Section 5 + 6- Oxygen Transfer Proteins and Zinc Metalloenzymes

Question 1

How is energy produced?

Answer 1

Respiration in which O2 is reduced to water

Question 2

What protiens help energy production?

Answer 2

• enzymes

- reactions up to 10^12 times faster than model

Question 3

How is oxygen normally activated?

Answer 3

• by coordination to a metal centre
• iron or copper are common
• increases rate of reaction

Question 4

What are Cytochromes P450?
Where are they found?
Why 450?

Answer 4

• a family of metalloenzymes
• function as monoxygenases - found in mammalian tissues, birds, fish, plants, insects, yeasts, bacteria,
• name from CO adducts characteristic absorption bands at 450 nm.

Question 5

What are oxygenases?

Answer 5

• enzymes that inset oxygen into other molecules
• a monooxygenase inserts one oxygen
• deoxygenate inserts two oxygens

Question 6

What does Cytochromes P450 catalyse?

Answer 6

• aromatic and aliphatic hydroxylation reactions
• other oxidation reactions
• essential for biosynthesis, metabolism and detoxification

Question 7

How does a reaction with Cytochromes P450 work?

Answer 7

• One oxygen atom is inserted

- One oxygen atom is reduced to H2O by a reducing agent

Question 8

What is the general structure of Cytochromes P450

Answer 8

• Similar to Hb and Mb
• Based on a Haem Ring
• Iron coordinated by S(Cys)

Question 9

Give the steps of the Cytochromes P450 catalytic cycle

Answer 9

• cytochrome P-450 contains a low-spin Fe(III) centre with an addition ligand, probably water, in the sixth coordination site
•  RH binding and H2O loss to give high-spin Fe(III) state (2). Redox potention changes from -300 to -170 mV
• One electron reduction to high-spin Fe(II) (3)
•  Binding of O2 and one-electron transfer from Fe(II). ν(O-O) = 1140 cm-1
•  Acceptance of electron to give peroxy state (5) (rate limiting step)
•  Protonation to give hydroperoxy state (6)

- Further protonation leading to heterolytic O-O bond cleavage and generation of a
formally oxoiron(V) species or Fe(IV)=O species with the porphyrin ring existing
as a radical cation (7)

- Oxygen atom transfer from (7) to substrate to give alcohol product and
regeneration of resting state

Question 10

What happens during a peroxide shunt?

Cytochromes P450

Answer 10

ingle oxygen atom donors such as hydrogen peroxide, alkyl hydroperoxides, peracids, and iodosylbenzenes can also be used to generate oxygenated products by bypassing the intermediates (3)–(5).

Question 11

Why is methane hard to hydroxylate?

Answer 11

•  High C-H bond energy
•  No dipole moment
•  No additional functionality to assist in binding in protein active site

Question 12

How is methane converted to energy?

Where is this useful?

Answer 12

• using MMO (methane monooxygenase)

- important in methanotrophic microorganisms where methane is the sole energy source

Question 13

What is the reaction for MMO?

Answer 13

CH4 + O2 + NADH + H+ + MMO goes to CH3OH+H2O+NAD

Question 14

What is NAD?

Answer 14

The coenzyme for MMO

Question 15

What is the structure of the hydroxylase component of MMO?

Answer 15

• 2 Diiron centres
• Bridging hydroxo group
• Fe(III) resting state for both irons

Question 16

Describe the MMO catalytic cycle

Answer 16

1. Reduction to diferrous Fe(II)state
2. O2 addition to give peroxy intermediate
3. Proton donation to outer oxygen of peroxy-ligand, leading to H2O elimination
   and generation of a Fe(V)-Fe(III) state [or Fe(IV)-Fe(IV)]
4. Fe(V) accepts a hydrogen from methane to give a CH3 radical and Fe(IV)-OH
   5.Fast generation of methanol via donation of OH

Question 17

What is Tyrosinase?

Answer 17

• Tyrosinase is a monooxygenase enzyme which catalyses the hydroxylation of monophenols to diphenols
• can also act as a two-electron oxidase catalysing the oxidation of diphenols to quinines.

Question 18

What is the structure of Tyrosinase?

Answer 18

• Dinuclear site with two Cu(I) centres
• Like Hc
• O-O streching frequency of 755 cm-1

Question 19

Describe the catalytic cycle Tyrosinase

Answer 19

1. Addition of oxygen to give di-Cu(II) oxy form
2. Coordination of phenolic substrate in axial position
3. Ortho-hydroxylation of the phenol
4. Intramolecular electron transfer to give quinone product and regenerate
   active site

Question 20

What are the uses of Zinc?

Answer 20

• catalytic processes
• structural engineering of proteins
• regulation processes

Question 21

How does zinc exist under physiological conditions?

Answer 21

• dicationic Zn(II) - closed shell d10 configuration

- diamagnetic and colourless

Question 22

What are the important characteristics of Zn (II) in biological systems?

Answer 22

1. Strong Lewis acid.
2. NOT redox active.
3. Ready formation of low coordinate binding sites (> acidity)
4. Accessible coordination numbers of 4, 5, 6.
5. Easily deformed coordination geometry.
6. Easily undergoes mono-ligand substitution chemistry

Question 23

What is a unique feature of monzinc sites?

Answer 23

• water molecule that can be activated by ionisation, polarisation or be ready for dislacement

Question 24

How does Zn coordination change the pKa of water?

Answer 24

free H2O - pKa = 15.7
metal bound in [Zn(H2O)6]2+ - pKa ≈ 10

metal bound in [ZnL3(H2O)] - pKa ≈ 7

the lower the coordination number of the zinc the more acidity there is for the attached water

Question 25

How is the ‘transparency’ of Zn(II) overcome?

Answer 25

• replacing Zn(II) with Co(II) which has a similar ionic radius and can tolerate similar coordination environments.
• often possible to replace Zn2+ in a protein by Co2+ without greatly perturbing the protein conformation.
• As Co2+ is a d7 metal centre, its UV spectra can be used to monitor its coordinate geometry and changes in the UV spectra with pH can be used to study the deprotonation of any attached H2O molecules.

Question 26

Where is carbonic anhydrase (II) found?

What does it catalyse?

Answer 26

• found predominately in red blood cells
• catalyses the reversible hydration of CO2:

CO2 + H2O goes to HCO3- + H+

Question 27

Why is CO2 hydration important?

Answer 27

• removal of CO2 from actively metabolizing sites

Question 28

How does CA-II change the rate of reaction?

Answer 28

CA-II increases the rate of hydrolysis by a factor of 10^7

Question 29

What is the structure of CA-II

Answer 29

• the zinc atom lies near to the bottom of a 15 Å deep cleft.
• 3 His and one water coordinated

Question 30

Give the mechanistic steps of CA-II

Answer 30

1. Hydrophobic pocket next to Zn2+ captures CO2
2. Nucleophilic attack on the carbon of CO2
3. Conformational rearrangement at Zn centre
4. Productdisplacementbysubstitutionwithwater
5. His-64 as proton acceptor via a chain of hydrogen bonded water molecule (rds)

Question 31

What is CPA?
Where is it found?
What does it catalyse?

Answer 31

• Carboxypeptidase A
• found in the pancreas
• helps assimilate ingested proteins

Question 32

How is CPA selective?

Answer 32

• The cleavage of proteins only occurs at the C-terminal amino acid
• high selectivity for substrates in which the C-terminal amino acid contains a large aliphatic or Ph substituent.

Question 33

Describe the structure of CPA

Answer 33

• CPA has a molecular weight of approx 34,500
• contains 307 amino acids and one zinc atom.
• Near the surface of the protein is a pocket containing the Zn2+ cation ligated by one bidentate Glu and two His residues.
• The 5-coordinate sphere is completed by a water molecule.
• Also vital to the mechanism are a guanidium ion of Arg-127 and the carboxylate group of Glu-270.

Question 34

What is the proposed mechanism for CPA?

Answer 34

1. Peptide is “manoeuvred” into position using hydrophobic and H-bonding interactions
2. Arg-127 polarises the carbonyl group activating it towards nucleophilic attack by the Zn bound “activated” H2O
3. Cleavage of the C-N bond facilitated by Glu-270
4. Protontransferandpeptideandnewamino-acidleave