Supporting Information Effect of distal histidines on ... · 1 Supporting Information Effect of distal histidines on hydrogen peroxide activation by manganese reconstituted myoglobin

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


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


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)


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


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)


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


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)


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



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


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



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.


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


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


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)


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


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


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)


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


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


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)


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


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


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)


13

ESI(+)-MS of different mutants:

Wild-type Mb:

L29H Mb:


14

F43H Mb:

H64F Mb:


15

L29H/F43H Mb:

L29H/H64F Mb:


16

F43H/H64F Mb:

L29H/F43H/H64F Mb:


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Supporting Information Effect of distal histidines on ... · 1 Supporting Information Effect of distal histidines on hydrogen peroxide activation by manganese reconstituted myoglobin