Supplementary Information
The Catalase Activity of Diiron Adenine Deaminase
Siddhesh S. Kamat, Gregory P. Holmes-Hampton, Ashima Bagaria, Desigan Kumaran, Shane E.
Tichy, Tarun Gheyi, Xiaojing Zheng, Kevin Bain, Chris Groshong, Spencer Emtage, J. Michael
Sauder, Stephen K. Burley, Subramanyam Swaminathan, Paul A. Lindahl, and Frank M. Raushel
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Figure S1: Intact mass analysis of Cac0887 using LC-ESI-MS showing a heterogeneous mixture
due to post-translational modification. The expected mass was 64640 Da.
2
Figure S2: Identification of His105 and His219 oxygenation sites in Cac0887. MS2 spectra of
the tryptic peptides 94GTTIIVDPHEIANVCGMDGIK115 (A) and 194IVDGHAPSVR203 (B).
Identified oxygenation sites are colored in red.
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Figure S3: Intact mass analysis of Cac0887 using LC-ESI-MS showing an improved peak after
using the iron chelator during fermentation.
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O2 evolved (µM)
100
80
60
40
20
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
[FeII/FeII]-ADE (µM)
Figure S4: Total concentration of O2 evolved following addition of 250 µM H2O2 to varying
amounts of [FeII/FeII]-ADEec at pH 8.0, 30 oC. The ratio of O2 evolved relative to the initial
enzyme concentration is 64.
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Relative catalase activity (A/Ao)
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
[Fe2+]/[apo-ADEec]
Figure S5: Catalase activity following addition of FeII to apo-ADEec. The maximal activity is
obtained at two enzyme equivalents of iron per monomer of ADEec.
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absorption [%]
0.10
0.00
-0.10
-0.20
-10
-5
0
5
velocity [mm/s]
10
Figure S6: Mössbauer spectrum of 205 µM Fe/Fe ADE with 15 equivalents of HOCl (~ 3 mM).
Spectrum was collected at 5K with a 700G field applied parallel to the radiation.
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Figure S7: EPR spectrum of 200 µM Mn/Fe ADE. EPR parameters: temperature, 10 K;
microwave 9.46 GHz, 0.02 mW.
8
0
A
-1
absorption [%]
-2
0.0
B
-0.5
-1.0
0.0
C
-0.5
-1.0
0.0
D
-0.5
-1.0
-4
-2
0
2
velocity [mm/s]
4
Figure S8: Mössbauer spectra of (A) 200 µM Fe/Fe-ADE. (B) 200 µM Mn/Fe-ADE. (C) 200
µM Mn/Fe-ADE with 1equivalent of H2O2. (D) 200 µM Mn/Fe-ADE with 10 equivalents of
H2O2. The spectra were collected at 5 K, 400 G (A-C) or 700G (D) magnetic field applied
parallel to the radiation. The data can be fit with a single quadrupole doublet with δ= 1.39
mm/sec, ΔEq= 3.27 mm/sec.
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