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1
Supporting Information
Effect of distal histidines on hydrogen peroxide
activation by manganese reconstituted myoglobin
Yuan-Bo Cai, a Xiao-Han Li,
a Jing Jing,
a and Jun-Long, Zhang*
a
Table of Contents
1 Spectral changes of F43H MnIII
Mb in the reaction with 100 equiv. H2O2 in pH
7.4 ……………………………………………………………………………..…2
2. Kinetic data and spectral changes of MnIII
Mb in the reaction with H2O2 ………..3
3. Spectral changes of ABTS oxidation for MnIII
Mb at pH 7.4 ………………..….9
4. ESI(+)-MS of different mutants ………………………………………………..13
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
2
Spectral changes of F43H MnIII
Mb in the reaction with 100 equiv. H2O2 in pH
7.4
300 400 500 600 700
0.2
0.4
0.6
0.8
Ab
so
rba
nce
Wavelength (nm)
Figure S1. Spectral changes of F43H Mn
IIIMb in the reaction with 100 equiv. H2O2 in pH 7.4
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
3
Kinetic data and spectral changes of MnIII
Mb in the reaction with hydrogen
peroxide:
Conditions: the concentration was 29 μM for MnIII
Mb protein and 580 μM for H2O2, the reaction
temperature was 20 oC.
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Ab
so
rba
nce
Wavelength (nm)
(a)
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Ab
so
rba
nce
Wavelength (nm)
(b)
Figure S2 Spectral change of wild-type MnIII
Mb in the reaction with H2O2 at a) pH 7.4 and b) pH
0 100 200 300 400 500 600
0.4
0.5
0.6
0.7
0.8
0.9
Data: wtMn51_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 3.014E-6
R^2 = 0.99985
y0 0.85966 ±0.00077
A1 -0.47405 ±0.00095
t1 128.21647 ±0.74001
Ab
so
rba
nce
(4
13
nm
)
Time (s)
0 50 100 150 200 250
0.40
0.45
0.50
0.55
0.60
Data: Data7_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 2.6927E-6
R^2 = 0.99906
y0 0.58886 ±0.0008
A1 -0.21042 ±0.00102
t1 66.43045 ±0.92964
Abso
rba
nce
(4
13
nm
)
Time (s)
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4
5.1. Insets: trace for the formation of MnIV
=O Por and the exponetial fitting function, kobs=1/t1
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Abso
rba
nce
Wavelength (nm)
(a)
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ab
so
rba
nce
Wavelength (nm)
(b)
Figure S3 Spectral change of F43H MnIII
Mb mutant in the reaction with H2O2 at a) pH 7.4 and b)
pH 5.1. Insets: trace for the formation of MnIV
=O Por and the exponetial fitting function, kobs=1/t1
0 10 20 30 40 50 60
0.45
0.50
0.55
0.60
0.65
Data: Data1_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 3.5817E-6
R^2 = 0.99921
y0 0.64233 ±0.00127
A1 -0.29282 ±0.00388
t1 13.89786 ±0.34049A
bso
rba
nce
(4
13
nm
)
Time(s)
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
5
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ab
so
rba
nce
Wavelength (nm)
(a)
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
Ab
so
rba
nce
Wavelength (nm)
(b)
Figure S4. Spectral change of L29H/F43H MnIII
Mb mutant in the reaction with H2O2 at a) pH 7.4
and b) pH 5.1. Insets: trace for the formation of MnIV
=O Por and the exponetial fitting function,
kobs=1/t1
0 20 40 60 80 100 120
0.42
0.44
0.46
0.48
0.50
0.52
0.54
Data: Data1_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 2.6612E-6
R^2 = 0.99754
y0 0.53129 ±0.00096
A1 -0.13369 ±0.00177
t1 27.59761 ±0.87832
Ab
so
rba
nce
(4
13
nm
)
Time (s)
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
6
300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
1.0
Ab
so
rba
nce
Wavelength (nm)
Figure S5 Spectral change of L29H MnIII
Mb mutant in the reaction with H2O2 at pH 7.4.
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Abso
rba
nce
Wavelength (nm)
Figure S6. Spectral change of H64F MnIII
Mb mutant in the reaction with H2O2 at pH 7.4.
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
7
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Abso
rba
nce
Wavelength (nm)
Figure S7. Spectral change of F43H/H64F MnIII
Mb mutant in the reaction with H2O2 at pH 7.4.
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Ab
so
rba
nce
Wavelength (nm)
Figure S8. Spectral change of L29H/H64F MnIII
Mb mutant in the reaction with H2O2 at pH 7.4.
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
8
300 400 500 600 700 800
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Abso
rba
nce
Wavelength (nm)
Figure S9. Spectral change of L29H/F43H/H64F MnIII
Mb mutant in the reaction with H2O2 at pH
7.4.
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
9
Spectral changes of ABTS oxidation for MnIII
Mb at pH 7.4:
Conditions: the concentration was 29 μM for protein and 580 μM for ABTS and H2O2, the
reaction temperature was 20 oC.
500 600 700 800 900
0.0
0.2
0.4
0.6
0.8
Ab
so
rba
nce
Wavelength (nm)
Figure S10. Spectral change of ABTS oxidation at pH 7.4 for wild-type MnIII
Mb.
500 600 700 800 900
0.0
0.5
1.0
1.5
2.0
Ab
so
rba
nce
Wavelength (nm)
Figure S11. Spectral change of ABTS oxidation at pH 7.4 for F43H MnIII
Mb mutant.
0 100 200 300 400 500
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Data: FHMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00003
R^2 = 0.99948
y0 0.77526 ±0.00331
A1 -0.79447 ±0.00379
t1 124.79738 ±1.77815
Ab
so
rba
nce
(7
34
nm
)
Time (s)
0 200 400 600 800 1000
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Data: wtMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00002
R^2 = 0.98296
y0 0.14053 ±0.00184
A1 -0.12746 ±0.003
t1 193.66956 ±11.51
Ab
so
rba
nce
(7
34
nm
)
Time (s)
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
10
500 600 700 800 900
0.0
0.1
0.2
0.3
0.4
Ab
so
rba
nce
Wavelength (nm)
Figure S12. Spectral change of ABTS oxidation at pH 7.4 for H64F MnIII
Mb mutant.
500 600 700 800 900
0.0
0.2
0.4
0.6
0.8
1.0
Abso
rba
nce
Wavelength (nm)
Figure S13. Spectral change of ABTS oxidation at pH 7.4 for L29H MnIII
Mb mutant.
0 2000 4000 6000 8000
0.01
0.02
0.03
0.04
0.05
0.06
Data: FHMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 2.8809E-6
R^2 = 0.98791
y0 0.0715 ±0.00214
A1 -0.05888 ±0.002
t1 4479.71147 ±298.31802
Ab
so
rba
nce
(7
34
nm
)
Time (s)
0 500 1000 1500 2000 2500 3000 3500 4000
0.05
0.10
0.15
0.20
0.25
Data: LHMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00001
R^2 = 0.99716
y0 0.262 ±0.0026
A1 -0.22882 ±0.00234
t1 1404.60453 ±37.41793
Abso
rba
nce
(7
34
nm
)
Time (s)
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
11
500 600 700 800 900
0.0
0.2
0.4
0.6
Ab
so
rba
nce
Wavelength (nm)
Figure S14. Spectral change of ABTS oxidation at pH 7.4 for L29H/H64F MnIII
Mb mutant.
500 600 700 800 900
0.0
0.5
1.0
1.5
2.0
Ab
so
rba
nce
Wavelength (nm)
Figure S15. Spectral change of ABTS oxidation at pH 7.4 for L29H/F43H MnIII
Mb mutant.
0 50 100 150 200 250
0.020
0.025
0.030
0.035
0.040
0.045
0.050
Data: LHHFMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 2.522E-7
R^2 = 0.99744
y0 0.05397 ±0.00065
A1 -0.04017 ±0.00048
t1 103.65178 ±4.40975
Ab
so
rba
nce
(7
34
nm
)
Time (s)
0 100 200 300 400 500 600 700 800
0.0
0.2
0.4
0.6
0.8
Data: HFMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00003
R^2 = 0.9996
y0 0.9759 ±0.00613
A1 -0.96139 ±0.00518
t1 353.27722 ±5.21806
Ab
so
rba
nce
(7
34
nm
)
Time (s)
Electronic Supplementary Material (ESI) for MetallomicsThis journal is © The Royal Society of Chemistry 2013
12
500 600 700 800 900
0.0
0.1
0.2
0.3
0.4
Ab
so
rba
nce
Wavelength (nm)
Figure S16. Spectral change of ABTS oxidation at pH 7.4 for F43H/H64F MnIII
Mb mutant.
500 600 700 800 900
0.0
0.3
0.6
0.9
Ab
so
rba
nce
Wavelength (nm)
Figure S17. Spectral change of ABTS oxidation at pH 7.4 for L29H/F43H/H64F MnIII
Mb mutant.
0 2000 4000 6000 8000
0.02
0.04
0.06
0.08
0.10
Data: FHMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00002
R^2 = 0.97382
y0 0.09387 ±0.00204
A1 -0.07863 ±0.00192
t1 2347.09636 ±155.02613
Ab
so
rba
nce
(7
34
nm
)
Time (s)
0 500 1000 1500 2000 2500 3000 3500
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Data: LFHMn74_B
Model: ExpDec1
Equation: y = A1*exp(-x/t1) + y0
Weighting:
y No weighting
Chi^2/DoF = 0.00002
R^2 = 0.99835
y0 0.42143 ±0.0064
A1 -0.40163 ±0.0059
t1 2057.40866 ±59.60735
Ab
so
rba
nce
(7
34
nm
)
Time (s)
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13
ESI(+)-MS of different mutants:
Wild-type Mb:
L29H Mb:
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F43H Mb:
H64F Mb:
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L29H/F43H Mb:
L29H/H64F Mb:
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F43H/H64F Mb:
L29H/F43H/H64F Mb:
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