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Subject Chemistry

Paper No and Title 15,

Module No and Title 19, Carriers

Module Tag CHE_P15_M19_e-text

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

TABLE OF CONTENTS

1. Learning Outcomes ...... 3 2. Introduction ...... 4 3. Oxygen Carriers ...... 4 4. ...... 4 4.1 Structure ...... 4 4.1.1 Active site structure of deoxyhemocyanin ...... 5 4.1.2 Active site structure of oxyhemocyanin ...... 5 5. ...... 6 5.1 Structure ...... 6 5.1.1 Active site structure of deoxyhemerythrin ...... 6 5.1.2 Active site structure of oxyhemerythrin ...... 7 6. ...... 7 6.1 Structure ...... 8 7. Summary ...... 10

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

1. Learning Outcomes

After studying this module, you shall be able to

 Know the need for oxygen carriers in living systems.  Learn about the different kinds of oxygen carriers in and invertebrates.  Learn about the structure of oxygen carriers: hemocyanin, hemerythrin and hemoglobin.  Understand the role of transition metal ions in these oxygen carriers.

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

2. Introduction

Most living organisms need dioxygen to perform respiration, which is breakdown of food products to release energy. There are two principal ways in which dioxygen is supplied to the cells. One mode of transportation is the plasma. However, dioxygen has a very low solubility in water (7.6 mg/L at 20°C and 1atm). The other way of transportation of dioxygen is with help of dioxygen carrying molecules in the blood plasma. This mechanism is capable of transporting large amount of dioxygen. Only about 1.5% of the dioxygen transported by the blood is dissolved directly in the blood plasma, rest is done by oxygen carriers.

3. Oxygen Carriers

Oxygen carriers are special types of which have a transition metal from 3d series bound to a . Only those transition metals can form dioxygen complexes with O2 which have vacant site/s for binding and exist in higher oxidation states also. The common metal ions are , and vanadium and the commonly encountered stoichiometries are 1:1 or 1:2 . The metal binds reversibly to dioxygen without the metal or being irreversibly oxidized. More broadly, a complex which can uptake dioxygen and can release it reversibly under ordinary conditions can be called a biological oxygen carrier. The extent of binding will depend on temperature, partial pressure of O2 and pH.

nML + O2 ⇌ MLn O2

In the above ML acts as a dioxygen carrier. In order for transition metal complexes to form dioxygen complexes there must be a vacant site (or sites) for binding and an accessible higher oxidation state (or states).

There are three known classes of dioxygen transport . These are: 1. 2. Hemerythrins 3. The Hemoglobin- family

4. Hemocyanin

Hemocyanins (or haemocyanins) are oxygen carrying proteins/oxygen carriers in invertebrates such as molluscs (eg , snails, ) and (eg scorpions, crabs, etc). It is extracellular protein and is present in .

4.1 Structure

Although the oxygen binding sites and function of molluscan and hemocyanins are quite similar, they are different in molecular structure at all levels. Arthropod hemocyanins are multiples of hexamers, each hexamer made of monomers (Figure 1) Molluscan hemocyanin occur as larger assemblies. CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

Figure 1: Single Oxygenated Functional Unit from the hemocyanin of an octopus

4.1.1 Active site structure of deoxyhemocyanin

Hemocyanin is a copper containing . Each monomer contains two cuprous ions [Cu(I)] that reversibly bind one dioxygen. An empty cavity is present between the two cuprous ions to accommodate the dioxygen. The Cu(I)- Cu(I) bond distance is 460 pm. The coordination number of each Cu(I) is three and is satisfied by three residues from the protein. This results in a distorted trigonal pyramidal geometry. Two phenylalanine residues which are in close proximity to the histidines residues provide a hydrophobic environment at the active site.

4.1.2 Active site structure of oxyhemocyanin

The binding of dioxygen to the Cu(I) at the active site leads to the following changes  Cu(I) is oxidized to Cu(II). 2-  O2 is reduced to O2 .  Colour of protein changes from colorless to blue.  Coordination number of copper changes to five from three.  Geometry of copper changes to square pyramidal from trigonal pyramidal. The equatorial plane has two histidyl nitrogens, the bound and the third histidyl nitrogen is axially coordinated to copper.  The Cu-Cu distance decreases to 360pm. In hemocyanin oxidative addition of dioxygen occurs. Evidence for the peroxo linkage comes from . The v(O-O) stetch is observed at 744 cm-1 confirming the presence of peroxo linkage ( Theoretical stretching frequency for peroxo is 740 to 930 cm-1). Experiments using asymmetrically labeled dioxygen 18O-16O proved conclusively that the coordination of dioxygen to Cu(II) is symmetrical (Figure 2).

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

Figure 2: Binding of Dioxygen to hemocyanin

5. Hemerythrin

Hemerythrin (Figure 3) is a reversible oxygen binding metalloprotein found in blood cells of a few . It is colorless in the deoxy form and on oxygenation the color changes to purple-red.

Figure 3: Single Oxygenated Hemerythrin protein

5.1 Structure

It is an oligomeric protein generally found in an octomeric form. The dimeric, trimeric and tetrameric forms of hemerythrin are also known.

5.1.1 Active site structure of deoxyhemerythrin

Each monomeric unit contains an active site which has two high spin ferrous ions [Fe(II)]. The ferrous ions are bridged together by a hydroxyl group and two carboxyl groups from an aspartate residue and a glutamate residue of the protein chain of 115 amino acid residues. One of the ferrous is hexacoordinated with an octahedral geometry and the other is pentacoordinated with a distorted trigonal bipyramidal geometry. The remaining coordination sites of hexacoordinated ferrous and pentacoordinated ferrous are satisfied by three and two imidazole nitrogens respectively from histidines residues of the protein chain (Figure 4) CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

Figure 4: Active site of deoxyhemerythrin

5.1.2 Active site structure of oxyhemerythrin

One monomeric unit of hemerythrin binds one dioxygen. The dioxygen adds to hemerythrin in an oxidative manner resulting in the formation of Fe(III) and peroxide. The dioxygen adds to the coordinatively unsaturated ferrous. The oxidative addition is followed by the shifting of proton - from the bridged OH to the bound peroxide resulting in the formation of hydroperoxo (HO2 ) group. The hydroperoxo group is hydrogen bonded with the µ-oxo group.

Figure 5 : Active site of oxyhemerythrin

Support for the above structures has been experimentally obtained. By use of radioisotope experiments, it was established that dioxygen binds asymmetrically in oxyhemerythrin. Single crystal X-ray diffraction study of oxyhemerythrin showed end –on coordination of dioxygen to only one iron.

6. Hemoglobin

Hemoglobin is a globular, iron containing metalloprotein with a quaternary structure (64*55*50 Å). It is found in red blood cells (RBC) of mammals and other animals. It transports dioxygen from the lungs to the tissues, where it is used to oxidize glucose. This process serves as a source of energy required for cellular metabolic processes. Hemoglobin also plays a role in transport of carbon dioxide and hydrogen ions. Hemoglobin increases the dioxygen carrying capacity of 1L of blood from 5mL to 250mL. The dioxygen binding capacity of hemoglobin is 1.34mL O2 per gram CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

of hemoglobin. Each RBC has 640 million molecules of hemoglobin. Hemoglobin synthesis occurs in the developing RBCs in the bone marrow. Hemoglobin has many firsts to its credit.  It is the first protein to have been crystallized (1849).  The first protein with a recognized physiological purpose (O2 transport, 1864; CO2 transport, 1904).  One of the first proteins to have its molecular weight and primary sequences established (1930).  One of the first proteins to have its tertiary and quaternary structures determined by X-ray crystallography (1960).

6.1 Structure

The structure of hemoglobin was solved by groups of Andrew Kendrew and Max Perutz. It is a tetramer with four subunits (Figure 6).

Figure 6: Structure of human hemoglobin. The proteins α and β subunits are in red and blue, and the iron-containing groups in green.

Each subunit of hemoglobin contains a single molecule of heme (Figure 7) and a polypeptide chain. Heme is Fe(II)- Protoporphyrin complex. are heterocyclic compounds formed by fusion of four pyrrole rings, linked by methine bridges. Iron is present at the centre of the protoporphyrin ring.

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

Figure 7: Structure of Heme

Different types of hemoglobin are present during fetal and adult life, differing in the kind of polypeptide chain. 1. Adult hemoglobin (α2β2) represented as HbA or HbA1 is the normal hemoglobin in adults. It has two α polypeptide chains and two β polypeptide chains. The α chain has 141 residues and β has 146 residues. 2. Minor adult hemoglobin (α2δ2) represented as HbA2 has two α and two δ chains. 3. (α2γ2) represented as HbF is composed of two α and two γ chains.

A normal adult has all three kinds of hemoglobin in the approximate amounts of about 97.5% HbA, 2% HbA2 and 0.5% HbF. The secondary structure of about 75% of amino acids in α or β chains is helical. Each polypeptide chain contains heme in the heme pocket. The four subunits are arranged in a tetrahedral manner and the individual polypeptide chains folded such that maximum number of polar or charged residues are on the outside surface of the subunit and the non-polar residues are inside. The exposed polar residues make the protein soluble in aqueous medium and the interior non-polar residues result in the formation of a hydrophobic pocket in which heme is present. This hydrophobic environment hinders the oxidation of ferrous ion. The hemoglobin tetramer can be represented as two identical dimers, each being αβ. Each α chain is in contact with both β chains. There are few interactions between the two α chains and the two β chains. The two polypeptide chains are held tightly within each dimer by three kinds of interactions namely hydrophobic, ionic and hydrogen bonding. The dimers are held together by only ionic bonds and can move with respect to each other.

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS

7. Summary

 Oxygen carriers are special metalloproteins which have a transition metal from 3d series bound to a protein.  Three known classes of dioxygen transport proteins are Hemocyanins, Hemerythrin and the Hemoglobin- Myoglobin family.  Hemocyanins are copper containing dioxygen transport proteins present in molluscs and 2- arthropods. Hemocyanins carry dioxygen as O2 .  Hemerythrin is found in marine invertebrates. It has two high spin ferrous ions and carries dioxygen as peroxide.  Hemoglobin is an iron containing metalloprotein with a quaternary structure. It acts as dioxygen carrier in mammals and animals and also plays a role in transport of carbon dioxide and Hydrogen ions.  Hemoglobin has four subunits, each subunit having a polypeptide chain and a heme unit. Heme is Fe(II)- Protoporphyrin complex.

CHEMISTRY PAPER No. 15: BIOINORGANIC CHEMISTRY MODULE No. 19: OXYGEN CARRIERS