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