Dioxygen Management Proteins

B.Sc. Chemistry (Honours) Semester – VI (under CBCS) Examination, 2023

Paper – CEMA – DSE – B4 (Dissertation)

Submitted by

Vedant Saraswat

C.U. Roll Number – 203051-21-0041

C.U. Registration Number – 051-1111-0191-20

Department of Chemistry

Charuchandra College

Affiliated to

Calcutta University

Under the guidance of

Dr. Parimal Rauth and Dr. Debolina Mitra

Year of Submission – 2023

Abstract:
Most organisms require molecular oxygen in order to survive. The oxygen is used in a host of
biochemical transformations, although most is consumed in the reaction that is the terminal (or
primary) step of oxidative phosphorylation to form ATP. The transportation of oxygen from
the air or water to the cells of an organism is vital. In the three chemically distinct dioxygen
carrier proteins that have evolved and are found today, the dioxygen binding site in the protein,
the active site, is an iron or copper complex. For hemoglobins, the most widely distributed
family of dioxygen carriers, the active site has long been known to consist of an iron porphyrin
(heme) group embedded in the protein. Almost all hemoglobins share the basic structure,
hemocyanin and hemerythrin, the other two biological dioxygen carriers, feature pairs of
copper atoms and iron atoms, respectively, at the active sites. This review, resumes the best
successes in the chemical characteristics of this extraordinary group of natural occurring
molecules and clarifies the potential of these molecules in many fields of application1.
Table of Contents:
1. Introduction
2. Porphyrin
2.1. Metalloporphyrin
2.2. Heme
2.3. Hemoglobin: Structure & Function

2.3.1 History of hemoglobin Research

3. Dioxygen Binding and Management

3.1. Interaction between heme and dioxygen

3.2. The binding of dioxygen to myoglobin

4. The physiology of hemoglobin and myoglobin

5. Other Biological Dioxygen carriers

5.1. Hemerythrin

5.2. Hemocyanin

6. Conclusion

7. Future Directions

8. Acknowledgement

9. References
 1. Introduction:
The porphyrins are an important class of naturally occurring macrocyclic compounds found in
biological compounds that play a very important role in the metabolism of living organisms.
They have a universal biological distribution and were involved in the oldest metabolic
phenomena on earth. Some of the best examples are the iron-containing porphyrins found as
heme (of hemoglobin) and the magnesium-containing reduced porphyrin (or chlorine) found
in chlorophyll. Without porphyrins and their relative compounds, life as we know it would be
impossible and therefore the knowledge of these systems and their excited states is essential in
understanding a wide variety of biological processes, including oxygen binding, electron
transfer, catalysis, and the initial photochemical step in photosynthesis. The porphyrins have
attracted considerable attention because are ubiquitous in natural systems and have prospective
applications in mimicking enzymes, catalytic reactions, photodynamic therapy, molecular
electronic devices and conversion of solar energy.2 In particular, numerous porphyrins based
artificial light-harvesting antennae, and donor acceptor dyads and triads have been prepared
and tested to improve our understanding of the photochemical aspect of natural photosynthesis.
The porphyrins play important roles in the nature, due to their special absorption, emission,
charge transfer and complexing properties as a result of their characteristic ring structure of
conjugated double bonds.

1
 2. Porphyrin:
2.1. Metalloporphyrin:
Four pyrrole rings of porphin carrying substituents other than hydrogen are called porphyrins.
The complexes in which a metal ion is held in the porphyrin ring system are called
metalloporphyrins. It is in the latter form that the porphyrins complex with metal ions, usually
dipositive, to form metalloporphyrin complexes. Some of the simplest metalloporphyrin are
the various cytochromes. In terms of overall structure and molecular weight they are anything
but simple3. However, the inorganic chemistry of several of them is very simple coordination
and redox chemistry. The active centre of the cytochromes is the heme group.

Figure-1: Structure of the porphyrin molecule4

It consists of a porphyrin ring chelated to an iron atom. The porphyrin ring consists of a macro
cyclic pyrrole system with conjugated double bonds (Figure1) and various groups attached to
the perimeter. We shall not be concerned with the nature and variety of these substituents
except to note that by their electron-donating or electron-withdrawing ability they can “tune"
the delocalized molecular orbitals of the complex and thus vary its redox properties. The
porphyrin can accept two hydrogen ions to form the +2 diacid or donate two protons and
become the -2 dianion.
From the covalent bond radii, we can estimate that a bond between a nitrogen atom and an
atom of the first transition series should be about 200 pm long. The size of the "hole" in the
centre of the porphyrin ring is ideal for accommodating metals of the first transition series. The
porphyrin system is fairly rigid and the metal-nitrogen bond distance does not vary greatly
2
from 193-196 pm in nickel porphyrins to 210 pm in high spin iron(II) porphyrins. The rigidity
of the ring derives from the delocalization of the π electrons in the pyrrole rings. Nevertheless,
if the metal atom is too small, as in nickel porphyrinates. the ring becomes ruffled to allow
closer approach of the nitrogen atoms to the metal. At the other extreme, if the metal atom is
too large, it cannot fit into the hole and sits above the ring which also becomes domed.

Figure – 2: Formation of tetra substituted Porphyrin5

The order of stability of complexes of porphyrins with +2 metal ions is that expected on the
basis of the Irving-Williams series, except that the square planar ligand favours the
configuration of Ni2+. The order is Ni2+ > Cu2+ > Co2+ > Fe2+ > Zn2+. The kinetics of formation
of these metalloporphyrins has also been measured and found to be in the order Cu 2+> Co2+ >
Fe2+ > Ni2+.6 If this order holds in biological systems, it poses interesting questions related to
the much greater abundance of iron porphyrins. What might have been the implications for the
origin and evolution of biological systems if the natural abundance of iron were not over a
thousandfold greater than those of cobalt and copper?

3
 2.2. Heme:
The porphyrin ring or modifications of it are important in several quite different biological
processes. The reason for the importance of porphyrin complexes in a variety of biological
systems is probably twofold: (1) They are biologically accessible compounds whose functions
can be varied by changing the metal, its oxidation state, or the nature of the organic substituents
on the porphyrin structure; (2) it is a general principle that evolution tends to proceed by
modifying structures and functions that are already present in an organism rather than
producing new ones de novo.

Figure – 3: The heme group: Type A hemes are found in cytochrome a; Type B hemes are
found in hemoglobin, myoglobin, peroxidase, and cytochrome b; Type C hemes are found in
cytochrome c: chloroheme is found in chlorocruorin.7

The heme group is a porphyrin ring with an iron atom at the centre (Figure 1). The oxidation
state8 of the iron may be either +2 or +3. And the importance of the cytochromes lies in their
ability to act as redox intermediates in electron transfer. They are present not only in the
chloroplasts for photosynthesis but also in mitochondria to take part in the reverse process of
respiration. The heme group in cytochrome c has a polypeptide chain attached and wrapped
around it. This chain contains a variable number of amino acids, ranging from 103 in some fish
and 104 in other fish and terrestrial vertebrates to 112 in some green plants. A nitrogen atom
from a histidine segment and a sulphur atom from a methionine segment of this chain are
coordinated to the fifth and sixth coordination sites of the iron atom.10 Thus, unlike the iron in
hemoglobin and myoglobin (Figure 3), there is no position for further coordination.
Cytochrome c therefore cannot react by simple coordination but must react indirectly by an
electron transfer mechanism. It can reduce the dioxygen and transmit its oxidizing power

4
towards the burning of food and release of energy in respiration (the reverse process to
complement photosynthesis).

Figure – 4: Synthesis of Heme9

The importance of cytochrome c in photosynthesis and respiration indicates that it is probably

one of the oldest (in terms of evolutionary history) of the chemicals involved in biological
processes. An interesting “family tree" of the evolution of living organisms can be constructed
from the differences in amino acid sequences in the peptide chains between the various types
of cytochrome c found, for example, in yeasts, higher plants, insects, and humans. Despite
these differences, however, it should be noted that cytochrome c is evolutionarily conservative.
Cytochrome c from any eukaryotic species will react with the cytochrome oxidase of any other
eukaryotic species, plant or animal, though at reduced rates.

5
 2.3. Hemoglobin: Structure & Function

In 1959, Perutz elucidated the structure of horse hemoglobin using x-ray crystallography
techniques, and in 1963 revealed the structure of human hemoglobin.11 Adult hemoglobin
consists of two α-globins and two β-globins which each contain a heme group. The heme
is a porphyrin ring with an iron atom at its center. The tertiary structure refers to a
polypeptide backboard with multiple structural components, while the quaternary structure
refers to the interaction of multiple polypeptides within the samestructure. There are two
conformations of the heme globins; the T-state and the R-state. In the R-state, its
oxygenated form, the heme lies in an isolated packet where it is tightly wedged between
the 16 side chains of the globin. This allows no movement of the iron from the plane of the
porphyrin ring. The iron forms 6-coordinates: four bonds to the porphyrin ring, one bond
to histidine, and the sixth to the oxygen. In the deoxygenated T-state, the iron is in its 5
coordinated form, and the iron becomes displaced from the porphyrin plane by 0.4 Å.12
The T-state is obstructed by the distal valine on the ligation side, and this obstruction cannot
be cleared without a change in the polypeptide structure. Within these two different
conformations, the globins determine the relationship of the iron within the porphyrin ring.
As pointed out by Perutz and others, the dominant structural change between all four
globins can be described as the α1β1 and α2β2 dimers remaining rigid while the contacts
between α1β2, α2β1, α1α2, and β1β2 move relative to eachother.13 During deoxygenation,
the α1α2, α1β1, and α2β2 heme distances remain stationarywhile the α1β1 and α2β2 act as
hinges for the β1β2 heme distance increases 5Å (Figure 4 and 5).14 The increase of the
hemoglobin during the oxy-to-deoxy transition has been termed a “molecular lung in
reverse.”

6
 Figure – 5: Molecular Lung in Reverse: Oxyhemoglobin

The α-globins are shown in transparent black, and the β-globins are shown in transparent blue.
The heme groups are represented in solid black.
Table – 1: Heme Iron Distances in hemoglobin

Structure Distance

Feβ1 - 34.6 Å
Feβ2

Feα1 - 25.5 Å
Feβ2

Feα1 - 34.7 Å
Feα2

The heme iron distances remain consistent during the oxy-deoxy transition of hemoglobin,
except for the Feβ1 - Feβ2 heme distances, which increase 5 Å.

7
 Figure – 6: Molecular Lung in Reverse: Deoxyhemoglobin

The α-globins are shown in transparent black, and the β-globins are shown in transparent
blue. The heme groups are represented in solid black.

Table – 2: Heme Iron Distances in Deoxyhemoglobin

Structure Distance

Feβ1 - 39.5 Å
Feβ2

Feα1 - 24.3 Å
Feβ2

Feα1 - 34.2 Å
Feα2

The heme iron distances remain consistent during the oxy-deoxy

transition ofhemoglobin, except for the Feβ1 - Feβ2 heme distances,
which increase 5 Å.

8
Figure – 7: 1H NMR spectra of hemoglobin15

Figure – 8: UV spectra of Hemoglobin and Myoglobin16

9
 2.3.1. History of hemoglobin Research:
Hemoglobin has been of great interest to the scientific community for the past two hundred
years. In 1825, Engelhart showed that the iron-to-protein ratio in hemoglobin was similar
across multiple species.17 In 1878, Bert gave the first crude measurements of oxygen binding
affinity.18 In 1890, Hüfner crystallized both oxyhemoglobin and deoxyhemoglobin. When the
deoxyhemoglobin crystal was exposed to oxygen, the crystals shattered, indicating a change
in the size and conformation of the hemoglobin.
Gasometric measurements in 1909 and 1922 were used by Barcroft, Haldane, and
coworkers to study the thermodynamics of hemoglobin.19 In 1924, Adair’s dialysis
experiments determined the molecular weight of hemoglobin. At the time, the molecular
weight of each globin was known to be 16,000, but the multiple of the globins wasunknown.
Adair determined the four globins of hemoglobin by comparing the molecular weight of
each globin to the molecular weight of hemoglobin and the percent iron in the protein. In
1924, the refined gasometric measurements by Adair and others were the first attempts to
determine sequential binding constants, the Adair constants, to be determined for
mammalian hemoglobins.20 With Adair’s work, the tetrameric nature of hemoglobin was
discovered, the molecular mass of the tetramer protein found, and the initial groundwork
for the binding constants laid:

1 𝐾1 [𝑥]+2𝐾1𝐾2[𝑥]2+3𝐾1𝐾2𝐾3[𝑥]3+4𝐾1𝐾2𝐾3𝐾4[𝑥]4
̅ = * Equation - 1
𝑌
4 1+𝐾1[𝑥]+𝐾1𝐾2[𝑥]2+𝐾1𝐾2𝐾3[𝑥]3+𝐾1𝐾2𝐾3𝐾4[𝑥]4

̅ represents the oxygen saturation of the hemoglobin and x represents the oxygen
Where 𝑌
concentration.

Gasometric techniques can be described using a Van Slyke apparatus.21 The Van Slyke
apparatus uses a round bottom flask filled with a solution of deoxygenated hemoglobin. A
measured amount of air with oxygen is introduced to the flask, and given time to equilibrate.
A small drop in the pressure indicated the binding of oxygen to hemoglobin. The gasometric
measurements take considerable amounts of time, and are prone to high percent errors for the
Adair constants.

10
Following the development of spectrophotometers, the ground work for tonometric methods,
thin-layers and automated methods were developed in which the oxygen concentration was
determined by dilution factors, calibrated flow methods, or by oxygen electrodes.18-21 These
techniques provided more precise data points, allowed multiple measurements per sample,
and required much less protein, resulting in the replacement of the gasometric methods.

11
 3. Dioxygen Binding and Management:
The respiratory system is an organ system in the body that functions in gas exchange with the
environment. Exchange of gases like carbon dioxide (CO2) and dioxygen (O2) are essential for
sustaining life forms. O2 is necessary in aerobic metabolism for oxidative phosphorylation
(synthesis of ATP) at the electron transport chain (ETC). ATP is the energy source needed for
muscular contraction in mammals. ATP synthesis requires oxygen as an electron acceptor in
the ETC, therefore oxygen must be readily available for use in metabolically active muscles.
Since muscles need large quantities of O2, it is transported by proteins in the blood and stored
in muscle tissue. One of these proteins is myoglobin. Myoglobin is a hemoprotein found in the
skeletal muscle of mammals that functions in oxygen storage and diffusion22. The heme in
myoglobin can reversibly bind a O2 molecule to regulate the transportation of O2 from red
blood cells to mitochondria when skeletal muscles are metabolically active.

3.1. Interaction between heme and dioxygen:

While all of the biochemical uses of the heme group are obviously important, the one that has
perhaps attracted the most attention because of its central biological role and its intricate
chemistry is the binding of the dioxygen molecule, O2. Hemoglobin picks up the dioxygen
from the lungs or gills and transports it to the tissues where it is stored by myoglobin. The
function of hemoglobin in the red blood cells is obvious, that of myoglobin is subtler. Besides
being a simple repository for dioxygen, it also serves as a dioxygen reserve against which the
organism can draw during increased metabolism or oxygen deprivation. Other suggested
functions include facilitation of dioxygen flow within the cell and a “buffering'' of the partial
pressure within the cell in response to increasing or decreasing oxygen supply.23
Dioxygen is far from a typical ligand. It probably resembles the carbon monoxide, nitrosyl and
dinitrogen ligands more than any others. None of these has a significant dipole moment
contributing to the σ bond, but the electronegativity difference between the atoms in CO and
NO enhances π* interactions. Dinitrogen and dioxygen lack this advantage, but may be
considered soft ligands with some π- bonding capacity. Iron(II), d6, is not a particularly soft
metal cation, but the “softening” (symbiotic) action of the tetrapyrrole ring system probably
facilitates dioxygen binding. Note that the heme group binds the truly soft ligand carbon
monoxide even more tightly, resulting in potentially lethal carbon monoxide poisoning.

12
However, there is another potentially fatal flaw in the binding of dioxygen by heme:
irreversible oxidation. If free heme in aqueous solution is exposed to dioxygen, it is converted
almost immediately into a µ-oxo dimer known as hematin. The mechanism of this reaction has
been worked out in detail. The reactions are as follows, where the heme group is symbolized
by PFeII . The first step is the binding of the dioxygen molecule, as in hemoglobin:
PFeII + O2 PFeIIO2 PFeIIIO2-
The bound dioxygen can now coordinate to a second heme, forming a µ-peroxo complex:
PFeIIO2 + FeII PFeIII-O-O-FeIIIP
Cleavage of the peroxo complex results in two molecules of a ferryl complex with the iron in
the +4 formal oxidation state:
PFeIII-O-O-FeIIIP 2PFeIII-O 2PFeIV=O →↔
Finally, attack of the ferryl complex on another heme results in the formation of hematin:
2PFeIV=O + PFeII PFeIII-O-FeIIIP

Figure – 9: Perspective view of picket-fence dioxygen adduct. The apparent presence of four
different 02 atoms results from a four-way statistical disorder of the oxygen atoms on
different molecules responding to the X-ray diffraction.24

Obviously, living systems have found a way to frustrate these reactions, otherwise all of the
heme would be precipitated as hematin rather than shuttling electrons in the cytochromes or
carrying dioxygen molecules in oxyhemoglobin (and storing them in oxymyoglobin). There
may be more than one mechanism in effect here, but certainly the primary one is steric
hindrance: The globin pan of the molecule prevents one oxoheme from attacking another heme.
13
This was first illustrated over thirty years ago by embedding the heme group in a polymer
matrix that allowed only restricted access to the iron atom: The embedded heme will reversibly
bind dioxygen.25 More recently this same result has been achieved by "picket-fence" hemes
and related compounds (Figure 9) that reversibly bind dioxygen and not only confirm the steric
hypothesis with regard to the stability of hemoglobin, but allow detailed structural
measurements to be made of a heme model compound. Thus the angular or bent coordination
of dioxygen to heme (in hemoglobin and myoglobin) was first indicated by the structure shown
in Figure 9. It has since been confirmed in myoglobin and haemoglobin.

3.2. The Binding of dioxygen to myoglobin:

Myoglobin is a protein of molecular weight of about 17.000 with the protein chain containing
153 amino acid residues folded about the single heme group (Figure 9). This restricts access to
the iron atom (by a second heme) and reduces the likelihood of formation of a hematin-like
Fe(III) dimer. The microenvironment is similar to that in cytochrome c, but there is no sixth
ligand (methionine) to complete the coordination sphere of the iron atom. Thus there is a site
to which a dioxygen molecule may reversibly bind.

Note how the differences in structure between the dioxygen-binding molecules (myoglobin,
hemoglobin, and cytochrome oxidase) and the electron carriers (various cytochromes,
including cytochrome oxidase which performs both functions) correlate with their specific
functions. In myoglobin and hemoglobin, the redox behaviour is retarded, and there is room
for the dioxygen molecule to coordinate without electron transfer taking place.26

Myoglobin contains iron(ll) in the high spin state. Iron(II) is d6 and. when high spin, has a
radius of approximately 92 pm in a pseudo-octahedral environment (the square pyramidal
arrangement of heme in myoglobin and hemoglobin may be considered an octahedron with the
sixth ligand removed), and the iron atom will not fit into the hole of the porphyrin ring. The
iron(II) atom thus lies some 42 pm above the plane of the nitrogen atoms in the porphyrin ring
(Figure 10). When a dioxygen molecule binds to the iron(II) atom, the latter becomes low spin
d6 (cf. the extremely stable Co+3 complexes with 2.4▲ LFSE). The ionic radius of low spin
iron(II) with coordination number six is only 75 pm, in contrast with the 92 pm of high spin
iron. Why the difference?
14
Figure – 10: Close-up of the heme group in myoglobin and hemoglobin. Note that the iron
atom does not lie in the plane of the heme group.

Recall that in octahedral complexes the eg orbitals are those aimed at the ligands. If they contain
electrons, which they do in the high spin case (t42g, e2g), they will repel the ligands as opposed
to the low spin case which allows unhindered access of the ligands along the coordinate axes.
Thus the effective radius of the iron atom is greater (along the x. y, and z axes) in the high spin
state than in the low spin state. The result is that the iron atom shrinks upon spin pairing and
drops into the hole in the porphyrin ring. All of the ligands (including the proximal histidine)
are able to approach the iron atom more closely. The net effect in myoglobin is minimal, but
the process is an important one for the transmission of dioxygen from the lungs to the tissues
by hemoglobin. The spin pairing of the normally paramagnetic dioxygen molecule is also of
interest, though often overlooked.27

15
 4. The physiology of hemoglobin and myoglobin:

In vertebrates dioxygen enters the blood in the lungs or gills where the partial pressure of
dioxygen is relatively high [21% oxygen = 0.21 x 1.01 x 103 Pa (760 mm Hg) = 2.1 x 104 Pa
(160 mm Hg)] under ideal conditions; in the lungs with mixing of inhaled and non-exhaled
gases, the value is closer to 1.3 x I04 Pa (100 mm Hg). It is then carried by red blood cells
(Figure 11) to the tissues where the partial pressure is considerably lower [of the order of 2.5
xI03 to 6.5 x I03 Pa (20-50 mm Hg)]. The reactions are as follows:
Lungs (gills) Hb + 4O2 -► Hb(O2 )4
Tissues Hb(O2)4 + 4Mb -► 4Mb(O2) + Hb

Figure – 11: Dioxygen binding curves for (I) myoglobin and for hemoglobin al various partial
pressures of carbon dioxide: (2) 20 mm Hg; (3) 40 mm Hg; (4) 80 mm Hg. Note that myoglobin
has a stronger affinity for dioxygen than hemoglobin and that this effect is more pronounced
in the presence of large amounts of carbon dioxide.28

Note that hemoglobin has an ambivalent function: It should bind dioxygen tightly and carry as
much as possible to the tissues, but once there it should, chameleon-like, relinquish it readily
to myoglobin which can store it for oxidation of foodstuffs. Hemoglobin serves this function
admirably as shown by Figure 11:
(I) Myoglobin must have a greater affinity for dioxygen than hemoglobin in order to effect the
transfer of dioxygen at the cell.

16
(2) The equilibrium constant for the myoglobin-dioxygen complexation is given by the simple
equilibrium expression:

[ MbO2 ] Equation - 2
KMb =
[Mb][O2]

If the total amount of myoglobin ([Mb] + [MbO2]) is held constant (as it must be in the cell)
while the concentration of oxygen is varied (in terms of partial pressure), the curve shown in
Figure 11 is obtained. Myoglobin is largely converted to oxymyoglobin even at low oxygen
concentrations such as occur in the cells.
(3) The equilibrium constant for the formation of oxyhemoglobin is somewhat more
complicated. The expression for the curve in the range of physiological importance in the
tissues is:

KHb = [Mb(O2)4] Equation - 3

[Hb][O2]2.8

The 2.8 exponent for dioxygen results from the fact that a single hemoglobin molecule can
accept four-dioxygen molecules and the binding of the four is not independent. It is not the
presence of four heme groups to bind four dioxygen molecules per se that is important. If they
acted independently, they would give a curve identical to that of myoglobin. It is the
cooperativity of the four heme groups that produces the curves shown in Figure 11. The
presence of several bound dioxygen molecules favours the addition of more dioxygen
molecules: conversely, if only one dioxygen molecule is present, it dissociates more readily
than from a more highly oxygenated species. The net result is that at low dioxygen
concentrations hemoglobin is less oxygenated (tends to release O2), and at high dioxygen
concentrations hemoglobin is oxygenated almost to the same extent as if the exponent were 1.
This results in a sigmoid curve for oxygenation of hemoglobin (Figure 11). This effect favours
oxygen transport since it helps the hemoglobin become saturated in the lungs and deoxygenated
in the capillaries.
(4) There is a pH dependence shown by hemoglobin. This is known as the Bohr Effect.
Hemoglobin binds one H+ for every two dioxygen molecules released. This favours the
conversion of carbon dioxide, a metabolite of the tissues, into the hydrogen carbonate ion
(HCO3-) promoting its transport back to the lungs.29

17
 5. Other Biological dioxygen carriers:
5.1. Hemerythrin:
Hemerythrin is a nonheme, dioxygen-binding pigment utilized by four phyla of marine
Dioxygen Carriers invertebrates. Its chief interest to the chemist lies in certain similarities to
and differences from hemoglobin and myoglobin. Like both of the latter, hemerythrin contains
iron(II) which binds oxygen reversibly, but when oxidized to methemerythrin (Fe3+ ) it does
not bind dioxygen. There is an octameric form with a molecular weight of about 108.000 that
transports dioxygen in the blood. In the tissues are lower molecular weight monomers, dimers,
trimers, or tetramers.30 And just as hemoglobin consists of four chains each of which is very
similar to the single chain of myoglobin, octameric hemerythrin consists of eight subunits very
similar in quaternary structure to myohemerythrin.

Figure – 12: Structure of Hemerythrin

A major difference between the hemoglobins and hemerythrins is in the binding of dioxygen:
Each dioxygen-binding site (whether monomer or octamer) contains two iron(II) atoms, and
the reaction takes place via a redox reaction to form iron(III) and peroxide (O22- ).
Oxyhemerythrin is diamagnetic, indicating spin coupling of the odd electrons on the two
iron(III) atoms. Mossbauer data indicate that the two iron(III) atoms are in different
environments in oxyhemerythrin. This could result from the peroxide ion coordinating one iron
atom and not the other, or from each of the iron atoms having different ligands in its
coordination sphere. The first evidence concerning the nature of the ligands came from an X-
ray study of methemerythrin.31 It indicated that the two iron atoms have approximately
octahedral coordination and are bridged by an oxygen atom (from water, hydroxo, or oxo),
aspartate, and glutamate.

18
The remaining ligands are three histidine residues on one iron atom and two histidines on the
other. This is a rather small difference, but it can be reconciled with the other data, and so until
recently the consensus has tended to favour a simple peroxo bridge between the two iron atoms.

5.2. Hemocyanin:
Note that the hydrogen atoms cannot be located at this level of resolution and so the hydrogen
bond shown is merely one suggestion for the possible stabilization of the peroxide ion.
No other oxygen-containing pigment is confusingly named hemocyanin, which contains
neither the heme group nor the cyanide ion; the name simply means "blue blood”: Whereas
hemoglobin turns bright red upon oxygenation, the chromophore (Cu I/CuII) in colourless
deoxyhemocyanin turns bright blue. Hemocyanin is found in many species in the Mollusca and
Arthropoda. The gross molecular structures of the hemocyanins in the two phyla are quite
different, though both bind dioxygen cooperatively, and spectroscopic evidence indicates that
the dioxygen-binding centres are similar. The dioxygen binding site appears to be a pair of
copper atoms, each bound by three histidine ligands. The copper is in the +1 oxidation state in
the deoxy form and +2 in the oxy form.
The structure of oxyhemocyanin has recently been determined. It presents yet a third mode of
binding between oxygen-carrying metal atoms and the dioxygen molecule. The latter oxidizes
each copper(I) to copper(II) and is in turn reduced to the peroxide ion (O22-). The two copper(II)
atoms are bridged by the peroxide ion with unusual µ-η2:η2 bonds, i.e., each oxygen atom is
bonded to both copper atoms.32
The parallels and differences among hemoglobin, hemerythrin. and hemocyanin illustrate the
ways in which evolution has often solved what is basically the same problem in different ways
in different groups of animals.
Ligand field stabilization energy (LFSE) refers to the increased stabilization of a complex that
occurs due to splitting of the d-orbitals. Rather than having 5 degenerate (same energy) orbitals,
the orbitals split so that some are lowered in energy and others are raised in energy. In
oxyhemocyanin, Cu(II) is found in a distorted tetrahedral geometry. Typically, d9 metal
complexes, such as Cu(II), prefer to be in a square planar environment, rather than tetrahedral.33
This is because more electrons can be placed in lower-energy orbitals in a square plane
compared to a tetrahedron. In figure 14, it can be noted that four of the five orbitals for the
square planar geometry are at a lower energy level relative to the tetrahedral geometry. There
is only one orbital at a high energy level, so it is more energetically favorable to have 8

19
electrons at the lower level and one electron at the high level. In the tetrahedron, on the other
hand, there are five electrons at a relatively high energy level, so overall there is less LFSE.

Figure – 13: Structures of Hemocyanin

This explains why d9 metals such as Cu(II) prefer to be in a square planar ligand field. While
many d9 metal complexes prefer the square planar environment, there is a significant number
of d9 metals that have structures that display a structure that is intermediate between a square
plane and tetrahedron. This type of intermediate structure is the case with Cu(II) in
oxyhemocyanin. When there is an uneven occupation of the t2 orbitals in the tetrahedral
geometry, it gives rise to Jahn-Teller (JT) distortion.

20
Figure – 14: D-Orbital Splitting Diagram for Cu(II) in Oxyhemocyanin

JT distortion occurs due to unequally distributed electrons in degenerate orbitals. Typically,

the distortion occurs in a way that places partially-filled orbitals at a slightly higher energy,
allowing the fully-filled degenerate orbitals to be lowered in energy. This occurs to increase
the overall stability of the molecule. d9 metals with a lower occupation in one of the t2 orbitals
have been shown to distort from a tetrahedron toward a square plane. This expected Jahn-Teller
distortion of a tetrahedron is consistent with the appearance of a distorted tetrahedral geometry
for Cu(II) bound to hemocyanin. As seen in the t2 antibonding orbital of the tetrahedral
arrangement in figure 14, there are two pairs of electrons that are paired, and one that is
unpaired. The unpaired electron shifts slightly up in energy so that the other two degenerate
orbitals will be lowered in energy, increasing the overall stability of the complex. Cu(I) and
Cu(II) are both found in geometries that are not typically preferred; if the copper ion is too
stable in a certain geometry, then it will become more difficult to change oxidation state. The
function of hemocyanin is oxygen transport, so it is vital that it can bind and release oxygen
quickly, changing oxidation states as it does so. If Cu(II) was in the preferred square planar
geometry, reduction to Cu(I) and release of oxygen would be less favorable. Similarly, if Cu(I)
were in a geometry that was strongly disfavored by Cu(II), oxidation would be less favorable,
so it would be more difficult to bind and transport oxygen.33

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 6. Conclusion:
In conclusion, dioxygen carriers play a crucial role in facilitating the transportation and
delivery of oxygen throughout various living organisms. These carriers are essential for the
survival and proper functioning of aerobic organisms, enabling them to efficiently utilize
oxygen for respiration and energy production. Several types of dioxygen carriers have been
identified in nature, each adapted to specific physiological needs and environmental conditions.
Hemoglobin, found in red blood cells of vertebrates, is one of the most well-known and
extensively studied dioxygen carriers. Its ability to reversibly bind and release oxygen ensures
efficient oxygen uptake in the lungs and oxygen delivery to tissues throughout the body.
Additionally, hemoglobin exhibits cooperativity, enhancing its oxygen-carrying capacity in a
regulated manner, responding to varying oxygen demands.34
Other dioxygen carriers, such as myoglobin, found in muscles and some respiratory pigments
like hemocyanin and hemerythrin, found in certain invertebrates, showcase diverse
mechanisms for oxygen transport tailored to their specific roles and physiological
requirements.
Furthermore, the fascinating adaptations seen in certain organisms, such as those living in
extreme environments or at high altitudes, have provided valuable insights into the evolution
and functional diversity of dioxygen carriers. These adaptations have allowed certain species
to thrive in conditions where oxygen availability is limited, demonstrating the versatility and
resilience of nature's oxygen transport systems.35

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 7. Future Directions:
Marked functional differences between the hemoglobin in solution and the hemoglobin within
the erythrocyte has shown and it is now important to determine the binding free energies and
enthalpies at the normal body temperature of 37ºC. Continued studies of adult human
hemoglobin ranging over pH and temperature should also be done with Parkhurst’s multiple
wavelength global analysis. This will give the complete thermodynamic description of oxygen
binding in normal human hemoglobin within the erythrocyte.
Thermodynamic studies of disease state erythrocytes, such as sickle-cell anaemia (HbS), are
expected to give new insight into the disease states of hemoglobin. HbS hemoglobin showed
high cooperativity with unusually large concentration-dependent Hill coefficients. However,
this conclusion has been questioned.36 The thermodynamics of oxygen binding with
hemoglobin S are highly concentration dependent. The spectrophotometry of hemoglobin S
required polymers and linkage theory to ensure the dimerization of hemoglobin does not occur
in solution while the hemoglobin S is in close interaction with other tetramers. Studies of HbS
within the erythrocyte would give clarity of the concentration-dependent thermodynamics of
sickle-cell anaemia.
In recent years, research has extended beyond natural dioxygen carriers, leading to the
development of synthetic and artificial carriers with potential applications in medicine,
biotechnology, and environmental remediation. These advancements hold promising prospects
for targeted oxygen delivery in medical treatments, oxygen storage, and transport in industrial
processes, and environmental solutions related to oxygen-sensitive ecosystems.
As our understanding of dioxygen carriers deepens, we continue to unravel the intricacies of
oxygen transport in living systems and the potential applications that could improve human
health and our ability to address environmental challenges. The study of dioxygen carriers
remains an exciting area of research, promising to unveil further discoveries that will contribute
to our knowledge of life's essential processes. Ultimately, appreciating the significance of these
carriers enhances our comprehension of the delicate balance between life and oxygen,
underscoring the vital interconnectedness of all living organisms with the Earth's atmosphere.37

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8. Acknowledgement:
I hereby acknowledge that myself Mr. Vedant Saraswat is very much grateful to my teacher
Dr. Parimal Rauth and Dr. Debolina Mitra for helping me to complete my Dissertation
project on the topic of Dioxygen Management Proteins and also I am very much thankful
to my classmates for helping me improving my project. The books, which I referred here
in this project are not only the best but also very much beneficial for any kind of fruitful
works. Also I am very much thankful to my H.O.D. sir (Head of the department) Dr.
Mominul Sinan for guiding me the procedure to complete this project.

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