Overview of Peroxidase Mechanism
As it applies to Dehaloperoxidase
NC State University
Heme iron is the binding
g site for
H2O, O2, and H2O2
The globin function requires that ferrous iron bind O2
reversibly.
The peroxidase function requires that ferric iron bind
and activate H2O2.
These functions appear to be diametrically opposed.
This is one of the interesting
interesting, and as yet unsolved
unsolved,
aspects of DHP structure and function.
The iron in heme is the binding site
f oxygen and
for
d peroxide
d
Heme is iron protoporphyrin IX.
Functional aspects in Mb
N
O
|||
O
N
Fe
N
N
O
O-
O
O-
The iron in heme is the binding site
f oxygen and
for
d peroxide
d
Heme is iron protoporphyrin IX.
Functional aspects in Mb
N
O
|||
C
N
Fe
1. Discrimination against
CO binding.
bi di
N
N
O
O-
O
O-
The iron in heme is the binding site
f oxygen and
for
d peroxide
d
Heme is iron protoporphyrin IX.
Functional aspects in Mb
N
N
Fe
1. Discrimination against
CO binding.
bi di
3+
N
N
2. O2 is the p
physiologically
y
g
y
relevant ligand, but it can
oxidize iron (autooxidation).
O
O-
O
O-
Peroxidase structure and
hemoglobin structure are distinct
Both peroxidases and globins are alpha-helical proteins,
but they have distinct folds and belong to different
families.
Cytochrome c Peroxidase
PDB: 1A2F
SCOP
Class: All  proteins
Superfamily:
Heme peroxidases
Family: CCP-like
Goodin and McCree
To be published
Horseradish Peroxidase
PDB: 2ATJ
SCOP
Class: All  proteins
Superfamily:
Heme peroxidases
Family: CCP-like
Hendrickson et al.
Biochemistry (1998)
37, 8054
Lignin Peroxidase
PDB: 1A2F
SCOP
Class: All  proteins
Superfamily:
Heme peroxidases
Family: CCP-like
Blodig
g et al.
Biochemistry (1998)
37, 8832
Myoglobin
PDB: 1A6G
SCOP
Class: All  proteins
Superfamily:
Globin-like
Family: Globins
Vojetchovsky,
j
y,
Berendzen,
Schlicting
Dehaloperoxidase
Proximal
Histidine
H90
Heme
Distal
Valine
V63
Shuttle
Histidine
H56
Levels of mechanism
1. We can examine the mechanism at the level of the
substrate, which is oxidized to form a radical.
2. We can examine the catalytic cycle that carries the
heme through three states and returns it to the ground
state on each successful enzymatic turnover.
3. We can examine the activation of the heme iron to
3
create the oxidizing species.
1. Mechanism for phenol oxidation by
HRP
HRP = Horseradish peroxidase
Heme Histidines
Heme,
Histidines, Arginine,
Arginine Calciums
2. Peroxidase Catalytic Cycle
+
X
HO
X
.
N
N
N
X
X
HO
N
Fe III
H2O2
N
X
H2O
N
Ferric
X
O
N
Fe
N
O
IV
N
N
F
Fe
N
N
N
X
N
Oxoferryl
Compound II
HO
X
X
IV
N
N
F
Fe
N
N
Or
N
.
+
.+
AA
O
IV
N
N
N
X
N
HO
X
X
Oxoferryl
Compound I
N
Oxoferryl
Compound ES
NC STATE UNIVERSITY
3. Poulos
Poulos-Kraut
Kraut mechanism
Distal histidine is required for peroxidase function
HN
H
N
3+
Fe
O H
O
Step 1: Peroxide binds to heme
Distal histidine is required for peroxidase function
HN
N
O H
+
H
O
Fe
Step 2: Protonation of histidine
Distal histidine is required for peroxidase function
HN
N
+
H
O H
O
Fe
Step 3: Reprotonation of H2O2
Distal histidine is required for peroxidase function
HN
N
+
H
O H
O
Fe
Step 4: Scission of the O-O
O O bond
Distal histidine is required for peroxidase function
HN
N
H
O H
O
Fe
+
Compound I
DHP oxidizes tribromophenol
UV-vis assay shown below
Dibromo
Quinone Tribromo
phenol