Lecture 6

Question 1

How is deoxygen reduced to water?

Answer 1

• through enzymes
• increase rate by up to 10^12 compared to the corresponding model reaction
• most use copper or iron centres to bind oxygen in order to activate it
• Characteristic CO absorption band at 450 nm

Question 2

General Features of Cytochromes- P450

Answer 2

• monoxygenases
• found in mammalian tissue, birds, fish,plants,insects,bacteria and yeasts
• essential to biosynthesis, metabolism and detoxification of harmful substances

Question 3

General Reactions of Cytochromes-P450

Answer 3

• one oxygen atom is inserted

- second being reduced to H20 in cooperation with a reducing agent such as NADH, Fe-S proteins or P450 reductase

Question 4

Structure of Cytochromes-P450

Answer 4

• Similar to Hb and Mb but with the iron coordinated to the protein by a cysteine sulphur

Question 5

Description of Cytochromes-P450 cycle

Answer 5

1) in the rest state CTP450 contains a low spin Fe(III) centre with an additional ligand (probably water) in the sixth coordination site.
2) RH binding and H2O loss to give high spin Fe(III). Redox potential changes from -300 to -170 mV
3) Binding of O2 and 1 e- transfer form Fe(III) weakening the O2 bond
4) Acceptance of electron to give peroxy state (RDS)
5) Protonation to give hydroperoxy state
6) Further protonation leading to heterolytic O-O bond cleavage and generation of a formally oxoiron(V) species Fe(V)=O with the porphyrin ring existing as a radical cation
7) Oxygen atom transfer from 7 to substrate resulting in alcohol product and regeneration of the resting state

Question 6

Peroxide Shunt

Answer 6

• In Cytochrome mechanism
• Single oxygen atom donors such as hydrogen peroxide, per acids, alkyl hydroperoxides and iodosylbenzenes can be used to generate oxygenated products, bypassing intermediates.

Question 7

Why is methane the hardest substrate to hydroxylate?

Answer 7

• high CH bond energy
• No Dipole moment
• no additional functionality to assist in binding the protein to an active site

Question 8

What is used to hydroxylate methane?

Answer 8

• monooxygenase
• methane to methanol conversion
• CH4 + O2 + NADH + H+ —-> CH3OH + H2O + NAD+
• NAD coenzyme

Question 9

What is the structure of MMO?

Answer 9

• two diiorn centres bridges by a hydro group

- Differs from the active site of Hr, with Fe(III) irons in the resting state

Question 10

Steps in the proposed MMO mechanism

Answer 10

1) Reduction to Fe(II) State
2) O2 addition to give peroxy intermediate
3) Proton donation (from amino acid chain) to outer peroxy oxygen, causing H2O elimination, leading to Fe(III)-Fe(V) system [or Fe(IV)-Fe(IV)] - v reactive species
4) Fe(V)=O accepts hydrogen from CH4 to give a CH3 radical and Fe(IV)-OH (reducing electron deficiency)
5) Fast generation of methanol via OH donation

Question 11

What does tyrosinase catalyse?

Answer 11

• hydroxylation og monophenols to diphenols

- can act as two-electron oxidases catalysing the oxidation of diphenols to quinines

Question 12

Structure of tyrosinase

Answer 12

• dinuclear active site

- Two Cu centres

Question 13

Mechanism of tyrosinase

Answer 13

1) Addition of oxygen to give a Di-Cu(II) oxy form
2) coordination of phenolic substrate in axial position
3) ortho hydroxylation of the phenol
4) Intramolecular e- transfer to give quinine product and regenerate the active site