Copper Binding to Premature
Galactose Oxidase: Biogenesis
of the Tyr-Cys Cofactor
Alta Howells
Dooley Group
Chemistry and Biochemistry
The Dooley Group
Copper Metalloproteins
Amine Oxidase
Galactose Oxidase
RCH2NH2 + O2  RCHO + NH3 + H2O2
Nitrous Oxide Reductase
NO3-  NO2-  NO  N2O  N2
RCH2OH + O2  RCHO + H2O2
Why Study Metalloproteins?
A perfect example…
• 1/3 of all proteins
require metals for
their function
– Fe, Cu, Co, Zn
Cytochrome c Oxidase
PDB: 1OCC
Copper Proteins
• Produces reactive oxygen
species
H2O2, Superoxide,
Hydroxyl
• Genetic diseases
• Green house gases
• Biotechnology
Galactose Oxidase
RCH2OH + O2  RCHO + H2O2
Implication of GO
• Secreted from Polyporus circinatus Fr.
• Believed to break down tree lignin
• Biomedical applications
 Sensor for colon cancer
 Disaccharide tumors
 Bioassays for D-Galactose and Lactose
• Synthesis of carbohydrates
Structure-Function Relationship
How the Structure is Generated
The pieces that make it a whole
Without those pieces
LOSS OF FUNCTION
Structure
Crystal Structure and Active Site
of Galactose Oxidase
Y495
Y272
H581
H2O
H496
Crystal Structure
C228
W290
Essential Cofactors
Cu
Tyr272-Cys228
Maturation of Galactose Oxidase
• Four post-translational modification events:
?
Previous Research
• Tyr-Cys crosslink is formed with Cu and oxygen
or excess Cu(II) (Rogers)
• Cu(I) processes at a faster rate than Cu(II)
(Whittaker)
Rogers, Melanie. Biochemistry, 2008, 47, 39
Whittaker, Mei M. JBC, 2003, 278, 22090
Previous Research
• Cu(II)SO4 titration into
Premat-GO in anaerobic
condition
• 406 nm band increases over
time and then decays
• Yellow complex is formed
Rogers, Melanie. Biochemistry, 2008, 47 (39)
Current Work on Cu(II) Binding
• Binding affinity of Cu(II)
• Titrate Cu(II)(Gly)4 complex into Premat-GO in
anaerobic conditions
• Use UV-Vis and CD spectroscopy
• Observe the change in abs (UV-Vis) and molar
ellipticity (CD) as we are titrating in Cu(II)
• Rate of formation of certain intermediates
Experiment 1: Cu(II)(Gly)4 to Premat-GO in aerobic conditions
Concentration= .307mM Premature GO
~ 20.9mg/ml
1500
1000
0
-500
-1000
Change in Molar Ellipticity at 490nm
1200
-1500
-2000
1000
-2500
375
400
425
450
475
Wavelength nm
500
525
550
Molar Ellipticity
Molar Ellipticity
500
.307mM ApoGO
.5:1 Cu:ApoGO 20min.
.5:1 Cu:ApoGO 40min.
1:1 Cu:ApoGO 60min.
1:1 Cu:ApoGO 80min.
1.5:1 Cu:ApoGO 100min.
1.5:1 Cu:ApoGO 120min.
2:1 Cu:ApoGO 140min.
2:1 Cu:ApoGO 160min.
800
600
400
200
0
0.15
0.20
0.25
0.30
0.35
0.40
[Cu(II)]mM
0.45
0.50
0.55
0.60
Experiment 2: Cu(II)(Gly)4 to premat-GO in anaerobic
conditions
Protein Concentration= .247mM Premature GO
0 mol Cu: 1mol ApoGO
~ 16.8mg/mL
.2 mol Cu: 1 mol ApoGO
.4 mol Cu: 1 mol ApoGO
.6 mol Cu: 1 mol ApoGO
.8 mol Cu: 1 mol ApoGO
1 mol Cu: 1 mol ApoGO
1.5 mol Cu: 1 mol ApoGO
2 mol Cu: 1 mol ApoGO
4000
2000
1000
0
Change in Molar Ellipticity at 425 nm
-1000
-2000
3500
-3000
3000
-4000
2500
-5000
300 350 400 450 500 550 600 650 700 750 800
Wavelength nm
Issues:
• Signal to noise ratio
• Protein precipitation
Molar Ellipticity
Molar Ellipticity
3000
2000
1500
1000
500
0.8 : 1 (Cu : protein)
0
-500
-1000
0.00
0.05
0.10
[Cu(II)]mM
0.15
0.20
0.25
Experiment 3: Cu(II)(Gly)4 complex to PrematGO with anaerobic conditions
Concentration= .241mM Premature GO
~ 16.4 mg/mL
2000
1000
500
0
-500
-1000
Change in Molar Ellipticity at 425nm
-1500
-2000
2200
425nm
2000
1800
375 400 425 450 475 500 525 550 575 600 625
1600
Wavelength nm
1400
1200
Issues:
• Signal to noise ratio
• Incubation time
• Temperature affecting kinetics
Molar Ellipticity
Molar Ellipticity
1500
ApoGO
.1 Cu(II) : 1 ApoGO
.2 Cu(II) : 1 ApoGO
.3 Cu(II) : 1 ApoGO
.4 Cu(II) : 1 ApoGO
.5 Cu(II) : 1 ApoGO
.6 Cu(II) : 1 ApoGO
.7 Cu(II) : 1 ApoGO
.8 Cu(II) : 1 ApoGO
1000
800
600
400
200
0
-200
-400
Peaks at a ratio of
.5 mol Cu(II): 1 mol Premat-GO
-600
0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20
[Cu(II)]mM
Experiment 4: Determining Incubation Time
Incubation on ice (4 C)
At 25 C
0.5 mol Cu(II) : 1 mol ApoGO
Conc. Protein = 11.3mg/mL
Abs
ApoGO Abs
Time 0 Abs
10 mins Abs
20 mins Abs
30 mins Abs
40 mins Abs
50 mins Abs
60 mins Abs
70 mins Abs
80 mins Abs
90 mins Abs
0.10
0.21
0.05
Buffer
ApoGO Abs
Time 0 Abs
5 mins. Abs
10 mins. Abs
15 mins. Abs
20 mins. Abs
25 mins. Abs
30 mins. Abs
35 mins. Abs
40 mins. Abs
45 mins. Abs
50 mins. Abs
55 mins. Abs
60 mins. Abs
Buffer + Cu
0.18
0.15
Absorbance
Absorbance
0.15
0.5 mol Cu(II) : 1 mol ApoGO
Conc. Protein = 17.7mg/mL
0.12
0.09
0.06
0.00
300
400
500
600
700
800
900
1000
0.03
Wavelength nm
Intermediate formation
in 40 minutes
Issues:
• O2 contamination
0.00
400
500
600
700
800
Wavelength nm
Intermediate formation
in 20 minutes
900
Current Research Summary
What has been achieved…
• Cu(II)(Gly)4 binds to premat-GO
• Yellow complex reaches maximum at
0.5:1 (Cu : Protein)
• Incubation time: 20 minutes at 25C
To obtain quality data…
• Reaction temperature
• Max protein concentration
• Maintain anaerobic condition
Acknowledgements