Heme: the catalytic center and a target in hemoproteins
Gerald Miwa Symposium
April 9, 2007
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)
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
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
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
+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
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)
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
Active ingredient of absinthe
O
CH3
α-Thujone Two-zone clock
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+
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
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
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”
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”
+ .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+
?
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
The blowtorch: enzyme maturation via autocatalytic heme modification
CYP4 P450 enzymes and human peroxidases
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
CYP4A prosthetic heme group
m/z = 632 amu (heme: m/z = 616 amu)
From pronase digestion
HPLCMS
N N
N NFe
OH
CO2HCO2H
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
Myeloperoxidase heme covalent links
In LPO, the residues are Glu 275 and Asp 125
Asp94
Met243
Glu242
S+
O
O
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
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
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
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
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
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
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
Structure of HRP-acetate complex (PDB 1h5a)
Å Å
Berglund, G. I., et al. (2002) Nature 463-468
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
Heme modification by autocatalytically generated
reactive species
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
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
Aerobic ROO. and anaerobic R.
vinyl additions
NOOCH3
.
N NCH3
.
NOOCH3
+
NCH3
+
NOOCH3
OH
NCH3
OH
NCH3
tert-BuOOHO2
tert-BuOOHno O2
H2O
-H+
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.
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
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
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.
Why covalent heme binding?
HOSCN, HOBr, HOCl in the HRP model
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
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
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
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
Do heme-protein covalent bonds protect the heme?
Lactoperoxidaseand the F41E HRP mutant
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
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
The F41E HRP mutation
F41E HRP
HRP and “F41E” superposition w/ LPO (in blue)
3 Me
WT HRP
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
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
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
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
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
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
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
U. Hoch
C. Colas
QuickTime™ and ahoto - JPEG decompressorneeded to see this picture.
L. Huang G.Wojciechowski
X. He
NIH
L. LeBrun
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are needed to see this picture.
A. Verras
O. Lardinois
L. Koo
C. Nishida
R. Ghiladi
19731981
1989
1996
2004
Next?My travels with Gerald
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