Supplementary Information
Isotopic fractionation associated with [NiFe]- and [FeFe]-hydrogenases
Hui Yang1, Hasand Gandhi2, Adam J. Cornish1,†, James J. Moran3, Helen W. Kreuzer3,
Nathaniel E. Ostrom2*, and Eric L. Hegg1*
1Department
of Biochemistry & Molecular Biology, Michigan State University, East Lansing, MI 48824-1319
2Department
of Integrative Biology, Michigan State University, East Lansing, MI 48824
3Chemical
and Biological Signature Sciences, Pacific Northwest National Laboratory, PO Box 999, MSIN P7-50,
Richland, WA 99352
†Current
address: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205
*Correspondence to:
E. Hegg (EricHegg@msu.edu), Michigan State University, Department of Biochemistry &
Molecular Biology, 510 Biochemistry Building, East Lansing, MI 48824-1319;
N. Ostrom (ostromn@msu.edu), Michigan State University, Department of Integrative Biology,
236C Natural Science Building, East Lansing, MI 48824.
Figure S1. Isotopic composition of H2 produced by different hydrogenases as a function
of time. In each graph, the different symbols represent biological replicate samples S1,
S2, etc. CrFeFe, Chlamydomonas reinhardtii [FeFe]-hydrogenase; CpFeFe, Clostridium
pasteurianum [FeFe]-hydrogenase; SoFeFe, Shewanella oneidensis [FeFe]-hydrogenase; SoNiFe,
Shewanella oneidensis [NiFe]-hydrogenase; DfNiFe, Desulfovibrio fructosovorans [NiFe]hydrogenase.
Figure S2. Best fit lines to the Rayleigh equation1 for the H2 consumption reaction:
𝛼P/R ln (𝑓 ∙
1 + 𝑅R,0
1 + 𝑅R,0 𝑅R
) = ln (𝑓 ∙
∙
)
1 + 𝑅R
1 + 𝑅R 𝑅R,0
When plotted using the coordinates shown above, the slope of each line is the isotopic
fractionation factor (α). S1, S2, and S3 represent different biological replicates. CrFeFe,
Chlamydomonas reinhardtii [FeFe]-hydrogenase; CpFeFe, Clostridium pasteurianum [FeFe]hydrogenase; SoFeFe, Shewanella oneidensis [FeFe]-hydrogenase; SoNiFe, Shewanella
oneidensis [NiFe]-hydrogenase.
1
J.M. Hayes. An Introduction to Isotopic Calculations. (Woods Hole Oceanographic Institution, Woods
Hole, MA 02543) 2004, http://www.whoi.edu/cms/files/jhayes/2005/9/IsoCalcs30Sept04_5183.pdf.
Table S1. δ2H data for production of H2 by different hydrogenases.
Name
Time
(h)
H2a
(μmol/mL)
δ2H (‰)
Average
CrFeFe hydrogenase
Cr Sample 1
1
3
Cr Sample 2
Cr Sample 3
Cr Sample 4
Cr Sample 5
mean ± SD
1
2
3
1
3
1
2
3
4
5
1
9/30/2010
1.65
2.36
10/1/2010
2.09
2.59
2.95
11/22/2010
1.56
1.74
1.18
1.12
1.42
1.20
1.12
11/23/2010
1.89
-546.0
-584.3
-565.1
-603.5
-614.7
-621.1
-613.1
-543.2
-563.5
-567.7
-570.6
-523.7
-578.1
-579.6
-548.9
-553.4
-563.9
-548.9
-568.9 ± 25.7
CpFeFe hydrogenase
Cp Sample 1
Cp Sample 2
Cp Sample 3
Cp Sample 4
Cp Sample 5
2
3
4
1
2
1
2
1
2
1
2
6/9/2011
1.55
2.93
1.21
8/4/2011
1.72
3.16
2.14
2.50
8/5/2011
1.41
1.86
0.89
1.03
-450.4
-505.5
-540.1
-498.4
-514.2
-492.9
-495.1
-465.6
-471.0
-349.6
-445.8
-498.7
-506.3
-494.0
-468.3
-397.7
Cp Sample 6
Cp Sample 7
1
2
3
1
2
3
0.60
1.40
2.03
2/6/2012
0.16
0.16
0.30
-427.1
-461.3
-472.1
-518.7
-519.0
-520.9
mean ± SD
-453.5
-519.5
-476.9 ± 41.5
SoFeFe hydrogenase
9/30/2010
SoFe Sample
1b
SoFe Sample
2
SoFe Sample
3
1
0.35
-632.4
2
3
0.41
0.45
8/5/2011
-639.9
-639.1
-637.1
1
2.62
-645.5
-645.5
-588.67
-590.26
-601.28
-593.40
1
2
3
SoFe Sample
4
1
2
11/7/2012
0.55
0.56
0.68
0.55
0.57
-590.64
-594.52
mean ± SD
-592.58
-617.2 ± 28.1
SoNiFe hydrogenase
8/5/2011
SoNi Sample
1
SoNi Sample
2
SoNi Sample
3
3
0.78
-633.3
-633.3
3
0.81
-630.7
-630.7
1
2
3
SoNi Sample
4
1
2
mean ± SD
11/7/2012
0.32
0.34
0.39
0.32
0.34
-617.57
-620.19
-629.13
-621.32
-618.91
-622.30
-620.11
-626.6 ± 6.39
DfNiFe hydrogenase
Df Sample 1
Df Sample 2
Df Sample 3
Df Sample 4
Df Sample 5
Df Sample 6
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
4
7/21/2009
0.63
0.84
1.00
0.59
0.76
1.09
8/6/2009
0.55
0.81
0.95
0.53
0.80
0.89
11/23/2010
0.51
0.66
0.96
12/1/2010
0.90
1.01
1.13
1.25
-728.5
-735.1
-735.6
-730.4
-731.8
-736.6
-762.7
-757.6
-759.3
-763.1
-757.4
-751.7
-722
-733.6
-743.9
-737.3
-735.8
-737.8
-744.7
mean ± SD
-733.1
-732.9
-759.9
-757.4
-733.2
-738.9
-742.6 ± 12.7
TrNiFe hydrogenase
9/9/2009
TrNiFe
Sample 1
TrNiFe
Sample 2
mean ± SD
1
0.22
-723.69
2
3
4
0.25
0.28
0.31
-725.46
-724.32
-723.05
1
0.12
-725.13
2
3
4
0.14
0.16
0.18
-726.37
-726.56
-726.86
-724.13
-726.23
-725.18 ± 1.05
a
The H2 concentrations were calculated by comparing the peak heights of the samples
generated via GC-IRMS to those of the standards with known amounts of H2.
Concentrations have not been corrected for sampling loss (~1% per injection).
Table S2. Raw data from the H2 uptake reaction catalyzed by the CrFeFe, CpFeFe,
SoFeFe, and SoNiFe hydrogenases. In the table, f represents the fraction of substrate (H2)
remaining in the reaction as quantified by the isotope ratio mass spectrometer.
δD
-706.63
-699.90
-690.30
time (min)
15
230
411
f
0.858
0.816
0.713
CrFeFe Uptake
Sample 2
-702.13
-700.39
-691.75
44
244
424
0.848
0.832
0.723
CrFeFe Uptake
Sample 3
-706.24
-705.49
-697.35
90
257
437
0.858
0.826
0.710
CpFeFe Uptake
Sample 1
-717.32
-703.75
-700.43
19
213
357
0.829
0.739
0.690
CpFeFe Uptake
Sample 2
-714.75
-706.30
-701.55
21
225
370
0.829
0.713
0.674
CpFeFe Uptake
Sample 3
-713.09
-708.98
-696.17
35
199
373
0.829
0.752
0.658
SoFeFe Uptake
Sample 1
-700.16
-697.82
-690.86
10
198
355
0.845
0.781
0.716
SoFeFe Uptake
Sample 2
-699.60
-693.42
-691.27
26
211
368
0.845
0.774
0.706
SoFeFe Uptake
Sample 3
-701.74
-696.48
-690.30
40
224
380
0.855
0.765
0.700
CrFeFe Uptake
Sample 1
SoNiFe Uptake
Sample 1
-701.99
-696.13
-689.41
10
192
351
0.858
0.784
0.703
SoNiFe Uptake
Sample 2
-698.32
-693.16
-687.08
23
205
364
0.858
0.774
0.700
SoNiFe Uptake
Sample 3
-703.93
-696.74
-692.62
36
218
376
0.858
0.755
0.684
Calculation of δH2 after equilibration with H2O
The fractionation factor for the H2-H2O exchange is defined as
α = RH2O/RH2
where RH2O and RH2 are the isotope ratios for the H2O and H2, respectively.
The isotope ratio of H2O (RH2O) can be calculated using the known δ value of Michigan
tap water, where
δH2O = -60‰
RH2O = (δH2O/1000 + 1)*RH2O(standard)
The RH2O(standard) is known to be 155.76*10-6 for the H isotope, and RH2O is calculated to
be 1.46*10-6.
According to Horibe et al.[31], the equilibrium fractionation factor (α) for the H2-H2O
exchange is defined as
α = 1.0473 + 201036/T2 + 2.060*109/T4 + 0.180*1015/T6
where T is the temperature in Kelvin.
At 25 °C, the α value is calculated to be 3.83. Therefore:
RH2 = RH2O/α = 1.46*10-6/3.83 = 3.81*10-5
This R value gives rise to a δH2 value following equilibration with Michigan tap water to
be -755‰.