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Carrie M. WilmotAssociate Professor
Oxygen activation in copper amine oxidase
University of Michigan
April 2008
Carrie M. WilmotAssociate Professor
Post-translationally modified amino acid cofactors
Okeley & Van der
Donk
Chemistry & Biology
7, R159-R171
(2000)
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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)
Carrie M. WilmotAssociate Professor
Role of copper in catalysis
Postulated mechanism for molecular oxygen reduction in all CuAOs was;
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
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).
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
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.
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
Johnson B.J. et al. (2007) J. Biol. Chem. 282: 17767-76.
Methods
low oxygen environment
Data collection statistics
Carrie M. WilmotAssociate Professor
Johnson & Wilmot,
unpublished
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
Carrie M. WilmotAssociate Professor
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)
Carrie M. WilmotAssociate Professor
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)
Carrie M. WilmotAssociate Professor
Cu2+
HisHis
His
H2N O
OOH2
protonated iminoquinone
~ 350 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 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
Carrie M. WilmotAssociate Professor
His
HisHis
OH
H2N OH
HO
OO
HOOH
semiquinone
~ 360, 435 & 465 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
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.
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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?
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
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
Carrie M. WilmotAssociate Professor
• Dr. Simon Phillips, Dr. Mike McPherson, Dr. Peter Knowles (Leeds)