of 11
S1
Supporting Information
Nitric Oxide Modulates Endonuclease III Redox Activ
ity
by a 800 mV Negative Shift upon [Fe
4
S
4
] Cluster Nitrosylation
Levi A. Ekanger,
1
Paul H. Oyala,
1
Annie Moradian,
2
Michael J. Sweredoski,
2
and Jacqueline K. Barton
1
*
Contribution from the
1
Division of Chemistry and Chemical Engineering and
the
2
Proteome Exploration Laboratory, Beckman Institute,
California Institute of Technology, Pasadena, CA 91
125
*E)mail: jkbarton@caltech.edu
Page
Contents
S1
Table of Contents
S2
UV–Vis Spectroscopy of Nitrosylation with and
without dsDNA
S2–S3
CW and Electron)Spin Echo EPR Spectroscopy
S4
Representative UV–Vis Spectra of Deoxymyoglobi
n Experiments
S4
UV–Vis Spectroscopy of Nitrosylation using
14/15
NO
S4–S5
HYSCORE Simulations
S6)S8
Q)band HYSCORE Spectroscopy
S9
Trypsin)Digested Native and Nitrosylated EndoI
II
S10
Representative Gels from Electrophoretic Mobi
lity Shift Assays
S11
Wide)Potential Sweep on DNA)Modified Gold Ele
ctrodes
Ekanger et al.
Supporting Information
S2
UV–Vis Spectroscopy of Nitrosylation with and witho
ut dsDNA
Figure S1.
UV–vis spectra of native (black) and nitrosylated E
ndoIII prepared in the absence
(solid red) or presence (dashed red) of dsDNA (Endo
/dsDNA ratio of 1:10).
CW EPR Spectroscopy
Figure S2.
Continuous)wave EPR spectrum of native EndoIII (20
μM by cluster) prior to
nitrosylation, demonstrating a diamagnetic spectrum
consistent with the presence of [Fe
4
S
4
]
2+
.
Ekanger et al.
Supporting Information
S3
Figure S3.
(A) Continuous)wave EPR spectra and (B) single)inte
gration EPR spectra of EndoIII
nitrosylation before (red) and after (blue) incubat
ion with dithionite in 20 mM phosphates, 150
mM NaCl, 0.5 mM EDTA, 10% glycerol, pH 7.5. Acquisi
tion parameters included a temperature
of 60 K and microwave power of 204 μW.
Figure S4.
(A)
Continuous)wave EPR spectra and of EndoIII nitrosyl
ation before (black, top
trace) and after (black, bottom trace) incubation w
ith dithionite in 20 mM phosphates, 150 mM
NaCl, 0.5 mM EDTA, 10% glycerol, pH 7.5 with simula
tions of [(Cys)
2
Fe(NO)
2
]
(red, top trace)
and [(μ)Cys)
2
Fe
2
(NO)
4
]
)
(red, bottom trace) species using parameters in Ta
ble 1 of the main text.
Arrows indicate the field positions at which X)band
HYSCORE spectra were acquired.
Acquisition parameters included a temperature of 60
K and microwave power of 204 μW. (B)
Pseudomodulated Q)band electron)spin echo (ESE) det
ected EPR spectra of EndoIII
nitrosylation before (black, top trace) and after (
black, bottom trace) incubation with dithionite in
the same buffer as detailed above with simulations
in red using parameters in Table 1 of the main
text. Arrows indicate the field positions at which
Q)band HYSCORE spectra were acquired.
Acquisition parameters: temperature = 20 K; microwa
ve frequency = 34.005 GHz; MW pulse
lengths π/2, π = 12, 24 ns; τ = 320 ns; shot repeti
tion time (srt) = 1 ms.
Ekanger et al.
Supporting Information
S4
Representative UV–Vis Spectra of Deoxymyoglobin Exp
eriments
Figure S5.
UV–vis spectra of conversions from (A) equine heart
metmyoglobin (dashed) to
deoxymyoglobin (solid) by treatment with sodium dit
hionite and (B) deoxymyoglobin to
nitrosomyoglobin by treatment with
15
NO (solid) or a
14
NO standard (dashed).
UV–Vis Spectroscopy of Nitrosylation using
14/15
NO
Figure S6.
UV–vis spectra of native EndoIII (black),
14
NO)nitrosylated EndoIII (red), and
15
NO)
nitrosylated EndoIII (dashed red).
HYSCORE Simulations
All EPR spectra (CW, HYSCORE) were simulated using
the EasySpin simulation toolbox
(version 5.2.16) with Matlab 2016b using the follow
ing Hamiltonian:


 












∙  ∙ 

∙  ∙ 
(1)
In this expression, the first term corresponds to t
he electron Zeeman interaction term
where


is the Bohr magneton,
g
is the electron spin g)value matrix with principle
components
Ekanger et al.
Supporting Information
S5
g
= [
g
xx
g
yy
g
zz
], and
is the electron spin operator; the second term cor
responds to the nuclear
Zeeman interaction term where


is the nuclear magneton,

is the characteristic nuclear g)
value for each nucleus (e.g.
1
H,
1
H,
31
P) and

is the nuclear spin operator; the third term
corresponds to the electron)nuclear hyperfine term,
where

is the hyperfine coupling tensor
with principle components

= [A
xx
A
yy
A
zz
]; and for nuclei with
 ≥ 1
, the final term
corresponds to the nuclear quadrupole (NQI) term wh
ich arises from the interaction of the
nuclear quadrupole moment with the local electric f
ield gradient (efg) at the nucleus, where

is
the
quadrupole coupling tensor. In the principle axis s
ystem (PAS),

is traceless and
parametrized by the quadrupole coupling constant


/
and the electric field gradient (efg)
asymmetry parameter

such that:
  


0 0
0 
!!
0
0 0 
""
# 
$
%
&'/(
)*(*,-)
/
−(1 − )
0
0
0
−(1 ) 0
0
0
2
2
(2)
where
$
%
&'
(
 2
(
2 − 1
)

""
and
 
3
44
,3
55
3
66
. The asymmetry parameter may have values
between 0 and 1, with 0 corresponding to an electri
c field gradient with axial symmetry and 1
corresponding to a fully rhombic efg.
The orientations between the hyperfine and NQI tens
or principle axis systems and the g)
matrix reference frame are defined by the Euler ang
les (α, β, γ).In general, the HYSCORE
spectrum for a given nucleus with spin

= ½ (
1
H) coupled to the
S
= ½ electron spin exhibits a
pair of peaks at frequencies
7
±
 9
:

± 7

9
(4)
Where
7

is the nuclear Larmor frequency and
;
is the hyperfine coupling. For nuclei
with
 ≥ 1
(
14
N,
2
H), an additional splitting of the
7
±
manifolds is produced by the nuclear
quadrupole interaction (P)
ν
±,>
?
 9 ν
@
±
AB(>
?
,-)

9
(5)
In HYSCORE spectra, these signals manifest as cross
)peaks or ridges in the 2)D
frequency spectrum which are generally symmetric ab
out the diagonal of a given quadrant. This
technique allows hyperfine levels corresponding to
the same electron)nuclear submanifold to be
differentiated, as well as separating features from
hyperfine couplings in the weak)coupling
regime (
|
;
|
< 2
|
7
*
|
) in the (+,)) quadrant from those in the strong c
oupling regime (
|
;
|
>
2
|
7
*
|
) in the (),)) quadrant. The (),)) and (+,)) quadra
nts of these frequency spectra are
symmetric to the (+,+) and (),+) quadrants, thus ty
pically only two of the quadrants are typically
displayed in literature. For systems with appreciab
le hyperfine anisotropy in frozen solutions or
solids, HYSCORE spectra typically do not exhibit sh
arp cross peaks, but show ridges that
represent the sum of cross peaks from selected orie
ntations at the magnetic field position at
Ekanger et al.
Supporting Information
S6
which the spectrum is collected. The length and cur
vature of these correlation ridges allow for
the separation and estimation of the magnitude of t
he isotropic and dipolar components of the
hyperfine tensor.
Q-Band HYSCORE Spectroscopy
Figure S7.
Q)band HYSCORE spectra and simulations of
14
N/
15
N hyperfine couplings of
[(Cys)
2
Fe(NO)
2
]
generated with (a)
14
NO (b)
15
NO and [(μ)Cys)
2
Fe
2
(NO)
4
]
generated with (c)
14
NO and (d)
15
NO. Top panels show the experimental spectra, with
intensities indicated by the
color map ranging from blue to red in order of incr
easing intensity. The bottom panels reproduce
Ekanger et al.
Supporting Information
S7
the experimental data in grey and overlay simulatio
ns from a relatively strongly coupled
14/15
N
nucleus (blue, denoted N
1
) and a relatively weakly coupled
14/15
N nucleus (red, denoted N
2
).
Specific simulation parameters are detailed in Tabl
e 1. Acquisition parameters: temperature = 20
K; microwave frequency = 33.986 GHz (a,b), 34.000 G
Hz (c,d); magnetic field = 1193.6 mT (g
= 2.034) (a,b), 1209 mT (g = 2.009) (c,d), MW puls
e lengths π/2, π = 12, 24 ns; τ = 100 ns; T
1
=
T
2
= 100 ns; WT
1
= WT
2
= 16 ns; shot repetition time (srt) = 1 ms.
Figure S8.
Q)band HYSCORE spectra and simulations of
14
N hyperfine couplings of
[(Cys)
2
Fe(NO)
2
]
generated with
14
NO. For each spectrum, the top panels show the expe
rimental
spectra, with intensities indicated by the color ma
p ranging from blue to red in order of
increasing intensity. The bottom panels reproduce t
he experimental data in grey and overlay
simulations from a relatively strongly coupled
14
N nucleus (blue, denoted N1) and a relatively
weakly coupled
14/15
N nucleus (red, denoted N2). Specific simulation pa
rameters are detailed in
Table 1 of the main text. Acquisition parameters: t
emperature = 20 K; microwave frequency =
33.986 GHz; MW pulse lengths π/2, π = 12, 24 ns; τ
= 100 ns; T
1
= T
2
= 100 ns; WT
1
= WT
2
= 16
ns; shot repetition time (srt) = 1 ms.
Ekanger et al.
Supporting Information
S8
Figure S9.
Q)band HYSCORE spectra and simulations of
14
N/
15
N hyperfine couplings of [(μ)
Cys)
2
Fe
2
(NO)
4
]
generated with
14
NO (top row) and
15
NO (bottom row). For each spectrum, the
top panels show the experimental spectra, with inte
nsities indicated by the color map ranging
from blue to red in order of increasing intensity.
The bottom panels reproduce the experimental
data in grey and overlay simulations from a relativ
ely strongly coupled
14/15
N nucleus (blue,
denoted N1) and a relatively weakly coupled
14/15
N nucleus (red, denoted N2). Specific
simulation parameters are detailed in Table 1 of th
e main text. Acquisition parameters:
temperature = 20 K; microwave frequency = 34.000 GH
z; MW pulse lengths π/2, π = 12, 24 ns;
τ = 100 ns; T
1
= T
2
= 100 ns; WT
1
= WT
2
= 16 ns; shot repetition time (srt) = 1 ms.
Ekanger et al.
Supporting Information
S9
Trypsin-Digested Native and Nitrosylated EndoIII
Figure S10.
(A) Logarithm intensity of peptides either non)modi
fied or modified by tyrosine
nitration for native (black) and nitrosylated (red)
EndoIII. (B) Treated/control ratio
demonstrating tyrosine nitration was not observed s
pecific to NO exposure.
Ekanger et al.
Supporting Information
S10
Representative Gels from Electrophoretic Mobility S
hift Assays
Figure S11.
Electrophoretic mobility shift assay gels of (a) na
tive and (b) nitrosylated EndoIII.
Ekanger et al.
Supporting Information
S11
Wide-Potential Sweep on DNA-Modified Gold Electrode
s
Figure S12.
Cyclic voltammograms of buffer (20 mM phosphates, 1
50 mM NaCl, 0.5 mM
EDTA, 10% v/v glycerol, pH 7.5) on DNA)modified gol
d electrodes using a standard (black) or
wide (dotted) potential sweep with a scan rate of 1
00 mV/s.