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

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

A. Verras

O. Lardinois

L. Koo

C. Nishida

R. Ghiladi

19731981

1989

1996

2004

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