kDa) 65 16.7 108 400-20000 Number of subunits 4 1 8 many

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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3. Myoglobin

Myoglobin, a relatively small globular monomeric protein consists of a single polypeptide chain bound non-covalently to a heme molecule (a prosthetic group consisting of a protoporphyrin ring and a central iron atom) and primarily functions as an intracellular oxygen storage site in cardiac and skeletal muscle fibres of vertebrates. The special oxygen storing property of muscle myoglobin permits diving mammals such as seals and whales to remain submerged in water for long periods. Apart from storing oxygen, myoglobin is also involved in the regulation of cellular oxygen in the repairing tissues and transport of oxygen to mitochondria for the oxidative phosphorylation process.

3.1 Structure of Myoglobin Myoglobin stands out to be the first protein to have a three-dimensional structure revealed through X-Ray crystallography by John Kendrew in 1957.

Figure 2: (a) 3-D structure of myoglobin of sperm whale (Physeter catodon) and (b) Structure of the active site of myoglobin

It consists of a single polypeptide chain of 153 amino acids called “globin” compactly folded into eight α-helicies that form a hydrophobic pocket containing a heme prosthetic group deeply embedded within, which is responsible for carrying oxygen molecules to muscle tissues (Figure 2). Each heme group contains one central coordinately bound iron atom that is normally (present as Fe2+) surrounded by four nitrogens of the planar porphyrin rings. The centrally bound iron atom is capable of forming two additional bonds, one on each side of the heme plane. While the fifth coordination site is occupied by a nitrogen atom from a histidine side chain “often referred as the proximal histidine group” on one of the amino acids in the protein, the sixth coordination site remains available for binding of dioxygen. The iron atom in the oxygen free form of myoglobin often called as “deoxymyoglobin” lies about 0.4 Ao out of plane of the CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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protoporphyrin rings as it is too large to fit into the cavity. When O2 binds, the iron atom is partially pulled back toward the porphyrin plane. The compact structure of myoglobin is stabilized by hydrogen and ionic bonds as well as by the hydrophobic interactions. Further, four units of myoglobin join together to form hemoglobin which acts as an efficient oxygen carrier but the mechanism of oxygen binding is bit more complex (Detailed discussion has been provided in other modules)

3.2 Mechanism of oxygen binding in myoglobin The binding of oxygen by myoglobin is directed by the mass action of oxygen which implies that when oxygen is present in large quantity, the formation of oxygenated myoglobin occurs, while on the other hand when the oxygen quantity becomes scarce, this form of myoglobin (i.e. oxymyoglobin) dissociates. In other words, it means that the oxygen binding process in myoglobin occurs reversibly which may be reflected by the simple equilibrium reaction:

Mb + O2 MbO2

Keq = ([Mb] [O2]) / [Mb-O2] O2 dissociation from Mb commonly described by its fractional saturation, YO2, and [O2] in partial pressure, pO2. YO2= [Mb-O2]/ [Mb] + [Mb-O2] Substitution from the equilibrium expression gives: YO2= ([pO2]/ [p1/2 + (pO2)] Thus, as described by this equation, the O2 binding curve for myoglobin follows a hyperbolic pathway. When the degree of saturation of myoglobin with oxygen is plotted against oxygen pressure, a steady rise is observed until complete saturation is approached and the curve levels off (Figure 3). As evident from this figure, myoglobin has a very high affinity for molecular oxygen, it is 50% saturated with oxygen at a partial pressure of just 1-2 torr and 95% saturated at about 20 torr. In fact, it is the high affinity of myoglobin that permits it to store oxygen efficiently in resting muscles at relatively lower pressure (approximately around 40 torr) and even in exercising muscles, where the partial pressure of oxygen (pO2) is around 20 torr. However, only in case of vigorous physical exercises, myoglobin releases a significant proportion of the stored oxygen as the pO2 drops considerably becoming less than 5 torr.

Figure 3: Oxygen dissociation curve of myoglobin

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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The O2 binding process by myoglobin is accompanied by a substantial structural change at the iron center: . Firstly, the radius of the iron atom shrinks considerably so much so that it fits into the plane of the porphyrin rings. . Secondly, a spin-pairing phenomenon occurs: The five-coordinate ferrous deoxy form (Fe2+) with a high spin is converted into the six coordinate oxy form containing a diamagnetic and low spin Fe3+(figure 4)

Figure 4: Illustration of structural changes occurring in myoglobin after oxygen is bound to it.

4. Hemocyanin

Hemocyanin is a complex respiratory metalloprotein present in the circulatory system of of certain molluscs and arthropods that serves as an oxygen carrier similar to the role of hemoglobin/myoglobin found in the blood of vertebrates. The oxygenation process in hemocyanin is performed by a binuclear copper centre which upon oxidation from Cu(I) to Cu(II) state changes color from clear to blue. The extent of its reversible binding with oxygen is a function of the partial pressure of oxygen. These metalloproteins are always found roaming free in blood plasma instead of confined within corpuscles, which is the source of the blue tinge of molluscs arthropods hemolymph (Figure 5). Besides being oxygen carriers, hemocyanins also function as strong immonogens in many species ranging from hagfish to rabbits.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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Figure 5: Octopus having copper based blue blood

Studies have revealed that hemocyanin has lower oxygen binding capacity in comparison to hemoglobin. Even though, some marine species utilize hemocyanin for oxygen transportation because in lower oxygen environments, such as at the bottom of oceans where these molluscs and arthropods live, it acts as a better oxygen carrier than hemoglobin.

4.1 Structure of Hemocyanin Hemocyanins are high molecular weight metalloproteins made up aggregates of several individual subunits, each containing two copper atoms embedded at the core that can bind oxygen molecule. The two copper-binding sites often designated Cu-A and Cu-B are complexed by three histidine residues that form the distorted pyramidal geometry of each atom. Further, two phenylalanine residues are present in close proximity to the histidine residues that act as hydrophobic core for the protection of active site. Owing to their aggregation tendency, these subunits may be arranged in chains or clusters with weights exceeding 1500 kDa. Although significant differences are observed between of structure and assembly of subunits mollusc and arthropod hemocyanins but the binding mechanism and active site are nearly identical. In arthropods, hemocyanin is made up of aggregates of multiples of hexamers in which each monmer subunit has a molar mass of about 72 kDa. On the contrary, the basic structure of a mollusc hemocyanin is a decamer of subunit which is an enormous polypeptide chain of about 350-450 kDa (Figure 6). It should be noted that arthropods hemocyanin is composed of subunit polypeptide which have a single active site, whereas in molluscans hemocyanin, the subunit contains seven or eight functional units, each with an active site.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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Figure 6: Structure of arthropods and molluscans hemocyanin 4.3 Function of Hemocyanin The function of the hemocyanin molecule revolves around a pair of copper atoms embedded at its core which act as oxygen binding sites. Upon oxygenation, the copper is oxidized from its Cu(I) state in the deoxy form to Cu(II) state (Figure 7). Once oxygen molecule binds between copper atoms, their distance increases which results in conformational change of the whole protein unit and hence there occurs a geometrical change from trigonal pyramidal to a distorted tetrahedral. This geometrical change upon oxidation of central copper atoms accounts for the change in color from clear to blue. Further, most hemocyanins bind with oxygen non-cooperatively, but in few hemocyanins of horseshoe crabs and some other species of arthropods, cooperative binding is observed. In case of cooperative binding, conformational change as a result of binding of oxygen on one unit in the hemocyanin would further increase the affinity of the neighboring units.

Figure 7: Illustration of structural change occurred when oxygen molecule binds with hemocyanin

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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5. Hemerythrin

Hemerythrin is one of the essential oxygen carrying proteins commonly found in the phyla of marine invertebrates such as sipunculids and brachiopods. Hemerythrin contains Fe (II) that binds oxygen reversibly; however after getting oxidized to methemerythrin (Fe3+) it loses the affinity to bind to further oxygen molecules. Astonishingly, unlike the other oxygen binding proteins such as hemoglobin and myoglobin, hemerythrin does not contain any heme unit and also differs fundamentally in its evolution, physiology and molecular structure. The most intriguing question that often strikes the mind of a chemist is that how does nature adapt a non-heme iron site to reversibly bind molecular oxygen.

5.1 Structure of hemerythrin

Figure 8: Structure of hemerythrin

Hemerythrin typically exists in the form of an octamer (with a molecular weight 108,000 dalton) containing apparently eight identical 13,500 dalton subunits, with each subunit composed of a four-α-helix fold bound to a binuclear iron center capable of binding a single molecule of O2 (Figure 8). The active site of this metalloprotein (deoxy form) fundamentally comprises of two iron atoms bridged by carboxylate side chains of a glutamate and aspartate residues. The co- ordination sphere of the two iron atoms (one of which is hexa-coordinate whereas the other is penta-coordinate) is completed by five terminally bound histidine units and a bridging hydroxo group. Upon oxygenation (i.e. interaction with O2), the deoxy form of hemerythrin is converted to the oxy form, in which an η1-hydroperoxo group binds to the available coordination site and forms a hydrogen bond to the bridging oxo unit. While the deoxy form of this protein is colorless and comprises of two Fe2+ ions per subunit, the oxy form is bright reddish violet in colour and contains two Fe3+ ions per subunit.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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5.2 Mechanism of oxygen binding and transport

The process involved in the dioxygen binding of hemerythrin is quite unusual. Extensive biomimetic studies have been carried out for exploring the mechanism of dioxygen activation and transport in non-heme diiron centres of living aerobic systems. These studies have revealed that hemerythrin binds oxygen reversibly as depicted in the Figure 9. When dioxygen is bound to the binuclear non-heme iron protein hemerythrin (Hr), transfer of one electron from each Fe(II) center and a proton from the bridging hydroxide of the diferrous site (deoxyHr) occurs to the oxygen binding of a single iron center (Fe2), resulting in the formation of a oxo-bridged diferric site which has a terminal hydroperoxide (oxyHr). As a result of the O2 addition, the Fe-O bond distances shorten, which further supports the formation of the “Oxo Bridge.” There is experimental evidence that a proton on the bridging hydroxide ion of the deoxy form is transferred to the bound peroxo ion thereby stabilizing it, which is accompanied by the concomitant oxidation of both the iron atoms to Fe (III).

Figure 9: Dioxygen binding by hemerythrin

The various forms of hemerythrin have been described according to oxidation and ligation states of the iron centre: Fe2+—OH—Fe2+ deoxy (reduced) Fe2+—OH—Fe3+ semi-met Fe3+—O—Fe3+—OOH− oxy (oxidized) Fe3+—OH—Fe3+— (any other ligand) met (oxidized)

5. Summary

. Most living species perform respiration in the presence of oxygen which requires biological oxygen carriers for transport and storage of molecular oxygen. . Besides transportation of dioxygen, myglobin also exhibits special oxygen storing property which permits diving mammals such as seals and whales to remain submerged in water for long periods.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen

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. Despite lower oxygen binding capacity, molluscs and arthropods utilize hemocyanin for oxygen transportation because it acts as a better oxygen carrier than hemoglobin in lower oxygen environments, such as at the bottom of oceans. . Hemerythrin is a non-heme iron protein commonly found in the phyla of marine invertebrates such as sipunculids and brachiopods.

CHEMISTRY Paper No. 15: Bioinorganic Chemistry Module No. 20: Transport and storage of dioxygen