Heme: the catalytic center and a target in hemoproteins
Transcript of Heme: the catalytic center and a target in hemoproteins
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Heme: the catalytic center and a target in hemoproteins
Gerald Miwa Symposium
April 9, 2007
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Hemefrom Greek haima, blood
N
N N
NFe
CO2H CO2HHans Fischer (1881-1945)Synthesis of heme 1929Nobel 1930
Fe(II), Fe(III), Fe(IV), Fe(V)
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Basic hemoprotein manifold
N
N N
NFeII
X
Y
N
N N
NFeIII
X
Y
N
N N
NFeII
X
OO.
N
N N
NFe
X
O
+.N
N N
NFeIV
X
O
e-/O2e-
e-/2H+H2O2
+.
N
N N
NFeIV
X
O
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N
N N
NO
N
N N
N H2O2 or O2/NAD(P)H
Cmpd I
N
N N
NO
+.
Cmpd II
e- transfer: RXH -> RX.)
FeIII FeIV
FeIV
e-
Peroxidase vs monooxygenase reactions
O transfer: RH -> ROH
FeO
FeO
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HOH
D
C8H17
HOH
OH
C8H17
X YO O
enzyme
X YO
enzyme
Corey, Gregoriou (1959) J. Am. Chem. Soc. 81, 3127-3133
Early view of enzymatic hydrocarbon hydroxylation
E. J. CoreySynthetic methodology
Nobel 1990
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+HDD
H
D
H HD
HDD
H
D
H OHD HD
DH
D
H DOH
Radical rebound mechanism of P450 hydroxylation
kH/kD ~ 12
+.N
N N
NFeIV
ON
N N
NFeIV
OHC H C.
N
N N
NFeIII
C OH
25%
Groves, McClusky, White, Coon (1978) BBRC 81, 154-160
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OHHO
..
Timing the radical recombination with a bicyclo[2.1.0]pentane radical clock
kr = 2.9 x 109 s-1 kt = 2.0 x 1010 s-1
krkt
Ortiz de Montellano & Stearns, JACS, 109, 3415 (1987)Bowry & Ingold, JACS, 113, 5699-5707 (1991)
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Recombination rates for variousradical clocks
. 2 x 1010 s-1
2 x 1013 s-1.
2 x 1010 s-1
. (CH2)nCO2H
n = 8, 10 2.6 x 1010 s-1
.OMe
CH2
C6H5
.
2 x 1012 s-1
Newcomb et al., 2000
Newcomb et al., 1995
Ortiz de Montellano & Stearns, 1987
Auclair et al, 2002
Cryle et al, 2004
kt kt
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Active ingredient of absinthe
O
CH3
α-Thujone Two-zone clock
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The α- / β-thujone radical clocks
O
CH3
.krβ = 1.0 x 108 s-1
O
.
CH3
O.CH3
O.
CH3
krα = 4.4 x 107 s-1
4HOβ
4HOαHe and Ortiz de Montellano J, Org. Chem. 69, 5684-5789 (2004)
OCH3HO
OH3C OH
O
CH3
OH
kr
kinv
kt
[Fe-OH]3+
kt
[Fe-OH]3+
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P450 hydroxylation rebound rate
O.CH3
O
.
CH3
OCH3
.
β-thujone
α-thujone
OCH3 OH
OCH3HO
OCH3
OH
ktαCam: (1.3 ± 0.2) x 1010 s-1
BM3: (7.0 ± 1.3) x 1010 s-1
ktβCam: (8.0 ± 1.2) x 1010 s-1
BM3: (12 ± 1.3) x 1010 s-1
krα
krβ
[Fe=OH]3+
[Fe=OH]3+
[Fe=OH]3+
ktα
ktβ
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Radical recombination rate and extentof inversion in oxidation of α-thujone
Subst k1 4OHαT/(x 1010 s-1) 4HOβT
1A2 - 1/3.62C9 - 1/32C19 - 1/32D6 0.42 2.5/12E1 - 1/3.23A4 0.53 4/1BM3 7 ± 1.3 15/1Cam 1.3 ± 0.2 10/1L244A 1.5 ± 0.4 1/1
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4TSreb
2CI
2Creb
FeO
SH
H
SHFeOH
SHFeO
HSHFeO
Alkyl reboundAlkyl rotation
R
4TSabs
2CR
RH
Hydrogen abstraction
4PRR
~1 kcal/mol
barrier free
LS
HS2TSabs
4CR
2P
4Creb4CI
Two-state hydrocarbon hydroxylation
C
dz2 (σ∗)
dπ (π*FeO)
dσ“a2u”
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O
FeIIIP
.RO
FeIVP
FeIVP
rebound
H-RO
FeIVP+.
Radical clock variability. Shaik two-state hypothesis of doublet vs quartet state oxidation
H R
H
rearrange
“doubletstate”
“quartetstate” .RrO
FeIVP
Hrebound
O
FeIIIP
H R
O
FeIIIP
H Rr
clock
.RO H“concerted”
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+ .N
N N
NFe+4
O N
N N
NFe+4
OH
N
N N
NFe+3
C H C. C OH
Intervention of a second oxidizing species?
N
N N
NFe+3
O
C H
OH
N
N N
NFe+3
OH
COH2+
N
N N
NFe+3
OHC+
?
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Is FeV=O a P450 catalytic species?
Newcomb, Zhang, Chandrasena, Halgrimson, Horner, Makris, Sligar (2006) J. Am. Chem .Soc. 128, 4580-4581.
NFeIII
N
NN
NFeIV
N
NNO
NFeV
N
NNO
+.N
FeIV
N
NNO
hνHOONO
2 e-/O2
Compound I decay not accelerated by lauric acid substrate
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The blowtorch: enzyme maturation via autocatalytic heme modification
CYP4 P450 enzymes and human peroxidases
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HPLC recombinant CYP4A proteins
03060
250 350 450
030
60
250 350 450Wavelength nm
4A1
Abs
orba
nce
Time (min)
50
30
10
280 nm400 nm
4A3
0 2 4 6 8 10
50
30
10
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CYP4A prosthetic heme group
m/z = 632 amu (heme: m/z = 616 amu)
From pronase digestion
HPLCMS
N N
N NFe
OH
CO2HCO2H
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N N
N NFe
CH2O2C-Glu
CO2HCO2H
CYP4A P450 heme covalent binding
4A1 EGHDTTASG4A2 EGHDTTASG4A3 EGHDTTASG4A8 EGHDTTASG4A11 EGHDTTASG4F1 EGHDTTASG4F3 EGHDTTASG4F4 EGHDTTASG4F5 GGHDTTASGBM3 FLIAGHETT
Hoch, Ortiz de Montellano (2001) J. Biol. Chem. 276, 11339-11346LeBrun, Xu, Kroetz, Ortiz de Montellano (2002) Biochemistry, 41, 5931-5937
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Myeloperoxidase heme covalent links
In LPO, the residues are Glu 275 and Asp 125
Asp94
Met243
Glu242
S+
O
O
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Lactoperoxidase prosthetic heme
0
45
90
135
0
35
70
105
0 10 20 30
nLPO
HOCH2
CH2OH
400 nm280 nm
N N
N NFe
CO2HCO2H
DePillis, Ozaki, Kuo, Maltby, Ortiz de Montellano (1997) J. Biol. Chem. 272, 8857-60
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LPO autocatalytic heme binding
Colas, Kuo, Ortiz de Montellano (2002) J. Biol. Chem. 277, 7191-7200
0
14
28
42
0
14
28
42
0 10 20 30
wt
0
15
30
45
0
10
20
30
0 10 20 30
wtrecombinantLPO
heme
+ 4 equiv H2O2
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Proposed carboxyl radical mechanismfor heme covalent binding
O
FeIV
O-O2C
+.FeIV
OFeIV
FeIII
.O2C
HO2C
CH3 CH3
CH2.CH2
+
-O2C-H2O
H+
Repeat sequence at 1- and 5-methyls
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N
N N
NO
+.FeIVN
N N
NO
FeIV
R C
O
OH R C
O
O.
Can hemoproteins oxidizecarboxylic acid groups?
?
Test system: HRP + H2O2 + CH3CO2H
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Heme adduct: HRP+ acetate + H2O2
020406080
100120
0 5 10 15 20 25
Abs
at 4
00 n
m
Time, min
adduct
heme
00.20.40.60.81
1.21.41.6
300 400 500 600 700
Abs
orba
nce
Wavelength (nm)
adduct
heme
100
0
Rel
ativ
e in
tens
ity
m/z500 600 700 800 900
674
706
Heme + CH3CO2- H = 674
Huang, Colas, Ortiz de Montellano (2004) J. Am. Chem. Soc. 126, 12865-12873
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Heme:
3.353.353.403.403.453.453.503.503.553.553.603.603.653.653.703.70
Acetyl-D3-heme:
3.353.353.403.403.453.453.503.503.553.553.603.603.653.653.703.70 9.909.9010.0010.0010.1010.1010.2010.2010.3010.3010.4010.40
Acetyl-heme:
3.353.353.403.403.453.453.503.503.553.553.603.603.653.653.703.70
9.909.9010.0010.0010.1010.1010.2010.2010.3010.3010.4010.40
γ δ
3/5 3/5
CO2H CO2H
N
N N
NFeIIIO
O
γ
δ β
1
3
58
NMR of δ-meso acetyl heme adduct
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8-Hydroxymethylheme is a minor product in the HRP-acetate reaction
Abs
at 4
00 n
m
Retention time (min)
0
100
200
300
400
500
0 5 10 15 20
acetate adduct
heme8-HOhemeunstable
m/zCO2H CO2H
N
N N
NFeIII
HO
standard
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Structure of HRP-acetate complex (PDB 1h5a)
Å Å
Berglund, G. I., et al. (2002) Nature 463-468
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H2O
FeIII
N N
N NRCO2
O
FeIV
N N
N N
.
+.
OFeIV
N N
N N
O RO
-O RO
HRP Compound I reaction with carboxylic acids
O
FeIV
N N
N NRCO2
H .
OFeIV
N N
N N FeIII
N N
N N
HO
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Heme modification by autocatalytically generated
reactive species
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N
N
N
NFe
CO2H
OOCH3
HO
CO2H
Structure and mass spectrum of modified heme from HRP F152M plus tert-BuOOH
m/zm/z = 680
in 18O2
684
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Por(FeIII) + tert-BuOOH Por+.(FeIV)=O (1)
Por+.(FeIV)=O + tert-BuOOH Por(FeIV)=O + tert-BuOO. (2)
tert-BuOO. + tert-BuOO. 2 tert-BuO. + O2 (3)
tert-BuO. CH3. + CH3COCH3 (4)
CH3. + O2 CH3OO. (5)
Por(FeIII) + tert-BuOOH Por(FeIV=O) + tert-BuO. (6)
Generation of CH3OO. radical from tert-BuOOH
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Aerobic ROO. and anaerobic R.
vinyl additions
NOOCH3
.
N NCH3
.
NOOCH3
+
NCH3
+
NOOCH3
OH
NCH3
OH
NCH3
tert-BuOOHO2
tert-BuOOHno O2
H2O
-H+
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Regiospecificity of radical additions to HRP heme
NCS., CH3.,
Cl., N3., Ph.,
CN., CH3CO2.
N
N N
N
CO2H
Fe
CO2H
CH3OO., NO2
., Br.
NCS., CH3.
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Dissociation energies for RH -> R. + H. at 298 K and site of addition to the heme
Substrate DH298 Site of(kcal/mol) addition
H-CN 126.3 ± 0.2 δ-mesoC6H5-H 112.9 ± 0.5 δ-mesoCH3CO2H 112 ± 3 δ-mesoCH3-H 104.99 ± 0.03 δ-meso, vinylH-Cl 103.15 ± 0.03 δ-meso, vinylCH3CH2-H 101.1 ± 0.4 δ-mesoH-NCS 96 ± 6 δ-mesoH-N3 92 ± 5 δ-mesoCH3OO-H 88 ± 1 vinylH-Br 87.54 ± 0.05 vinyltert-BuOO-H 84 ± 2 none (too large)H-NO2 79.1 ± 0.2 vinyl
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Heme N-alkylation: autocatalytically formed P450 heme adducts
R
RCH=CH2 RCH(OH)CH2-RC=CH RC(=O)CH2-ArNHNH2 Ar-RNHNH2 R-
NN
CO2Me
CO2Me
NN
N
NH2
N
N
N
N
CO2H CO2H
R
Substrate
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Formation of aryl-iron and N-aryl adducts
-e-X
N N
XXN N N N
N N
N NN
N
NX
N
N
N
FeIII FeIII
FeIVFeII
N=NH
Herman Emil Fischer (1852-1919)Discovered PhNHNH2Nobel 1902
Augusto, Kunze, Ortiz de Montellano (1982) J. Biol. Chem. 257, 6231-6241.
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Why covalent heme binding?
HOSCN, HOBr, HOCl in the HRP model
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N
N N
NFeIII
Br
Br
P P
OH
OH
N
N N
NFeIII
P P
+H2O2
Br-
Modified hemes from reaction of HRP with Br- / H2O2 at pH 5
020406080
0 5 10 15 20 25
Abs
at 4
00 n
mm
AU
Time, min
t = 5.3 min t = 11.5 min
M+ 808
N
N N
NFeIII
Br
Br
P P
OH
M+ 790
Huang, Wojciechowski, Ortiz de Montellano (2005) J. Am. Chem. Soc. 127, 5345-5353
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Modified hemes from reaction of HRP with Cl- / H2O2 at pH 5
M+ = 616 (heme) + Cl + OH M+ = 616 (heme) + Cl - H
0100200300400500
0 5 10 15 20 25
Abs
at 4
00 n
m,
mA
U
Time, min
8.4 min 18.1 min
Heme
orN
N N
NFeIII
Cl
P P
OH
N
N N
NFeIII
Cl
P P
OH
N
N N
NFeIII
P P
Cl
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HOBr
-H+
-H+
H2O
N
N N
NFeIII
P P
Electrophilic addition mechanism of heme vinyl group modification
Br-
HOBr
+.N
N N
NFeIV
P P
O
Br
N
N N
NFeIII
P P
Br
OH
N
N N
NFeIII
P P
+Br
N
N N
NFeIII
P P
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0
20
40
60
80
100
120
RXN Cmpd 2 Cmpd 6 Heme
HRP
Act
ivity
(%)
N
N N
N
ABTS oxidation by apo HRP reconstituted with heme or vinyl-modified hemes
FeIII
Br
Pr Pr
N
N N
NFeIII
Br
Br
Pr Pr
OH
2
6
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Do heme-protein covalent bonds protect the heme?
Lactoperoxidaseand the F41E HRP mutant
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0
20
40
60
80
100
0 2 10 20
Rel
ativ
e ac
tivity
, %
Reaction time, min
Lactoperoxidase remains active after reaction with H2O2 and Br- (or Cl-)
Asp125
Glu275
LPO covalently bound
H2O2 alone
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HPLC of LPO-Br- reaction after digestion
LPO / 20 eq H2O2 / 0.4 M Br- (20 min), then protease digestion.Peak 2 is incomplete digestion - not present with more trypsin.
LPO-Br reaction
010
20
30
40
0 5 10 15 20 25 30 35 40
AB
S at
400
nm
LPO Control
1,5-dihydroxyheme
0
20406080
0 5 10 15 20 25 30 35 40
AB
S at
400
nm
1
2
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The F41E HRP mutation
F41E HRP
HRP and “F41E” superposition w/ LPO (in blue)
3 Me
WT HRP
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Autocatalytic heme binding in F41E HRP
N
N N
NFe
CO2H CO2H
OH
m/e = 632.2
From protein digestionafter H2O2 pretreatment
before H2O2
afterH2O2
Colas and Ortiz de Montellano, J. Biol. Chem. 279, 24131-24140 (2004)
0
30
60
90
0 12 24
0
10
20
30
0 12 240
3
6
9
0
3
6
9
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HRP + 30 eq H2O2, 0.6 M Br-, 30 mindigested with trypsin-proteinase K
HRP control: Br- oxidation then trypsin-proteinase K
Abs
orba
n ce
at 4
0 0 n
m
5 10 15 20 25 30
Time (min)
N N
N N
CO2H CO2H
Fe
HOOH
Br
heme
m/z 728 m/z 790N N
N N
CO2H CO2H
Fe
Br OH Br
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HRP F41E oxidation of bromideA
bsor
ban c
e at
40 0
nm
F41E pretreated with 2 x 6 eq H2O2, then + 30 eq H2O2, 0.6 M Br-, 30 min
5 10 15 20 25 30Ti iTime (min)
N N
N N
CO2H CO2H
Fe
HO
heme
N N
N N
CO2H CO2H
Fe
BrHO
N N
N N
CO2H CO2H
Fe
HOOH HO
m/z 666 m/z 632 m/z 710
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N
N N
NFeIII
Br
Br
N
N N
NFeIII
H2O2
P PP P
OH
Br-
HRP
N
N N
NFeIII
P PLPO
O
O
O
O
H2O2
Br-
Protection of heme vinyl groups by heme-proteincovalent bonds
No heme modification
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Regiospecificity of radical and electrophile additions to HRP heme
N
N N
N
CO2H
Fe
CO2H
HOBr, HOCl,HOSCN
NoneN
N N
N
CO2H
Fe
CO2H
Cl., N3.,
CN., CH3.,
Ph., NCS., CH3CO2
.,
NCS., CH3.,
CH3OO., NO2
.
electrophilic radicalR., Ar., RCH=CH2,RCH=CH
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FeN
NN
N
Pr
Me
Pr
V Me
V
MeMe
Heme oxygenase reaction1st step
NADPHCPR, O2
OH
FeN
NN
N
Pr
Me
Pr
V Me
V
MeMe
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Electrophilic vs radical?O
NN
OH
NN
H+
FeIII
OH
NN
NN
OH
H
+
FeIII
ON
N
H-O.
NN
H+
FeIV
electrophilicaddition
radicaladdition
heterolysis
homolysis product
OH
NN
NN
OH
H
.FeIV
Wilks, Torpey, Ortiz de Montellano (1994) J. Biol. Chem. 269, 29553-29556Sharma, Kevorkiants, de Visser, Kumar, Shaik (2003) Angew. Chem. Int. Ed. 43, 1129-1132
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U. Hoch
C. Colas
QuickTime™ and ahoto - JPEG decompressorneeded to see this picture.
L. Huang G.Wojciechowski
X. He
NIH
L. LeBrun
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
A. Verras
O. Lardinois
L. Koo
C. Nishida
R. Ghiladi
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19731981
1989
1996
2004
Next?My travels with Gerald