Effects of Specific Mutations on Cupredoxin Protein Amicyanin Structure and Electron Transfer
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1) The document summarizes research on the effects of specific mutations on the copper protein amicyanin. 2) Mutation of tryptophan 45 decreases the structural stability of amicyanin, regardless of whether copper is present or not, by disrupting an interior hydrogen bond. 3) Mutation of histidine 95 to glycine decreases the rate of electron transfer between amicyanin and methylamine dehydrogenase, but adding an imidazole ligand can partially restore the electron transfer rate by substituting for the missing histidine side chain.
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Effects of Specific Mutations on Cupredoxin Protein Amicyanin Structure and Electron Transfer
- 1. Effects of Specific Mutations on the Structure and Function of the Copper Protein Amicyanin, a Biological Electron Transfer Mediator Brian Dow Davidson Lab Burnett School of Biomedical Sciences University of Central Florida
- 2. Cupredoxin Proteins • Type 1 Copper site Redox Potential (mV) – 2 Histidine, 1 Cysteine, 1 Methionine ligands 800 700 600 500 400 300 200 100 0 • Bacteria, fungi, plants • Mediate electron transfer 680 370 265 184 270 294
- 5. Cu(II) Amicyanin UV-Visible Absorption Spectra *Cu(I) Amicyanin does not have visible absorption
- 7. Trp45 Fluorescence Apoamicyanin (copper removed) Properties of Paracoccus denitrificans amicyanin Mazhar Husain, Victor L. Davidson, and Alan J. Smith Biochemistry 1986 25 (9), 2 431-2436 Amicyanin
- 8. Does Trp45 reciprocate electronic communication to the Copper in Amicyanin? ??? Trp45
- 10. Electronic Properties UV-Visible Absorption & Molar Absorptivity 2.0 Resonance Raman W45Y WT WT W45Y 1.5 Intensity Absorbance 5000 1.0 3000 0.5 0.0 4000 300 400 500 Wavelength (nm) 600 700 2000 200 400 600 Raman shift [cm-1] 800 1000
- 11. Kinetic Parameters of W45Y Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex Copper TTQ Heme
- 12. Kinetic Parameters of W45Y MADH-Amicyanin MADHAmicyanin-Cytochrome c551i WT W45Y WT W45Y Kcat 61 ± 2 s-1 66 ± 3 s-1 18 ± 2 s-1 13± 1 s-1 Km 2.3 ± 0.3 μM 3.2 ± 0.5 μM 1.3 ± 0.2 μM 1.0± 0.7 μM
- 14. Redox and pH Dependent Conformational Change His95 Met98 Oxidized Amicyanin Reduced Amicyanin +II +I Cys92 His53
- 15. pH-dependent Redox Potential +0.5 pKa in W45Y
- 16. Additional H-bond Met51 His95 His53 Met98 Cu(I) Cys92 3.5Å Reduced W45Y Amicyanin Reduced Wild Type Amicyanin *W45Y Cu(I) is less ordered
- 17. • Despite fluorescence quenching, there is no effect of Trp45 on copper • Possibility of decreased copper occupancy – Investigate stability of the copper site
- 18. Optical Melt 1.0 0.5 0.6 0.4 0.4 W45Y 53.9±0.1° C WT 66.4 ±0.2° C 0.2 0.0 30 Absorbance A595 0.8 0.3 0.2 0.1 40 50 60 70 80 0.0 300 Temperature (°C) When copper site is disrupted, absorbance at 595 nm is lost 400 500 600 Wavelength (nm) 700
- 19. Cu(I) Chelation Bathocuproine disulfonate (BCS) Absorbance 0.15 0.10 0.05 0.00 300 400 500 Wavelength (nm) Cu(I) amicyanin has no visible absorbance. Cu(I)-BCS complex 600 absorbs light at 470 nm.
- 20. Cu(I) Chelation Normalized A430 1.0 0.8 W45Y 1.5 s-1 WT 0.6 s-1 0.6 0.4 0.2 0.0 0 2 4 Time (min) 6
- 21. W45Y Copper Binding • Optical Melt shows 10° C decrease in Cu(II) site stability • Chelation of Cu(I) by BCS is 2.5x faster than WT • Is copper bound less tightly, or is structural stability affected? • Circular Dichroism to better quantitate thermal stability of oxidized, reduced, and apo (copper removed) amicyanin
- 22. Structural Thermal Stability Wild Type Amicyanin W45Y Amicyanin
- 23. Circular Dichroism Oxidized Reduced Apo WT Tm=60 ±1° C Tm=53 ±2° C Tm=55 ±1° C Tm=46 ±2° C Tm=50 ±1° C Tm=34 ±2° C W45Y
- 24. Lost Hydrogen bond Copper Tyr90 2.6Å W45Y Wild Type Amicyanin W45Y Amicyanin
- 25. W45Y Summary • Trp45 does not reciprocate electronic communication with the copper • W45Y introduces new H-bond; increases the pH-dependent redox potential; no affect on intrinsic redox potential • W45Y mutation decreases stability by disruption of interior H-bond • Stability is affected regardless of copper presence
- 26. Mediation of Biological Electron Transfer by External Chemical Ligands His95 His53 Met98 Cys92
- 27. H95G: Type 1 Copper Site Maintained WT WT H95G H95G H95G-Imidazole Extinction Coefficient 4.6mM-1cm-1 -1 4.6mM-1cm 5.6mM-1-1cm-1 5.6mM cm-1 4.7mM-1cm-1 Redox Potential 256 ±1 mV 256 ±1 mV 252 ±1 mV 252 ±1 mV 247±1 mV
- 28. Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex Copper TTQ Heme
- 29. Predicted Pathway of Electron Transfer from MADH to Amicyanin His53 His95 Cu(II) TTQ Kinetic and Thermodynamic Analysis of a Physiologic Intermolecular ElectronTransfer Reaction between Methylamine Dehydrogenase and Amicyanin Harold B. Brooks and Victor L. Davidson Biochemistry 1994 33 (19), 5696-5701 Pro94 Carbonyl Met98 Cys92
- 30. Electron Transfer Simulation from MADH to H95G Amicyanin His53 Cu(II) TTQ MADH-H95G Simulation Pro94 Gly95 Carbonyl Met98 Cys92
- 31. Electron Transfer Simulation from MADH to H95G Amicyanin via Imidazole His53 Imidazole Cu(II) TTQ MADH-Imidazole-H95G Simulation Pro94 Carbonyl Met98 Cys92
- 32. Electron Transfer Parameters His53 His53 His53 His95 Imidazole Cu(II)Met98 TTQ Pro94 Cys92 Carbonyl Cu(II) TTQ Met98 Pro94 Gly95 Cys92 Carbonyl MADH-H95G Simulation Met98 Cu(II) TTQ Pro94 Gly95 Cys92 Carbonyl MADH-Imidazole-H95G Simulation MADH-WT Amicyanin KET Kd Distance MADH-H95G Amicyanin MADH-Imidazole-H95G 10 s-1 6 s-1 ±0.4 8 s-1 ±1.0 4.5μM ±0.5 9.0μM ±1.6 4.5 μM ±1.6 7.9Å 12.0Å ±0.5 9.7Å ±0.2
- 33. H95G ET Summary • Preliminary data suggest that imidazole can substitute for the His95 side chain • ET is partially restored by the addition of imidazole as a ligand • A new way to probe ET mechanisms and applications – Amino acids, ligand wires, etc.
- 34. Acknowledgements • UCF College of Medicine Biomedical Sciences – Victor Davidson – Sooim Shin – Heather Williamson – Yu Tang – Esha Sehanobish • UCF Physics – Suren Tatulian – Alfons Schulte – Jason Matos • Argonne National Laboratories • Seoul National University of Science & Technology – Narayanasami Sukumar – Moonsung Choi
Editor's Notes
- T1 copper site proteins, system present in all organisms, ET mediators
- Show spectra,
- WT pKa 7.5W45Y pKa 8.0
- Hydrogen bond stabilizing flipped out form, stabilizing the reduced form
- WT pKa 7.5W45Y pKa 8.0
- LABEL RESIDUESDisordered copper, possible decreased occupancy/higher mobility
- Temperature at which T1 site is disrupted~10° C lowerNo changes in T1 site according to crystalIs affinity for copper lowered?
- Add directional arrowAdd text “CuI has no visible absorbance in Ami, but it does w/ BCS”
- Measure relative affinity for Cu(I) by BCS chelationCu(I) is chelated faster from W45YCu(I) held less tightly, as reflected in crystalAffinity for copper lost, or overall less stable?
- Is copper bound less tightly, or is structural stability affected?AzurinKd is 25fM (determined by ITC)
- Look at figure from paperDetermine if Cu binding is weaker or protein overall is less stableTm determined by CD of β-turnsLower thermal stability in Ox., Red., & ApoChange in copper affinity is due to overall lower stability
- LABEL RESIDUES
- ADD stability is affected regardless of presence or absence of copper
- Label residues
- Add referenceAdd triple complexpiture beforeHighlight entire electron pathway
- ADD Em, spectrum are the sameEstablsh that T1 site appears to be maintainedRemove lambda, and HAB
- Prelim data sugg that imi ligand can sub for his side chainAnother way to probe mech and might have et mediating applications