Copper Binding to Premature Galactose Oxidase: Biogenesis of the Tyr-Cys Cofactor Alta Howells Dooley Group Chemistry and Biochemistry

The Dooley Group Copper Metalloproteins NO 3 -  NO 2 -  NO  N 2 O  N 2 Nitrous Oxide Reductase RCH 2 NH 2 + O 2  RCHO + NH 3 + H 2 O 2 Amine Oxidase RCH 2 OH + O 2  RCHO + H 2 O 2 Galactose Oxidase

Why Study Metalloproteins? 1/3 of all proteins require metals for their function –Fe, Cu, Co, Zn Cytochrome c Oxidase PDB: 1OCC A perfect example…

Copper Proteins Produces reactive oxygen species H 2 O 2, Superoxide, Hydroxyl Genetic diseases Green house gases Biotechnology Galactose Oxidase RCH 2 OH + O 2  RCHO + H 2 O 2

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 RelationshipStructure How the Structure is Generated The pieces that make it a whole Without those pieces LOSS OF FUNCTION

H496 Y495 H581 Y272 C228 W290 H2OH2O Crystal Structure Essential Cofactors Cu Cu Tyr272-Cys228 Tyr272-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. Biochemistry, 2008, 47, 39 Whittaker, Mei M. JBC, 2003, 278, 22090

Previous Research Cu(II)SO 4 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

Experiment 2: Cu(II)(Gly) 4 to premat-GO in anaerobic conditions 0.8 : 1 (Cu : protein) Issues: Signal to noise ratio Signal to noise ratio Protein precipitation Protein precipitation

Experiment 3: Cu(II)(Gly) 4 complex to Premat- GO with anaerobic conditions425nm Peaks at a ratio of.5 mol Cu(II): 1 mol Premat-GO Issues: Signal to noise ratio Signal to noise ratio Incubation time Incubation time Temperature affecting kinetics Temperature affecting kinetics

Experiment 4: Determining Incubation Time Incubation on ice (4  C) At 25  C Intermediate formation in 20 minutes Intermediate formation in 40 minutes Issues: O 2 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 25  C To obtain quality data… Reaction temperature Max protein concentration Maintain anaerobic condition

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