Bioinorganic Chemistry

Question 1

What are the main hard and soft ligands used in the body? Give the relevant amino acids.

Answer 1

N is a hard donor and is usually donated from histidine. Carboxylate groups are hard donors and commonly are donated from aspartate and glutamate aa. OH is hard and can be donated from tyrosine. S is soft and is donated from either cysteine or methionine.

Question 2

Describe the Irving Williams series of +2 metal ion stability.

Answer 2

As the nuclear charge increases across the row, Z_eff/radius increases meaning a complex stability between the metal and ligand increases. Cu(II) is the most stable. However the series peaks before it reaches Zn(II) as the CFSE is no longer relevent as all the d orbitals are filled.

Question 3

How does biology bind specific metals into proteins?

Answer 3

The folded proteins pre-organise with ligands held in the perfect size to bind the desired metal. This becomes complex when metals with similar sizes but different properties are present.

Question 4

Draw a haem group binding to an iron atom,

Answer 4

First draw the nitrogens and the 90° bonds coming from them. Two nitrogens must have 2 bonds from the haem to have a neutral charge.

Question 5

What are the challenges with studying the active site of a metalloprotein? What are the solutions?

Answer 5

The active site of metalloproteins are shielded with around 30,000 Da of other atoms. This gives extremely complex spectra.

To resolve the metal binding site, a method must be chosen that amplifies the signal from the metal.

Question 6

Describe single crystal x-ray diffraction.

Answer 6

A single crystal of the metalloprotein is grown (very difficult), then a diffraction experiment is done to map the electron density in the structure. Assigning the electron density map can be difficult so the data is often augmented with other information to assign the contor map. This often depends on the resolution of the crystal data.

Question 7

How does time resolved single crystal x-ray diffraction work? Why is this useful?

Answer 7

The method works by suspending metalloprotein crystals in aqueous solution, which is then flowed through a laser that excites the crystal. An x-ray passed through the structures and gives off the diffraction pattern.

This method can give structures of metalloproteins that are excited in their mechanism of action.

Question 8

Describe the method of extended x-ray absorption fine structure (EXAFS).

Answer 8

Here metalloproteins can be resolved in solution or in solid state. A huge synchotron generates x-rays of increasing power that ionise core electrons from the metal. Once the ionisation threshold is reached, 1s electrons start to be ionised with no kinetic energy. As the x-ray power increases, the kinetic energy of the expelled electrons increases.

Electrons are backscattered by the nearby atoms which interfere with the absorption of incedent x-rays. Degree of interference depends on the size of the backscattered wave, distance to each surrounding atom and the energy of the initial x-ray.

Question 9

How do you interpret EXAFS spectra?

How would you draw the spectrum from a structure?

Answer 9

The raw data is fourier transformed twice to give a x-axis of distribution of atoms around the metal in all directions, and a y-axis of electron density.

Importantly there is a large decay of signal over time to almost nothing at 5 Å.

Create a list of atoms at increasing distance from the metal. Then add a column of the number of electrons in each atom. Draw a graph of electron density at increasing distance from the nucleus, remembering that the signal decays to 0 at 5 Å.

Question 10

Describe the process of anisotropic EPR and the information that can be gained from it.

Answer 10

In EPR, the unpaired electrons are detected by changing their spins with microwaves. Anisotripic EPR is where the g_⊥ and g_∥ signals are split from one peak into two with two different splitting constants, A_⊥ and A_∥. This is done by freezing samples to 20 K.

The g values tell us about the coordination geometry and the splitting tells us about the spin of the nucleus.

Question 11

Describe how the anisotropic EPR spectra of type 1, 2 and 3 copper enzymes are different, giving their A values.

Answer 11

Type 1 is involved in electron transfers and has closely spaced g_∥ peaks, approximately 5 mT.

Type 2 is an oxidation enzyme and has widely spaced g_∥ peaks, approximately 15 mT.

Type 3 is EPR silent.

Question 12

Describe Mossbauer spectroscopy and how it is used to study metalloproteins.

Answer 12

Mossbauer spectroscopy measures absorption of gamma rays by the atomic nuclei. This is based on the principle that nuclei have energy levels like electrons.

These transitions can depend on oxidation state and spin of the nucleus. Gamma rays are shot at the sample which is moved back and forth to induce a doppler shift. The absorption is measured as a fuction of sample speed.

Question 13

Describe how resonance raman spectroscopy is used to study metalloproteins.

Answer 13

Scattering must be enhanced from the metal, this is done by tuning the laser to the visible region as it resonates with the electron absorption of metals.

One example of use is studying the state of O₂ when bound to a carrier protein. The bond vibration decreases by over half when the oxygen binds to the metal.

Question 14

How can amino acid sequencing be used to study the metal environments of metalloproteins?

Answer 14

Firstly, we know which amino acids are capable to coordinating to metal ions. Secondly, in similar proteins, a consensus sequence is preserved, meaning the same amino acid sequence binds the same metal in similar proteins. The zinc finger protein has a -x-Cys-x-x-Cys-x- consensus sequence. Type 1 copper: -Cys-x₂-His-x₄-Met-.

This can help identify types of metalloproteins. (learn to be able to identify from sequence).

Question 15

Why is iron so important for life yet so difficult to get?

Answer 15

When life evolved mechanisms to survive the oceans were filled with Fe(II) with a low O₂ concentration in the atmosphere. Now almost all Fe(II) is oxidised to Fe(III) where it is extremely stable as Fe(OH)₃. Therefore organisms have to compete over what remains availible.

Question 16

Describe siderophores and give the key features of enterobactin.

Answer 16

Siderophores are chelating ligands produced by bacteria to specifically bind iron. The complex is recognised by the bacteria and absorbed, then the ligand is removed and the metal used in biosynthesis.

Enterobactin has three catechol (aryl group with two OHs) groups which coordinate to the metal in perfect octahedral geometry. There are three serine residues linked together by ester bonds in a ring which are broken to release the metal (chelating 10⁴⁹ stability to 10²⁰).

Question 17

How can siderophores adapt to avoid antibodies and attack other bacteria?

Answer 17

Methylation of catechols can prevent recognition by antibodies.

Bacteria can evolve to absorb iron from siderophores they don’t produce. To stop this, siderophores with targeted antibiotics can be produced to kill the stealing bacteria.

Question 18

Describe how the human body transports iron. Give the key features of the active site.

Answer 18

The body has a protein called transferrin (Tf) which has a quaternary structure made of 4 lobes. Two Fe(III) ions bind to the protein and the lobes fold in, kinetically blocking any other chelators. The stability is much lower than siderophores but since it is sterically hindered, it cannot reach the iron.

The active site is made up of hard and borderline donors (O and N). This forms a 7-coordinate structure around the iron. The structure is preorganised to allow for easy binding and to stop other metals binding.

Question 19

Describe how the human body stores iron.

Answer 19

The iron storage protein is called ferritin and is a large, hollow protein, assembled of four helical bundles. These proteins make up the coat of ferritin with 4 fold and 3 fold channels to pass through the coat. Fe(II) enters through these channels and is stabilised as Fe(III) in an Fe-O structure.

Question 20

What is the uptake-reduction model and why does it explain the carcinogenity of [CrO₄]^2-?

Answer 20

The uptake-reduction model is where a damaging species enters a cell by a different species’ pathways, and is then reduced to a stable form.

[CrO₄]^2- enters cells by the [SO₄]^2- pathways and is then reduced to Cr(III). This is stable inside the cell and is carcinogenic.

Question 21

What are the biological advantages of using zinc and why is it hard to study?

How can we overcome these challenges?

Answer 21

• Zn(II) is the only biologically significant oxidation state, redox stable
• Zn(II) is d¹⁰, can act as a lewis acid and kinetically labile
• Zinc is very hard to analyse spectroscopically

Cobalt is similar to zinc and is d⁷ and magnetic. The similarities are that there is no CFSE for T_d and O_h coordination geometries so we can use Co(II) as a Zn(II) substitute.

Question 22

Describe the discovery of the environment of the Zn(II) in carbonic anhydrase. Draw the catalytic mechanism and describe why it is possible with Zn(II).

Answer 22

Replacing the Zn(II) with Co(II) using EDTA means the protein can be studied in a UV-Vis spectrometer. The attenuation coefficient, ε, means the metal can only be in a non-centrosymmetric coordination environment. This is confirmed from the structure.

This is possible as the Zn(II) is acidic which is transferred to the water, inducing the deprotonation.

Question 23

What role does tetrahedral Zn(II) have in the function of liver alcohol dehydrogenase?

Answer 23

Ethanol is converted to ethanal. This occurs through the deprotonation of ethanol which is challenging. The Zn(II) protein helps this using its acidity to stabilise the ethoxide. This then reacts on with NAD⁺ to form ethanal and NADH.

Question 24

Describe how Zn(II) plays an important structural role in zinc finger protiens and how the structure can be analysed.

Answer 24

Zinc finger proteins are named as such as they have long extended finger regions which reach into the DNA helix. The Zn(II) coordinates to N and S atoms in the chain to pin the amino acids together with a long chain pointing away from the metal.

Replacing Zn(II) with Co(II) gives observable spectroscopic features which can be analysed to see the tetrahedral metal environment.

Question 25

Describe the challenges and capabilites of the body in processing O₂ to H₂O.

Answer 25

O₂ must be reduced by 4 electrons in a very short time to avoid the production of partially reduced O₂ (superoxide, peroxide and sometimes hydroxyl radicals). To do this, the body has 4 seperate but very fast electron transfers using cytochromes and FeS proteins. This all occurs in the mitochondria and converts Cu(I) to Cu(II) and Fe(II) to Fe(III). These metals have the O₂ linked between the metals.

Question 26

Give the labels for a, b and c of the Marcus curves for an electron transfer and taking the equations for both curves, derive an equation for the activation energy of the process.

Answer 26

a = ΔG of the reaction

b = activation energy of electron transfer

c = reorganisation energy, λ, to convert from the oxidised complex to the reduced complex

Curve equations: y = x², y - ΔG = (x - p)²

Setting them equal: x² = x² - 2px + ΔG + p²

Rearrange: x = (ΔG + p²)/2p

Sub into y = x²: y = (ΔG + p²)²/4p²

As p² = λ (sub p into y = x²): y = (ΔG + λ)²/4λ = b

Question 27

Given that the rate of electron transfer is quantified by:

k = Aexp(-(ΔG + λ)²/4λkT)

What is the key factor that must be minimised by the body?

Answer 27

λ must be minimised. This is the reorganisation factor and means that the reduced and oxidised form of the complex in the body need to be in very similar shapes.

Question 28

Describe the iron enviroments of cytochromes and rubredoxin and explain why they are effective at transferring electrons.

Answer 28

Cytochromes have a 4 N macrocycle around the Fe with two amino acid residues above and below the Fe. It transfers a single electron and has a rigid structure, meaning it has a very low reorganisation energy, λ.

Rubredoxin is a 4 S (deprotonated cysteine) protein. The protein holds the cysteines in place meaning there is low reorganisation energy, λ.

Question 29

Describe Ferredoxins and 4Fe-4S structures and give why they are effective at transferring electrons.

Answer 29

Ferredoxins are two Fe atoms bound by 4 S atoms, with the atoms sharing two S ligands. This is also how Cu_A is organised.

4Fe-4S form a cubic structure with 4 S ligands for each Fe.

Both these structures delocalise their electrons so they can be oxidised and reduced without large reorganisation.

Question 30

Describe how Cupredoxin is able to quickly transfer electrons between the Cu(I) and Cu(II) states.

Answer 30

Cu(I) has no CFSE (d¹⁰) and prefers T_d geometry.

Cu(II) is d⁹ and prefers square planar or distorted O_h geometries.

These geometries have high reorganisation differences leading to slow electron transfer. To overcome this, the copper atoms are held in an ENTATIC STATE which is halfway between T_d and square planar. This gives low reorganisation energy and fast electron transfer.

There is also a pi interaction between the Cu and the S ligand leading to electron delocalisation.