Carrie M. Wilmot Associate Professor Oxygen activation in copper amine oxidase University of Michigan April 2008

Carrie M. WilmotAssociate Professor

Oxygen activation in copper amine oxidase

University of Michigan

April 2008


Post-translationally modified amino acid cofactors

Okeley & Van der

Donk

Chemistry & Biology

7, R159-R171

(2000)


Roles of Copper Amine Oxidases (CuAOs)

• Prokaryotes and lower eukaryotes: Amines as N & C sources.• Plants: Development, 2o metabolism. Response to wounding.• Animals: Metabolism, regulation of glucose uptake, leukocyte

adhesion to vascular cell walls. Increased CuAO activity linked to congestive heart

failure, late-diabetic complications and inflammation in humans.


Physical properties of CuAOs• Homodimeric enzymes

• monomer

• 75 - 95 kDa

• ~700 amino acids

• Contain two cofactors

• the self-processed cofactor 2,4,5-trihydroxyphenylalanine quinone, or TPQ

• mononuclear Cu(II) (Type 2 or “non-blue” copper site)

P

O-

O

O

TPQ

24 5


P

O-

O

O

TPQ

P

OH

HO

NH2

Aminoquinol

P

O-

O

O

TPQ

Catalysis

reductive half oxidative half

RCH2NH3+ RCHO O2

H2O

H2O2

NH4+

P

OH

+ H2O + 2O2 + Cu(II)

P

O-

O

O

2

4 5+ H2O2 + Cu(II) + -OH

TPQTYR

Biogenesis

Catalytic chemistries: TPQ biosynthesis and amine oxidation

R group can be H to proteins

Catalyzes the conversion of primary amines to aldehydes

Carrie M. WilmotAssociate ProfessorLi R. et al. (1998) Structure 6: 293-307; Johnson B.J. et al. (2007) J. Biol. Chem. 282: 17767-76.

Hansenula polymorpha CuAO

1.7 Å resolution

HPAO


Active site of CuAOs

Cu-Wa:2.6-2.8Å

EquatorialCu ligands:

2.0-2.2Å

Numbering:Hansenulapolymorpha

CuAO (HPAO)

OOH

Cu2+

HisHis

His

OHOH

NH

OHC

R

H

Cu2+His

HisHis

OHOH

H2NOH

C

O

His

HisHis

OH

H2N OH

HO

H2O

H

R

CuAO catalytic mechanism

Cu2+

HisHis

OH2O

OO

His

NH2

OO

2+

His

HisHis

OH2O

NH

OC

H

HR

H2OR

Reductive half-reaction

semiquinone aminoquinol


Oxidative half-reaction• Substrate reduced enzyme exists in two forms;

Aminoquinol / Cu(II) >60%

Colorless

Semiquinone / Cu(I) <40%

Yellow; twin peaks @ 435, 465nm• Following anaerobic amine reduction:

Plant CuAOs have ~40% semiquinone / Cu(I)

Bacterial CuAOs have ~20% semiquinone / Cu(I)

Non-plant eukaryotes ~0% semiquinone / Cu(I)


Role of copper in catalysis

Postulated mechanism for molecular oxygen reduction in all CuAOs was;


Metal replacement studies in yeast HPAO Mills SA, Goto Y, Su Q, Plastino J, & Klinman JP. (2002) Biochemistry 41: 10577-

84.

• Co- and Cu-HPAO have very similar kinetic and chemical mechanisms. Ni(II) and Zn(II) substitutions are inactive.

• Co-HPAO, where the reduction potential for Co(II)/Co(I) makes Co(I) an unlikely intermediate in catalysis (e.g. –0.4 to –0.5V Co(I)/Co(II) vs SHE in methionine synthase), had a kcat(O2) almost identical to Cu-HPAO under substrate saturating conditions, effectively ruling out the requirement for a Cu redox change during HPAO catalysis.

• Co(III) was discounted as a kinetically relevant species as the O-18 KIE and kcat(O2) were identical for Cu-HPAO and Co-HPAO.

• The major difference is a decrease in O2 affinity in the Co-HPAO.• The primary role of the metal is suggested to be electrostatic

stabilization of the reduced dioxygen intermediates, and that redox changes at the metal are not required for catalysis.


Metal replacement studies in bacterial AGAO Kishishita S, Okajima T, Kim M, Yamaguchi H, Hirota S, Suzuki S, Kuroda S,

Tanizawa K, & Mure M. (2003) Role of copper ion in bacterial copper amine oxidase: spectroscopic and crystallographic studies of metal-substituted enzymes. J Am Chem Soc. 125: 1041-55.

• Co(II)-AGAO (Arthrobacter globiformis) and Ni(II)-AGAO both have kcat(O2) 100-fold down compared to Cu-AGAO.

• As with Co(II)/Co(I), the reduction potential of Ni(II)/Ni(I) (e.g. 1.16V vs. SHE in complexes with N/O ligands) disfavors a mechanism involving substantial +1 redox character.

• These studies did not rule out a mechanistic redox role for Cu(I) in the catalytic mechanism of the bacterial enzymes.

• The lower kcat(O2) for Co-AGAO and Ni-AGAO (1s-1) was attributed to molecular oxygen reduction by aminoquinol as the mechanistically relevant species. These kcat(O2) are comparable to the Cu-HPAO and Co-HPAO values (2s-1).


Model compound 6-amino-4-ethylresorcinol was found to consume oxygen at a rate of 18.6 M-1s-1

Model compound can activate O2

6-amino-4-ethylresorcinolOH

HO

NH2

Mills SA & Klinman JP (2000) J. Am. Chem. Soc. 122: 9897-9904

OOH

Cu2+

HisHis

His

OHOH

NH

OHC

R

H

Cu2+His

HisHis

OHOH

H2NOH

C

O

His

HisHis

OH

H2N OH

HO

H2O

H

R

CuAO catalytic mechanism

Cu2+

HisHis

OH2O

OO

His

NH2

OO

2+

His

HisHis

OH2O

NH

OC

H

HR

H2OR

OO

Cu2+His

HisHis

OHOH

H2NOH

OO

Cu2+His

HisHis

OH

OOH2N OH

HO

Cu2+

HisHis

His

OO

H2N OH

HO

OOCu2+

HisHis

His

H2N O

O HO OH

NH4+

H2O

HO OH

off-Cu

on-CuReductive half-

reaction

Oxidative half-reaction

Bacteria and plants

Non-plant eukaryotes


Hansenula polymorpha CuAO (HPAO)

• Yeast enzyme:― Preferred substrates are small aliphatic

primary amines.― Kinetically most similar to mammalian

enzymes (human and bovine plasma).― Turnover relatively slow, kcat= 3 s-1.― Among those with low propensity to form

measurable semiquinone/Cu(I) upon anaerobic substrate reduction.


aminoquinolsemiquinoneprotonated iminoquinone

UV/vis spectroscopic features

NH2

R

O

RH

Cu2+

HisHis

OH2O

OO

HisCu2+

His

HisHis

OHOH

H2NOH

E.T.

His

HisHis

OH

H2N OH

HO

OO

HOOH

Cu2+

HisHis

His

H2N O

OOH2

H2O

NH4+

Cu2+

HisHis

His

HN O

OOH2H

Cu2+

HisHis

OH2O

OO

His

deprotonated iminoquinoneTPQ

Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: 7117-27Hartmann C et al (1993) Biochemistry 32: 2234-41Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117: 8707-18

0

0.1

0.2

0.3

0.4

0.5

6 6.5 7 7.5 8 8.5 9

fractionofTPQdata

wtHPAO fraction of TPQsqD630N fraction of TPQsq

fracti

on o

f TP

Qs

q

pH

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

300 400 500 600 700 800

27nov06wtHPAOpH7CN

aerobic wtHPAOanaerobic wtHPAOMeaddnCNaddn1Meaddn1minCNaddn1_7minCNaddn2CNaddn2_4min

AU

pH 7.0

pH dependence of semiquinone content in HPAO

• Determined by anaerobic reduction with methylamine.

• Equilibrium is pulled to semiquinone/Cu(I) by the addition of CN- ions.

• Δ465 nm is used to quantitate semiquinone fraction.

Welford RW, Lam A, Mirica LM & Klinman JP (2007) Biochem. 46: 10817-27.



Enzymology in crystals• [protein] in crystals [protein] in the cell.• The crystal acts likes a porous cage that enables molecules,

such as substrates, to diffuse through the solvent channels.• Many enzymes retain catalytic activity in the crystal.• If there are no large conformational changes during

catalysis, many proteins remain crystalline during turnover.• Need the majority of the protein molecules in a crystal to be

in the same state to “see” that state in the structure.• desired intermediate must accumulate • must remain stable during X-ray data collection

• Depending on the system, spectroscopy can track the reaction in the crystalline protein.


Single crystal kinetics

Hadfield AT & Hajdu J (1993) J. Appl. Cryst. 26: 839-842. Sjogren T et al. (2002) J. Appl. Cryst. 35: 113-116.


TPQ

~ 480 nm

UV/vis spectroscopic features

NH2

R

O

RH

Cu2+

HisHis

OH2O

OO

His

Mure M & Klinman JP (1993) J. Am. Chem. Soc. 115: 7117-27Hartmann C et al (1993) Biochemistry 32: 2234-41Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117: 8707-18

Caveat: oxidation state of Cu

experimentally undetermined in crystal structures

Oxidized active site

2.6 Å2.4 Å

2.3 Å

• TPQ modeled in single conformation (although alternative conformations possible).

• O5 and D319 C=O separated by intervening water.

• Room in amine channel for 5 ordered waters.

• Axial Cu ligand is water (Wa).

O

O O

D319

TPQ

Wa

Johnson B.J. et al. (2007) J. Biol. Chem.

282: 17767-76.

Trapping intermediates in the crystal

• Crystal contacts often slow enzyme turnover without changing mechanism.

• Differential packing may lead to differential subunit reactivity.

• The asymmetric unit of HPAO contains 3 dimers (6 active sites).

90°

Active sites in the HPAO asymmetric unit

2 : 4


Johnson B.J. et al. (2007) J. Biol. Chem. 282: 17767-76.

Methods

low oxygen environment

Data collection statistics


Johnson & Wilmot,

unpublished


H2O

NH4+

Cu2+

HisHis

His

HN O

OOH2H

deprotonated iminoquinone

~ 450 nm

UV/vis spectroscopic featuresMure M & Klinman JP (1993) J. Am. Chem. Soc. 115: 7117-27Hartmann C et al (1993) Biochemistry 32: 2234-41Mure M & Klinman JP (1995) J. Am. Chem. Soc. 117: 8707-18

Methylamine reduction at pH 7.0: the deprotonated iminoquinone

• Cofactor orientation in all active sites identical to active orientation in oxidized structure.– all cofactors in “off-Cu”

conformation.• Water structure in amine

channel similar to that found in oxidized structure.

• Diatomic present at axial position of Cu

• Equatorial bound water is present.

2.4 Å

2.3 Å2.4 Å

0.36

0.38

0.4

0.42

0.44

0.46

300 400 500 600 700

wavelength (nm)

ab

so

rban

ce u

nit

s

pH 7.0

HN O

OH

Comparison to E. coli CuAO at steady stateECAO

HPAO

HPAO Wilmot CM et al (1999) Science 286: 1724-8



ECAO steady state species

OO-

Cu

Cu

O O2-

Superoxide

Peroxide

Cu

Peroxide

His526His689

2.8Å 3.0Å

Wilmot CM et al (1999) Science 286: 1724-8.

Aerobically trapped steady state species

(2.1Å resolution)


ECAO steady state species

Peroxide

• Water poised for attack at C5 of iminoquinone to release ammonia.

• Product aldehyde hydrogen bonded to Asp383, preventing it from ionizing and activating water.

• Proton transfer pathways to dioxygen;

(1) O2 of reduced TPQ dioxygen

(2) O4 of reduced TPQ Tyr369 water dioxygen

(HPAO: D3) (HPAO: D2)


Cu2+

HisHis

His

H2N O

OOH2

protonated iminoquinone

~ 350 nm


Methylamine reduction at pH 6.0:the protonated iminoquinone

• Direct H-bond between N5 and D319 C=O.

• Equatorial bound water present.

• Diatomic present at axial position of Cu.

• All cofactors in “off- Cu” conformation.

2.8 Å

2.6 Å

0.28

0.3

0.32

0.34

0.36

0.38

300 400 500 600 700

wavelength (nm)

ab

so

rban

ce u

nit

s

pH 6.0

H2N O

O


His

HisHis

OH

H2N OH

HO

OO

HOOH

semiquinone

~ 360, 435 & 465 nm


pH dependence of semiquinone content in HPAO

• Determined by anaerobic reduction with methylamine.

• Equilibrium is pulled to semiquinone/Cu(I) by the addition of CN- ions.

• Δ465 nm is used to quantitate semiquinone fraction.


Welford RW, Lam A, Mirica LM & Klinman JP (2007) Biochem. 46: 10817-27.

0

0.1

0.2

0.3

0.4

0.5

6 6.5 7 7.5 8 8.5 9

fractionofTPQdata

wtHPAO fraction of TPQsqD630N fraction of TPQsq

fracti

on o

f TP

Qs

q

pH

His

HisHis

NH2

HO

O

Methylamine reduction at pH 8.5:deprotonated iminoquinone

/semiquinone mix

• Cofactor has Cu-on and Cu-off occupancies– 50-60% of cofactor

directly ligated to Cu

• Water structure in amine channel consistent with pH 7 deprotonated iminoquinone (450 nm).

• Equatorial bound water disappears.

2.7 Å

2.3 Å

2.5 Å

0.3

0.31

0.32

0.33

0.34

0.35

0.36

0.37

0.38

300 400 500 600 700

wavelength (nm)

ab

so

rban

ce u

nit

s

pH 8.5

Cu2+

HisHis

His

HN O

O OH2H

Other sites in the asymmetric unit

Chain pH 6.0 pH 7.0 pH 8.5

A

B

C

D

E

F

protonated iminoquinone deprotonated iminoquinone semiquinone

H2N O

O

HN O

OH

NH2

HO

O

Cu1+

Cu2+ His

HisHis

OH2OH

H2NOH

His

HisHis

NH2HO

O

OO

O

O

OH

H2NOH

H2O

O

O

H2N O

O HO OH HOOH

Cu2+

HisHis

His

OH2

O O

O

H2O NH4

Cu2+ His

HisHis

H2OCu2+ His

HisHis

OH2OH

H2NOH

H2O

O

O

HN O

O HO OHH

Cu2+ His

HisHis

H2O Cu2+ His

HisHis

H2O

Revisited oxidative half-reaction mechanism

favored at high pH

favored at low pH

acidic basic

?

solved in ECAO


Summary

• Elevating pH in methylamine reduced HPAO results in structurally and spectroscopically distinct intermediates;– pH 6.0 protonated iminoquinone– pH 7.0 significant increase in deprotonated

iminoquinone– pH 8.5 reveals Cu-on cofactor orientation

concurrent with observable semiquinone-Cu(I) species• highest kcat in solution for HPAO ~ pH 8.5


Unaddressed questions

– Is semiquinone/Cu(I) always copper ligated?

– Is a copper ligated semiquinone species reactive with molecular oxygen?

– Or is it a non-reactive (off-pathway) species that builds up at high pH?

– Does the equatorial site have any role in the oxidative half-reaction?


Anaerobic substrate reduced ECAO + azide ions

Cu2+

Azide

Aminoquinol

(60%)

(40%)

(60%)

His526

His524

His689

0

0.2

0.4

0.6

0.8

360 460 560

Wavelength (nm)

Ab

sorb

ance

Crystal spectrumfollowing X-raydata collection


Aminoquinol / Cu2+, with Cu2+ / azide ion LMCT band @ 380nm

1.9Å resolution

Acknowledgements

• Wilmot Lab– Dr. Carrie Wilmot– Dr. Bryan Johnson– Dr. Arwen Pearson– Val Klema– Peder Cedervall

• Klinman Lab (UC Berkeley)– Dr. Judith Klinman– Dr. Richard Welford

• Beamline Staff (19-ID, Advanced Photon Source)– Dr. Steve Ginell

• Kahlert Structural Biology Lab– Ed Hoeffner

• Computer Staff– Dr. Patton Fast

• Financial Support– National Institutes of

Health– Minnesota Medical

Foundation– MN Partnership for

Biotechnology & Medical Genomics

– Minnesota Supercomputing Institute


• Dr. Simon Phillips, Dr. Mike McPherson, Dr. Peter Knowles (Leeds)

Documents

Carrie M. Wilmot Associate Professor Oxygen activation in copper amine oxidase University of Michigan April 2008