BCHM339 Summary and Questions

“Living in an oxygen env is injurious to your health”

Discuss this statement with particular respect to any harmful oxygen species and the role of
superoxide dismutase. Using diagrams where necessary, describe the essential features of
the active site structure of Cu2Zn2 superoxide dismutase and the so-called ping pong
mechanism of this enzyme. Include discussion of the experimental evidence that supports
this mechanism.

Discuss the strategy behind the design of the picket fence porphyrin with particular
reference to aspects that model the binding pocket of Hb. You should compare this model
complex with previous less successful ones.

Discuss whether you think the picket fence porphyrin is a successful model for Hb including
an account of the similarities and differences between the two molecules.

Describe the essential features of the active site of hemoglobin. Discuss the design strategy
of the “picket fence” porphyrin with particular reference to aspects which model the
binding pocket of haemoglobin. Include in your discussion the ways in which the picket
fence porphyrin a successful model for hemoglobin and describe the major differences and
how they were accounted for.

Discuss the function of carbonic anhydrase in biological systems, particularly with respect
to:
(i) the solubility of carbon dioxide in water;
(ii) the blood-buffer system.

Why is zinc so suited to the role it plays in carbonic anhydrase?

Briefly indicate the role of the siderophores in biological systems.

There are three main modes of iron binding in siderophores. Describe these and identify
and name the binding sites in the following siderophores.

The Mössbauer spectrum of Catalase Compound I is consistent with that of an Fe(IV)

complex but the UV-Vis spectrum is not typical of a normal metalloporphyrin

In what way does the UV-Vis spectrum differ from that expected?

How is this difference accounted for?

INTRO
Essential trace elements are common in many functions, in structural, charge carriers and
information transfer, formation, stabilisation and degradation of organics, electron transfer
and catalysis.
Protein Structure: Amino acid composed of amine and carboxylic ends, with R functional
group. If all three connect to the first carbon (alpha carbon) it is an alpha amino. A protein is
made up of aminos, with peptide (or amide) linkages between each monomer this is a C=O
to N bond. A protein is a polymer of these aminos, and there are 20 common aminos,
always mapped from the N terminus, and all have the same basic amino acid structure. They
differ in the variation on the R residue. The most important residues for metal ion
coordination are Glu and Asp (carbonyl), His (imidazole) Cys (CH2S) and Met (CH2-S-CH3).
Glu and Asp are hard donors, so bind to hard acceptors and metals like Fe3+ and can bridge
two metal ions.
His is an imidazole, and is an intermediate donor, and a good ligand for transition metals. It
can also form a bridge if deprotonated e.g. in superoxide dismutase.
Cys and Met are soft, good donors and are good for stabilising lower oxidation states, like
Cu+.

There are other non-protein ligands which are present, such as inorganic species, or
prosthetic groups such as Heme, and other porphyrin ring structures.

Porphyrin rings are 4,5 membered rings with conjugation between 4 pyrrole groups. It is
tetradentate and macrocyclic and will lose two protons to bind a metal ion in the centre.

Transition metals have several properties:

They have larger ionic radii and are softer than non-transitional metals and are therefore
often more ionic.
Transition metals are also not as hard, and are often classed as intermediate acceptors, and
will bind best to intermediate donors such as His.
They are also labile, with varying coordination numbers, variable oxidation states and their
properties can be tuned based on geometry and ligand type.
These properties make TM complexes very useful in industrial catalysis and can act in
catalytic biological systems. (Metalloenzymes) – where the metal ion is the active site.

pKa indicates the strength of an acid, the lower the pKa, the stronger the acid, as this
indicates how willingly it will give up a proton.

Transition metals form strong complexes and are therefore relatively static. Bulk metals on
the other hand are found with hard donors and are mobile, forming weak complexes.

Zinc is the second most abundant trace element, and only has one common ion – Zn2+. It is
therefore not redox active and is preferred in complexes where transition metal properties
are required but being redox active may be harmful to nearby biological systems, as well as
this, it is a Lewis acid. Examples of Zn containing metalloenzymes are Carbonic Anhydrase
and Alcohol Dehydrogenase. Zinc enzymes are either ionised or polarised, ionisation
involves deprotonated the H2O, to give OH-, whole the polarisation pathway involves a
substrate directly attaching to Zn.
Zinc also has structural roles, such as in insulin or zinc finger proteins, and is also a
stabilising metal ion in superoxide dismutase.

CARBONIC ANHYDRASE
A Zn catalytic enzyme, found in red blood cells, blood plasma and skeletal muscles. HCA II
was first recognised in 1940 and is found mainly in erythrocytes. It is involved in the
reversible hydration of dissolved CO2 to bicarbonate HCO3-. It acts as a blood buffer
system, and keeps the pH around 7.4
CO2 + H2O <-> HCO3- + H+
Without the enzyme, the rate of reaction is 10^-1 per second, with the enzyme, it increases
to 10^7.
It is used in biology for unloading CO2, in its transportation of CO2 from cells to lungs, and
pH balance, the enzyme turnover rate is 10^6 per second, indicating 10^6 molecules of CO2
per one molecule of carbonic anhydrase.
In the active site, Zn is at the bottom of a 15-angstrom cleft, and investigation was therefore
difficult, as EPR, UV and most spectro techniques didn’t work. They therefore replaced zinc
with Co2+ or Cd2+, which helped understand the environment (isomorphous replacement),
Co had 50% of activity, this is due to a similar Td geometry.
In the active site, Zn is ligated to 3 his residues and one H2O. In the resting state, it is Zn2+.
The enzyme has tuned the active site, so it is as acidic as possible, allowing switching
between coordination geometries.
In the cycle, Zn begins with 3 his and Oh attached, which nucleophilic attacks the CO2,
which binds to give the HCO3 anion, H2O is then added to form the cyclic species, where a
protein transfer occurs from the coordinate (closest to Zn) O to the terminal O. Then the
HCO3- is lost, and we still have H2O attached to the zinc, so a nearby His, picks up the H+,
returns Zn back to the starting form, and the H picked up by His is deprotonated by a basic
solvent.
This acts as a blood buffer as the entire cycle is reversible, therefore if the pH is too low and
the H+ conc is too high, the cycle will be reversed, producing more CO2 and less HCO3- and
H+.

ZINC FINGERS
Zinc fingers will intercalate into the major groove of DNA and are involved in binding to
specific DNA or RNA sites and control transcription. Zinc is used as it bonds strongly to
intermediate donors, and has no redox activity, which could alter DNA structure. It is
involved in the very strong bond between the TF and the DNA, and each domain is
organised into a configuration coordinated to Zinc. Zinc is therefore and essential part of the
protein, and the transcription process is specific to zinc, due to its geometry within the
active site. This geometry is important, as it will squeeze the rest of the finger domains into
shape. This is why Zinc is imp, as it provides the required Td geometry, and the bulk protein
has a very large effect on how side chains of particular amino acids will be held. We are
therefore tuning the active site using Zinc, so it functions.

SUPEROXIDE DISMUTASE
Copper Zinc Superoxide Dismutase disposes of the very toxic superoxide radical very quickly
in biological systems. They catalyse the disproportionation of the superoxide radical.
202.-  O2 + O22-
2Superoxide  Oxygen + Peroxide
Peroxide is then mopped up by catalase or peroxidase.
Aerobic organisms contain high concentrations of SOD, and it can be isolated from a wide
range of microorganisms.
Only 80% of oxygen is taken up in respiration, so SOD must mop up the superoxide radical
produced for it reacts with organic molecules. Superoxide is produced when Oxygen is
partially reduced.
Copper is used to having readily converting redox states (from +1 tot +2 to +3) +1 to +2
happens very readily. As Copper 2+ is the most available bivalent ion, and Copper +1 is the
most effective monovalent ion for binding organic molecules. Both +1 and 2 have equal
tendency to bind to organic molecules therefore the couple is very useful and adaptable
over a large range of redox potentials. Copper +1 will also act as a pi bond donor, so it will
bind easily to ligands like CO2 and O2.

From research, we have seen that Copper is detrimental to the function of SOD, whereas Zn
acts as a tuning metal ion, so will tune His bridge, so it binds to Copper 2+ in preference of
H+ but binds to H+ in preference of Copper +1. We have also seen, based on colour change
studies, that when binding 2 superoxide molecules, it will cycle between oxidation states.
Copper is bound to 3 His and H2O, then a His bridge, Zn is bound to His bridge, 2 His and
Asp.
When the superoxide binds to Copper in the active site, it is between oxidation states, as a
result a proton replaces Copper at the His group, and the now converted Oxygen leaves the
copper. This gives Copper +1 and a protonated His. Now Copper picks up another
superoxide and an H+, this leads to H+ bonding to the O atom, forming water and
converting Copper back to 2+, so will coordinate back to the His.

HEMOGLOBIN AND MYOGLOBIN

Hemoglobin is a oxygen transport protein in vertebrates, found in red blood cells. It is a
tetramer containing 4 subunits, each with one heme group. Myoglobin is used for dioxygen
storage, as it holds O2 in the tissue for when it is needed. Mb, unlike Hb is a monomer.

HB MB MODEL SYSTEMS
First was a Schiff base, which indicated O2 was bound end on, and proposed distal His had
to be there.
We found with energy levels that if an axial ligand was added to the heme, it forms a sq.
pyramidal arrangement, and raises the dz2 orbital above dxy. This is a prerequisite for
dioxygen binding.
Next proposal used TPP to prevent the protic cycle and dimerization. Picket fence model
had bulky groups facing up, which were non protic, to prevent imidazole binding to the 6 th
ligand point, allowing dioxygen to bind end on. Based on this, meff, Mossbauer, gamma
oxygen values were all very similar, with the difference in partial pressure for oxygen being
likely due to the functional need of oxymb to have less affinity, allowing transport and
release of O2 in the cell.
It was not perfect data however, and found the distal his was important, and it h bonds to
the O, guiding it into the pocket.
In the model also, the Fe is displaced form the plane when in +2 state, once O binds, pulled
into plane as now in 3+ state, this pulls up His, and following proteins within Hb and MB,
leading to cooperative effect, and altering O affinity for each of 4 heme groups, lowers the
first, then second affinity increases and so on, until 4th heme has 300-400 times the affinity
of the first.

IRON UPTAKE AND REGULATION

If you have an iron deficiency, there is anaemia, and too much is hemochromatosis. Iron
poisoning can also be treated by chelation therapy to remove excess via the kidneys. Iron
must be kept in homeostasis.
Uptake – absorption of fe through food or ingestion, in large mammals, due to being largely
insoluble, it must be taken up with ascorbic acid to make it easier to absorb and solubilise.
Transport –transferrins involved, which helps move iron through cell membranes, has a
dimer with each monomer binding Fe3+ ion. Occurs in a pac man action, where CO32- must
be taken up first.
Utilisation - is the incorporation into proteins such as Hemes
Storage – Ferritin and hemosiderin, safe storage of excess in a non-toxic and easily
accessible form.
Elimination – removal of controlled amounts from the system by excretion.

Ferritin - formed in response to Fe levels,provides an internal Fe store, Hemosiderin is

groups of ferritins complexed together.

SIDEROPHORES
Low molecular weight, they are fe chelating structures, produced by bacteria, they utilise
the chelate and macrocyclic effects. They can easily donate electrons and are therefore
good Lewis acids.

Enterobactin – Ring internal structure with 3 catecholate binding sites, binding constant is
10^45. The three sticks with catecholate come up to bind to Fe, therefore lowering the
entropy as the format is already preformed.

Desferroxamine – Has three hydroximate groups, these are made up of NO- and C=O, it
forms a claw shape to surround the Fe3+ ion, with the three hydroximates facing inwards.

Ferrochrome - macrocyclic, has some glycine side chains, but possesses 3 hydroximate
groups internally. It has a very high formation constant of 10^30

Pseudobactin – Possesses a mixture of ligands, with one catecholate, one hydroximate and
one alpha hydroxy acid which is a c=O with two lots of O-.

CATALASE
Catalases catalyse the disproportionation of peroxide:
2H2O2  O2 + 2H2O
Catalase is an enzyme in the liver that breaks down hydrogen peroxide into oxygen and
water. As a result, the oxygen gas bubbles escape and create a foam. It is common in nearly
all organisms that are exposed to oxygen.
A lack of catalase leads to a build-up of peroxide, this leads to greying and the prevention of
melanin production, seen when getting older.
Catalase is a tetramer with 4 long peptide chains over 500 aa long. It also has 4 fe containing
heme groups. There are lots of different catalases that work in a variety of pH ranges.
Human catalase the optimum pH is about 7, and they have individual optimum
temperatures.

In catalase, the 5th position possesses a Tyr residue with a phenolate group.
In the resting state, H2O is bound at the 6th position.
The resting state reacts with hydrogen peroxide to form compound one. When labelling the
two oxygens in the peroxide, we found they ended up in different places, one remains
bound, the other is part of compound one, which is 6 coordinates. The resting state has Fe
3+, while compound one has Fe 4+.