Copper Binding to Premature Galactose

Oxidase: Biogenesis of the Tyr-Cys Cofactor

Alta HowellsDooley Group

Chemistry and Biochemistry

The Dooley GroupThe Dooley GroupCopper MetalloproteinsCopper Metalloproteins

NO3- NO2- NO N2O N2

Nitrous OxideNitrous Oxide ReductaseReductaseRCH2NH2 + O2 RCHO + NH3 + H2O2

Amine OxidaseAmine Oxidase

RCH2OH + O2 RCHO + H2O2

Galactose OxidaseGalactose Oxidase

Why Study Metalloproteins?

• 1/3 of all proteins require metals for their function–Fe, Cu, Co, Zn

Cytochrome c OxidasePDB: 1OCC

A perfect example…

Copper Proteins• Produces reactive

oxygen speciesH2O2, 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 cancerDisaccharide tumors

Bioassays for D-Galactose and Lactose

• Synthesis of carbohydrates

Structure-Function Relationship

StructuStructurere

How the Structure is GeneratedHow the Structure is GeneratedThe pieces that make it a wholeThe pieces that make it a whole

Without those Without those piecespiecesLOSS OF LOSS OF FUNCTIONFUNCTION

H496

Y495

H581

Y272

C228 W290

H2O

Crystal StructureCrystal StructureEssential CofactorsEssential Cofactors CuCu Tyr272-Cys228Tyr272-Cys228

Crystal Structure and Active Site of Galactose Oxidase

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.Rogers, Melanie. Biochemistry, Biochemistry, 20082008, 47, 39, 47, 39

Whittaker, Mei M. Whittaker, Mei M. JBCJBC, , 20032003, 278, 22090, 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.Rogers, Melanie. Biochemistry, Biochemistry, 20082008, 47 (39), 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

0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.600

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[Cu(II)]mM

Change in Molar Ellipticity at 490nm

375 400 425 450 475 500 525 550

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Wavelength nm

.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.

Concentration= .307mM Premature GO ~ 20.9mg/ml

Experiment 2: Cu(II)(Gly)4 to premat-GO in anaerobic conditions

0.00 0.05 0.10 0.15 0.20 0.25-1000

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Change in Molar Ellipticity at 425 nm

0.8 : 1 (Cu : protein)0.8 : 1 (Cu : protein)Issues:Issues:• Signal to noise ratioSignal to noise ratio• Protein precipitationProtein precipitation

300 350 400 450 500 550 600 650 700 750 800-5000

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0 mol Cu: 1mol ApoGO .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

Mo

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Wavelength nm

Protein Concentration= .247mM Premature GO ~ 16.8mg/mL

Experiment 3: Cu(II)(Gly)4 complex to Premat-

GO with anaerobic conditions

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20

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Change in Molar Ellipticity at 425nm

425nm425nm

Peaks at a ratio ofPeaks at a ratio of.5 mol Cu(II): 1 mol Premat-GO.5 mol Cu(II): 1 mol Premat-GO

Issues:Issues:• Signal to noise ratioSignal to noise ratio• Incubation timeIncubation time• Temperature affecting kineticsTemperature affecting kinetics

375 400 425 450 475 500 525 550 575 600 625

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Concentration= .241mM Premature GO ~ 16.4 mg/mL

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Wavelength nm

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

Experiment 4: Determining Incubation Time

Incubation on ice (4 C)

At 25 C

Intermediate formationIntermediate formationin 20 minutesin 20 minutes

Intermediate Intermediate formationformationin 40 minutesin 40 minutes

300 400 500 600 700 800 900 1000

0.00

0.05

0.10

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Abs

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Wavelength nm

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

0.5 mol Cu(II) : 1 mol ApoGOConc. Protein = 11.3mg/mL

400 500 600 700 800 900

0.00

0.03

0.06

0.09

0.12

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0.18

0.21

Ab

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Wavelength nm

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

0.5 mol Cu(II) : 1 mol ApoGOConc. Protein = 17.7mg/mL

Issues:• O2 contamination

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 25C

To obtain quality data…• Reaction temperature• Max protein concentration• Maintain anaerobic

condition

AcknowledgementsAcknowledgements