effects of specific mutations on the structure and function of the copper protein amicyanin, a...
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Effects of Specific Mutations on the Structure and Function of the Copper Protein Amicyanin, a
Biological Electron Transfer Mediator
Brian DowDavidson Lab
Burnett School of Biomedical SciencesUniversity of Central Florida
Cupredoxin Proteins• Type 1 Copper site– 2 Histidine, 1 Cysteine, 1 Methionine ligands
• Bacteria, fungi, plants• Mediate electron transfer
Stellacyanin Azurin Pseudoazurin Amicyanin Plastocyanin Rusticyanin0
100
200
300
400
500
600
700
800
184
265 270 294370
680
Redo
x Po
tenti
al (m
V)
Overall Amicyanin Structure
Copper
Amicyanin Copper LigandsHis95
Met98
His53
Cys92
Copper
Cu(II)
Cu(II) Amicyanin UV-Visible Absorption Spectra
*Cu(I) Amicyanin does not have visible absorption
Trp45
Copper
10.1 Å
Trp45 Fluorescence
Properties of Paracoccus denitrificans amicyaninMazhar Husain, Victor L. Davidson, and Alan J. SmithBiochemistry 1986 25 (9), 2431-2436
Apoamicyanin(copper removed)
Amicyanin
Does Trp45 reciprocate electronic communication to the Copper in Amicyanin?
???
Trp45
W45Y Amicyanin
Electronic PropertiesUV-Visible Absorption & Molar
Absorptivity Resonance Raman
200 400 600 800 10002000
3000
4000
5000W45YWT
Raman shift [cm-1]
Inte
nsity
300 400 500 600 7000.0
0.5
1.0
1.5
2.0 WTW45Y
Wavelength (nm)
Abs
orba
nce
Kinetic Parameters of W45Y Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex
TTQCopper
Heme
Kinetic Parameters of W45YMADH-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
pH-dependent Redox Potential
Redox and pH Dependent Conformational Change
Met98His53
His95
Cys92
+II
+I
Oxidized AmicyaninReduced Amicyanin
pH-dependent Redox Potential
+0.5 pKa in W45Y
Additional H-bond
3.5Å*W45Y Cu(I) is less ordered
Reduced W45Y AmicyaninReduced Wild Type Amicyanin
Cu(I)
Met51
Met98
Cys92
His53
His95
• Despite fluorescence quenching, there is no effect of Trp45 on copper
• Possibility of decreased copper occupancy– Investigate stability of the copper site
Optical Melt
30 40 50 60 70 800.0
0.2
0.4
0.6
0.8
1.0
Temperature (°C)
A 595
When copper site is disrupted, absorbance at 595 nm is lost
300 400 500 600 7000.0
0.1
0.2
0.3
0.4
0.5
Wavelength (nm)A
bsor
banc
e
WT66.4 ±0.2° C
W45Y53.9±0.1° C
Cu(I) Chelation
300 400 500 6000.00
0.05
0.10
0.15
Wavelength (nm)
Abs
orba
nce
Cu(I) amicyanin has no visible absorbance. Cu(I)-BCS complex absorbs light at 470 nm.
Bathocuproine disulfonate (BCS)
Cu(I) Chelation
0 2 4 60.0
0.2
0.4
0.6
0.8
1.0
Time (min)
Nor
mal
ized
A43
0 W45Y1.5 s-1
WT0.6 s-1
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
Structural Thermal StabilityWild Type AmicyaninW45Y Amicyanin
Circular Dichroism
WT
W45Y
Oxidized Reduced Apo
Tm=60 ±1° C Tm=53 ±2° C Tm=55 ±1° C
Tm=46 ±2° C Tm=50 ±1° C Tm=34 ±2° C
Lost Hydrogen bond
2.6Å
Wild Type AmicyaninW45Y Amicyanin
Tyr90 W45Y
Copper
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
Mediation of Biological Electron Transfer by External Chemical Ligands
His95
Cys92Met98
His53
WT H95G H95G-Imidazole
Extinction Coefficient
4.6mM-1cm-1 5.6mM-1cm-1 4.7mM-1cm-1
Redox Potential
256 ±1 mV 252 ±1 mV 247±1 mV
WT H95G
Extinction Coefficient
4.6mM-1cm-1 5.6mM-1cm-1
Redox Potential
256 ±1 mV 252 ±1 mV
H95G: Type 1 Copper Site Maintained
Methylamine Dehydrogenase Amicyanin Cytochrome c551i Complex
TTQCopper
Heme
Predicted Pathway of Electron Transfer from MADH to Amicyanin
Kinetic and Thermodynamic Analysis of a Physiologic Intermolecular Electron-Transfer Reaction between Methylamine Dehydrogenase and AmicyaninHarold B. Brooks and Victor L. DavidsonBiochemistry 1994 33 (19), 5696-5701
TTQPro94
Carbonyl
His95
His53
Met98
Cys92
Cu(II)
Electron Transfer Simulation from MADH to H95G Amicyanin
TTQPro94
Carbonyl Gly95
His53
Met98
Cys92
Cu(II)
MADH-H95G Simulation
Electron Transfer Simulation from MADH to H95G Amicyanin via Imidazole
TTQPro94
Carbonyl
Imidazole
His53
Met98
Cys92
Cu(II)
MADH-Imidazole-H95G Simulation
MADH-WT Amicyanin
MADH-H95G Amicyanin MADH-Imidazole-H95G
KET10 s-1 6 s-1 ±0.4 8 s-1 ±1.0
Kd 4.5μM ±0.5 9.0μM ±1.6 4.5 μM ±1.6Distance 7.9Å 12.0Å ±0.5 9.7Å ±0.2
MADH-WT Amicyanin
MADH-H95G Amicyanin
KET 10 s-1 6 s-1 ±0.4
Kd 4.5μM ±0.5 9.0μM ±1.6Distance 7.9Å 12.0Å ±0.5
Electron Transfer Parameters
TTQ Pro94 Carbonyl
His95
His53
Met98
Cys92
Cu(II)
TTQPro94
CarbonylGly95
His53
Met98
Cys92
Cu(II)
TTQ
Imidazole
Pro94 Carbonyl
Gly95
His53
Met98
Cys92
Cu(II)
MADH-H95G Simulation MADH-Imidazole-H95G Simulation
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.
Acknowledgements• UCF College of Medicine
Biomedical Sciences– Victor Davidson– Sooim Shin– Heather Williamson– Yu Tang– Esha Sehanobish
• Argonne National Laboratories
– Narayanasami Sukumar
• UCF Physics– Suren Tatulian– Alfons Schulte– Jason Matos
• Seoul National University of Science & Technology
– Moonsung Choi