Heme peroxidase
Cytochrome c
peroxidase
HRP
ROOH + 2 H+ + 2 e→ ROH + H2O
AH2 + H2O2 → A +
2H2O
His
His
CPO
A-H + X- + H+ + H2O2
→AX + 2H2O
Cys-
Catalase
2 H2O2 →2H2O + O2
Tyr-
A.-F. Miller, 2008, pg
1
W•
Peroxidases
Oxidation of a variety of compounds by H2O2
AH2 + H2O2 → A + 2H2O
From plants: horseradish root and fig sap.
FeIIIheme is LS at high pH, HS at low pH.
Two-electron oxidation: HRP(FeIII)+ H2O2 →HRP-I + H2O
Green complex (complex I)
FeIV=O•Por• (HS)
One electron reduction: HRP-I + AH2 →HRP-II + AH•
complex II
FeIV=O (LS)
A.-F. Miller, 2008, pg
2
Catalase and CPO
Two-e- oxidation: Cat(FeIII)+ H2O2 →Cat(FeIV=OPor•) + H2O
Cat-I (FeIV=OPor•)+ H2O2 → FeIIIPor + H2O + O2
CytcP-I (FeIV=OPor•) +2Cytc(FeII) + 2 H+ → FeIIIPor + H2O
+2Cytc(FeIII)
HRP: Cytc(FeII) is replaced by alkylamines, sulfides.
CPO-I (FeIV=OPor•) +Cl- → FeIIIPor + OClhypochlorite (hypochlorous acid)
AH + OCl- →ACl + OHA.-F. Miller, 2008, pg
3
P450 intermediates
A.-F. Miller, 2008, pg
4
Schlichting et al. (2000) Science 287:1615-1622
Reduced P450•Cam
+ O2 ↓
A.-F. Miller, 2008, pg
5
Schlichting et al. (2000) Science 287:1615-1622
Wat901(T252)
D251 bkbn O flipped
T252 bkbn NH
→O2, H2O
A.-F. Miller, 2008, pg
6
Schlichting et al. (2000) Science 287:1615-1622
New waters upon O2 binding
FeII (dark gray/blue water) vs. FeII•O2 (light gray/cyan water)
A.-F. Miller, 2008, pg
7
Schlichting et al. (2000) Science 287:1615-1622
Before - after irradiation
at 1.5 Å
A.-F. Miller, 2008, pg
8
Schlichting et al. (2000) Science 287:1615-1622
Fobs-Fcalc(no O),
3.6-4.5 σ
O occupied at 0.6, B=13 Å2
Fe
1.0 14
Camphor C5 1.0 18
Wat903 near O on Fe, T252, O of Gly248
(departing H2O ?)
A.-F. Miller, 2008, pg
9
Schlichting et al. (2000) Science 287:1615-1622
T252 and D251. T is
conserved, mutation to
anything other than Ser (or
methoxy) uncouples
activity.
D is a carbonyl switch.
N255 sc stabilizes displaced
carbonyl of D251 that
makes room for O2 and
moves amide NH of T252
Wat902 : bucket brigade.
to face O2
Helix is strained (sc of T252 and bkbn Gly248)
A.-F. Miller, 2008, pg
10
Schlichting et al. (2000) Science 287:1615-1622