of 24
Nitric Oxide Modulates Endonuclease III Redox Activity by a 800
mV Negative Shift upon [Fe
4
S
4
] Cluster Nitrosylation
Levi A. Ekanger
,
Paul H. Oyala
,
Annie Moradian
,
Michael J. Sweredoski
, and
Jacqueline
K. Barton
*,†
Division of Chemistry and Chemical Engineering, Beckman Institute, California Institute of
Technology, Pasadena, California 91125, United States
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology,
Pasadena, California 91125, United States
Abstract
Here we characterize the [Fe
4
S
4
] cluster nitrosylation of a DNA repair enzyme, endonuclease III
(EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry,
electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein,
and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions
transforms the [Fe
4
S
4
] cluster into a dinitrosyl iron complex, [(Cys)
2
Fe(NO)
2
]-, and Roussin’s
red ester, [(
μ
-Cys)
2
Fe
2
(NO)
4
], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per
nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous
reports, but the Roussin’s red ester is an unreported product of EndoIII nitrosylation. Hyperfine
sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with
14
N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin’s red
ester. Whole-protein mass spectrometry of EndoIII nitrosylated with
14
NO and
15
NO support the
assignment of a protein-bound [(
μ
-Cys)
2
Fe
2
(NO)
4
] Roussin’s red ester. The [Fe
4
S
4
]
2+/3+
redox
couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but
nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on
gold despite having a similar DNA-binding affinity as the native protein. However, direct
electrochemistry of protein films on graphite reveals the reduction potential of native and
nitrosylated EndoIII to be 127 ± 6 and −674 ± 8 mV vs NHE, respectively, corresponding to a shift
of approximately −800 mV with cluster nitrosylation. Collectively, these data demonstrate that
DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by
[Fe
4
S
4
] cluster nitrosylation.
*
Corresponding Author
: jkbarton@caltech.edu.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b07362.
UV−vis spectroscopy of nitrosylation with and without dsDNA, CW and electron-spin echo EPR spectroscopy, representative UV−vis
spectra of deoxymyoglobin experiments, UV−vis spectroscopy of nitrosylation using 14/15NO, Q-band HYSCORE spectroscopy and
simulations of EndoIII nitrosylated using 14/15NO, trypsin-digested native and nitrosylated EndoIII, representative gels from
electrophoretic mobility shift assays, wide-potential sweep on DNA-modified gold electrodes (PDF)
Notes
Theauthors declare no competing financial interest.
HHS Public Access
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. Author manuscript; available in PMC 2018 October 14.
Published in final edited form as:
J Am Chem Soc
. 2018 September 19; 140(37): 11800–11810. doi:10.1021/jacs.8b07362.
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Graphical Abstract
INTRODUCTION
Nitric oxide (NO) is an endogenous reactive nitrogen species that plays an important role in
homeostatic regulation,
1
3
cytoprotective and cytotoxic pathways,
4
6
tumor progression,
7
10
and biofilm dispersal.
11
NO often elicits a response by coordinating metal centers of
metalloenzymes, such as in heme nitrosylation of soluble guanylyl cyclase during vaso-
dilation.
12
Non-heme Fe centers are also prone to nitrosylation. For example, the
transcription factors SoxR, NsrR, and FNR each contain a [Fe
2
S
2
] or [Fe
4
S
4
] cluster that
react with NO.
13
17
The [Fe
4
S
4
] clusters in transcription factors often play a functional role
by modulating protein conformation which, in turn, modulates DNA-binding affinity and
gene expression.
17
Nitrosylation of iron−sulfur clusters typically results in protein-bound
iron nitrosyl species such as the relatively common dinitrosyl iron complex,
[(Cys)
2
Fe(NO)
2
]
.
18
20
However, there is growing evidence that other iron nitrosyl species,
such as the Roussin’s red ester, [(
μ
-Cys)
2
Fe
2
(NO)
4
], a structural analogue of the red iron
nitrosyl salt reported by Roussin in 1858 (Figure 1),
21
are more common than previously
thought.
22
26
While recent studies have focused on the nitrosylation of transcription factors,
the [Fe
4
S
4
] clusters of DNA repair enzymes, such as endonuclease III (EndoIII) and DinG,
are also targets for NO reactivity.
27
,
28
The [Fe
4
S
4
] clusters in DNA repair enzymes were once thought to be strictly structural
motifs based on their relatively positive reduction potential. However, upon binding DNA,
the close proximity of the polyanionic phosphate backbone negatively shifts the [Fe
4
S
4
]
2+/3+
midpoint potential to approximately 80 mV vs normal hydrogen electrode (NHE),
thereby activating its redox activity in the DNA-bound form.
29
31
Our laboratory has
proposed that redox signaling between DNA-bound [Fe
4
S
4
]
2+
and [Fe
4
S
4
]
3+
clusters,
utilizing DNA-mediated charge transport (DNA CT), facilitates efficient DNA lesion
detection.
32
Given the importance of [Fe
4
S
4
] clusters for DNA CT in biology and their
propensity to react with NO, we are interested in studying how NO affects DNA CT by
cluster nitrosylation.
Our laboratory has explored DNA CT using EndoIII in
Escherichia coli
as a model [Fe
4
S
4
]
cluster DNA repair enzyme. The [Fe
4
S
4
] cluster of EndoIII reacts with NO, leading to
inactivation of DNA repair.
27
However, [Fe
4
S
4
] cluster nitrosylation of EndoIII has not been
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explored within the context of DNA CT. Here we report the effect of EndoIII [Fe
4
S
4
] cluster
nitrosylation on DNA CT using DNA-modified gold electrochemistry, protein film
voltammetry, electro-phoretic mobility shift assays, mass spectrometry of whole and trypsin-
digested protein, and UV−vis, continuous-wave (CW) and pulse electron paramagnetic
resonance (EPR), and circular dichroism spectroscopies. We connect our biophysical
observations within the DNA CT model to account for the electrochemical consequences of
[Fe
4
S
4
] cluster nitrosylation.
EXPERIMENTAL SECTION
General Procedures
All chemicals were of reagent-grade purity or better, purchased from Sigma-Aldrich unless
otherwise noted, and used as received unless otherwise noted. Water was purified on a Milli-
Q Reference Ultrapure Water Purification System (≥18.2 M
Ω
cm). Anaerobic atmospheres
(3–4% H
2
in N
2
, ≤1 ppm of O
2
) were maintained in vinyl chambers using Pd scrubbing
towers (Coy Laboratories).
EndoIII Overexpression and Purification.
Following reported procedures,
30
E. coli
EndoIII was overexpressed in BL21star-
(DE3)pLysS cells containing a pET11-His
6
-ubiquitin-
nth
construct. Purification was
performed at 4 °C using fast protein liquid chromatography (Bio-Rad). Protein was kept in
storage buffer (20 mM phosphates, 150 mM NaCl, 0.5 mM EDTA, 10% v/v glycerol, pH
7.5) at −80 °C until further use. Purity of isolated EndoIII was ≥95% as determined by SDS-
PAGE. EndoIII concentrations were determined by UV−vis using peptide and [Fe
4
S
4
]
cluster absorbances at 280 (
ε
= 19.94 × 10
3
M
−1
cm
−1
as calculated by ExPASy ProtParam,
Swiss Institute of Bioinformatics) and 410 nm (
ε
= 17.0 × 10
3
M
−1
cm
−1
),
33
respectively.
EndoIII Nitrosylation with Natural Abundance NO.
NO was introduced to reaction solutions through the proton-mediated decomposition of 1-
(hydroxy-NNO-azoxy)-L-proline, disodium salt (PROLI NONOate, Cayman Chemicals).
PROLI NONOate (~10 mg aliquot in vial) was dissolved in 400
μ
L of a solution of Ar-
sparged NaOH (0.01 M), quantified by UV−vis absorbance at 252 nm (
ε
=8.40 × 10
3
M
−1
cm
−1
),
34
and stored on ice under an anaerobic atmosphere until further use. EndoIII
nitrosylation reactions were prepared by bringing an aliquot of EndoIII (100
μ
M in storage
buffer) on ice into the anaerobic chamber. Within the anaerobic chamber, PROLI NONOate
(typically 80–85 mM in 0.01 M NaOH) was added directly to Ar-sparged storage buffer (pH
7.5) to initiate the decomposition of PROLI NONOate (3.75 mM), which was vortexed and
used within 5 min of preparation. PROLI NONOate in buffer was mixed with an appropriate
volume of storage buffer and EndoIII to achieve a 1:50 EndoIII/NO ratio (1 equiv of PROLI
NONOate decomposes to 2 equiv of NO). The final concentration of EndoIII was 60
μ
M
during nitrosylation in a total reaction volume of 83
μ
L. After 10 min and still within the
anaerobic chamber, the nitrosylation reaction solution was loaded onto a pre-rinsed Micro
Bio-Spin 6 size-exclusion column (Bio-Rad) and spun into a clean 1.5 mL tube following
the manufacturer’s protocol. For every nitrosylation reaction, a non-NO-exposed control was
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included and also purified by a size-exclusion column. Experiments and successive dilutions
or concentrations were performed using filtrate collected after size-exclusion purification.
UV−Vis, Circular Dichroism, and CW EPR Spectroscopies.
UV−vis data were acquired using either a Cary 100 Bio (Agilent) or DeNovix DS-C
spectrophotometer. Circular dichroism spectroscopy was performed using an AVIV
Biomedical spectrometer in the Beckman Institute Laser Resource Center. In an anaerobic
chamber, protein collected from size-exclusion purification was diluted to 10
μ
M in a total
volume of 800
μ
L and loaded into rectangular spectrophotometer cells with 2 mm path
length (Starna Cells). Spectra are normalized to ellipticity minima at 222 nm.
X-band CW-EPR spectroscopy was performed on an EMX X-band spectrometer (Bruker)
equipped with an ESR-900 cryogen flow cryostat (Oxford) and an ITC-503 temperature
controller. Samples (150
μ
L each) were loaded into 4 mm thin-wall precision quartz EPR
tubes (Wilmad LabGlass, 715-PW-250MM). To reduce dinitrosyl iron and Roussin’s red
complexes, nitrosylated EndoIII was incubated with sodium dithionite (1:25 protein/
dithionite ratio) for 30 min prior to freezing in liquid nitrogen while remaining capped under
an anaerobic atmosphere. An acquisition temperature of 60 K and microwave power of
0.204 mW were used for the ratiometric comparison of double integration spin intensities
because the iron nitrosyl signals were not saturated under these conditions. Signal saturation
was evaluated by ensuring signal intensity grew as the square root of microwave power.
Double integration spin intensities were measured using Matlab R2016a.
Synthesis of
15
NO and Quantification with Deoxymyoglobin.
To a 10 mL conical tube was added Na
15
NO
2
(400 mg, 5.72 mmol, 98 atom %
15
N) and
FeSO
4
·7H
2
O (2.00 g, 7.19 mmol). The tube was capped with a rubber septum and charged
with Ar. The dropwise addition of concentrated H
2
SO
4
(4 mL) into the capped reaction
vessel produced heat and vigorous gas evolution which was directed by cannula from the
reaction vessel into the bottom of a NaOH scrubbing tower (10 mL, 1 M). Gases from the
headspace of the scrubbing tower were further directed by a separate cannula and bubbled
into a 4 mL glass vial containing 2 mL of ice-cold degassed buffer (20 mM phosphates, 150
mM NaCl, 0.5 mM EDTA, 10% v/v glycerol, pH 7.5). At the time of bubbling, the glass vial
was capped with a rubber septum, and a needle was used as a vent to relieve pressure in the
system. After gas evolution from the reaction vessel had ceased (2–3 min of gas bubbling),
the glass vial with buffer was brought into an anaerobic chamber remaining capped and on
ice. The concentration of
15
NO in the buffer was determined under an anaerobic atmosphere
using a deoxymyoglobin colorimetric method.
The method for determining the concentration of dissolved NO in buffer is adapted from a
reported procedure.
35
Equine heart metmyoglobin (27 mg, 1.53
μ
mol) was dissolved in 500
μ
L buffer (20 mM phosphates, 150 mM NaCl, 0.5 mM EDTA, 10% v/v glycerol, pH 7.5)
and reduced to deoxymyoglobin using sodium dithionite (7.6 mg, 43.7
μ
mol) under an
anaerobic atmosphere. Excess sodium dithionite was removed by size-exclusion purification
using Micro Bio-Spin 6 size-exclusion columns (Bio-Rad). The reduction of metmyoglobin
to deoxymyoglobin is evidenced by a shift in the Soret band from 418 nm for metmyoglobin
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to 434 nm for deoxymyoglobin. The UV−vis spectrum of deoxymyoglobin indicates no
observable contamination by oxymyoglobin (418, 542, and 580 nm) or metmyoglobin (408,
502, and 630 nm). The concentration of deoxymyoglobin was 3.62 mM determined using a
reported extinction coefficient of 114 mM
−1
cm
−1
at 434 nm.
35
,
36
Deoxymyoglobin was used to quantify the concentration of
15
NO dissolved in buffer by a
ratiometric comparison with
14
NO. PROLI NONOate was used to prepare a solution of
14
NO with excess deoxymyoglobin (
14
NO/deoxymyoglobin ratio of 1:1.3). A separate
solution was prepared at the same time containing an aliquot of
15
NO solution added to the
same quantity of deoxymyoglobin used for the
14
NO standard. After 1 h of equilibration
under an anaerobic atmosphere, UV−vis absorbances at 420 nm were used to calculate the
concentration of the 15NO solution based on the known concentration of
14
NO using eq 1.
15
NO
=
15
NO
A
420
/
14
NO
A
420
14
NO
(1)
EndoIII Nitrosylation with
15
NO.
A 33.3
μ
L aliquot of
15
NO (2.7 mM) in ice-cold buffer was added to EndoIII under an
anaerobic atmosphere. After 10 min, the reaction solution was purified by size-exclusion
purification on pre-rinsed Micro Bio-Spin 6 size-exclusion columns (Bio-Rad). Experiments
and successive dilutions or concentrations were performed using filtrate collected after size-
exclusion purification.
Pulse EPR.
Samples for hyperfine sublevel correlation (HYS-CORE) EPR spectroscopy were prepared
by concentrating nitrosylated EndoIII samples with or without dithionite treatment using
Amicon Ultra Centrifugal Filters (10k molecular weight cut off) to a concentration of 0.5
mM protein in 75
μ
L.
All pulse X-band (9.7 GHz) and Q-band (34 GHz) EPR and HYSCORE spectroscopy was
performed using a Bruker ELEXSYS E580 pulse EPR spectrometer equipped with a Bruker
MD4 (X-band) or D2 (Q-band) resonator. Temperature control was achieved using an ER
4118HV-CF5-L Flexline Cryogen-Free VT cryostat manufactured by ColdEdge equipped
with an Oxford Instruments Mercury ITC temperature controller.
Pulse X- and Q-band HYSCORE spectra were acquired using the 4-pulse sequence (
π
/2 −
τ
π
/2 − t
1
π
− t
2
π
/2 −
τ
− echo), where
τ
is a fixed delay, and
t
1
and t
2
are variable
delays independently incremented by Δ
t
1
and Δ
t
2
, respectively. Sixteen step phase cycling
was utilized to eliminate contributions from secondary/tertiary echoes. The time domain
spectra were baseline-corrected (third-order polynomial), apodized with a Hamming window
function, zero-filled to 8-fold points, and fast Fourier-transformed to yield the frequency
domain.
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All EPR spectra (CW, HYSCORE) were simulated using the EasySpin
37
simulation toolbox
(version 5.2.16) with Matlab 2016b (see SI for description of simulations).
Ferene Assay for Fe Quantitation.
The ferene assay was performed according to a published protocol.
38
Briefly, samples were
diluted 1:1 with HNO
3
(21.7% v/v) to a total volume of 200
μ
L. Samples were heated at
95 °C for 30 min, cooled at 4 °C for 10 min, and centrifuged. To each sample was added 600
μ
L of ammonium acetate (7.5% w/v), 100
μ
L of ascorbic acid (12.5% w/v), and 100
μ
L of 3-
(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5
,5
′′
,-disulfonate (ferene, 10 mM). Samples were
incubated under ambient conditions for 30 min before absorbance at 593 nm was recorded.
Calibration curves were prepared using Fe standard solution (1001 ± 2 mg/L Fe in 2% v/v
HNO
3
, TraceCERT Fe standard for ICP).
Protein Mass Spectrometry.
Whole protein was analyzed using an Agilent 1100 series liquid chromatography mass
selective detector (LC/MSD) system. Following size-exclusion purification, control and
nitrosylated protein samples were diluted 1:50 or 1:20 in formic acid (0.2% v/v),
respectively. Trypsin-digested protein samples were prepared by adding 5
μ
L of protein
solution (60
μ
M) to 45
μ
L of sodium deoxycholate (1% w/v) in Tris buffer (100 mM, pH
8.5). Disulfides were reduced by adding 0.5
μ
L of tris(2-carboxyethyl)-phosphine (TCEP,
0.5 M in water) and, after 20 min, protected by adding 1
μ
L of iodoacetamide (0.5 M in
water) followed by 20 min incubation in the dark under ambient conditions. To preserve
disulfide bonds in some samples, TCEP was omitted from the digestion protocol. After thiol
protection, 1
μ
L of lysyl endopeptidase (0.1
μ
g/
μ
L in 100 mM Tris buffer, pH 8.5) was
added to protein samples. After incubating for 2.5 h under ambient, dark conditions, all
samples were diluted by half with the addition of Tris buffer (100 mM, pH 8.5) followed by
the addition of 1
μ
L CaCl
2
(100 mM in water) and 1
μ
L trypsin (0.44
μ
g/
μ
L in 100 mM Tris
buffer, pH 8.5). After digesting overnight at 37 °C, samples received formic acid (5% v/v) to
bring the total sample volume to 95
μ
L and produce a cloudy, white precipitate which was
removed by centrifugation. Purification of the supernatant was performed by reverse-phase
high-performance liquid chromatography (HPLC), and solvent removed from purified
samples by vacuum centrifugation. Dried samples were dissolved in formic acid (0.2% v/v)
and were injected onto a capillary in-house packed C18 column and subjected to a 60 min
gradient by an Easy nLC 1200 HPLC. Chromatographically separated peptides were
analyzed using a Q Exactive mass spectrometer (ThermoScientific) coupled to a NanoFlex
ion source.
Synthesis and Characterization of Thiol-Modified DNA.
All oligonucleotides were purified by reverse-phase HPLC using a C-18 column (Agilent).
Disulfide-modified oligonucleotide was synthesized on a 3400 Applied Biosystems DNA
synthesizer using standard phosphoramidite chemistry (all phosphoramidites were purchased
from Glen Research), where the C6 S−S phosphoramidite was installed at the 5
terminus.
Disulfide-modified oligonucleotide was cleaved from the solid support, purified by HPLC,
and reduced to hexanethiol-modified oligonucleotide (5
HS-C
6
-GTG CTG CAA CGT
GTC TGC GC 3
) with dithiothreitol in buffer (50 mM Tris, 50 mM NaCl, pH 8.4). Thiol-
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modified oligonucleotide was purified by size-exclusion chromatography (GE Illustra
NAP-5 column), HPLC, and characterized by matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (MALDI−TOF, Bruker Autoflex); MALDI−TOF (
m/z
): [M
+ H]
+
calculated for C
200
H
264
N
73
O
124
P
20
S, 6323.1; found, 6323.2. The complementary
strand (5
GCG CAG ACA CGT TGC AC 3
) was purchased from IDT and purified by
HPLC. Oligonucleotides were further purified after HPLC by ethanol precipitation. Thiol-
modified oligonucleotide was annealed to its complement (50
μ
M oligonucleotides in
Arsparged buffer containing 5 mM sodium phosphates, 50 mM NaCl, pH 7.0) by heating at
90 °C for 5 min followed by cooling to ambient temperature over 90 min. Thiol-modified
double-stranded DNA was stored under Ar at −20 °C until further use.
DNA-Modified Gold Electrochemistry.
The fabrication of multiplexed gold chips has been previously described.
39
,
40
Multiplexed
gold chips were cleaned in acetone and isopropanol with sonication, dried under an Ar
stream, and ozone-cleaned for 25 min (UVO Cleaner). Immediately after cleaning, a rubber
gasket (Buna-N) and acrylic clamp were affixed to the chip surface, and thiol-modified
dsDNA (20
μ
L, 50
μ
M) was added to each quadrant to form a self-assembled monolayer on
the gold electrode surface. After incubation with thiol-modified DNA for 18–21 h, quadrants
were washed with buffer (5 mM sodium phosphates, 50 mM NaCl, pH 7.0) and incubated
with 6-mercaptohexanol (1 mM in 5 mM sodium phosphates, 50 mM NaCl, pH 7.0). After
45 min, the electrode was washed three times with Ar-sparged storage buffer and brought
into the anaerobic chamber for electrochemical analyses. A three-electrode setup was
employed consisting of DNA-modified gold working electrodes, Ag/AgCl reference
electrode (MW-2030, RE-6, BASi) with a gel tip (1.0% UltraPure Invitrogen Agarose
prepared with 3 M NaCl), and a Pt wire auxiliary electrode. The total volume on the
multiplexed chip was 200
μ
L. A scan rate of 100 mV/s was used for cyclic voltammograms,
and potentials were converted to the NHE scale.
Direct Electrochemistry on Edge-Plane Graphite.
Protein films were prepared on an edge-plane pyrolytic graphite electrode (Pine Research
Instrumentation). Prior to protein deposition, the electrode was cleaned by abrasion on
sandpaper (400 grit) followed by sonication in isopropanol then water. Clean surfaces were
confirmed by performing electrochemical experiments with protein storage buffer that did
not contain glycerol. Micro Bio-Spin (Bio-Rad) columns were used to exchange sample
buffer with protein storage buffer that did not contain glycerol to enable dry film formation.
Protein films were deposited onto the electrode surface under an anaerobic atmosphere by
sequential drying of protein aliquots (3 × 25
μ
L). Drying was accomplished by placing the
upright electrode with aliquot in the airstream of a Pd catalyst fan box equipped with a
single catalyst pack (Coy Laboratories) for approximately 20 min per aliquot. Dried protein
films were secured with 10
μ
L Nafion (5% aqueous dispersion) to prevent film dispersal as
previously described.
41
After protein and Nafion depositions, a droplet (50
μ
L) of protein
storage buffer without glycerol was added on top of the film. A three-electrode setup was
employed by submerging reference and auxiliary electrodes in the droplet. The reference
and auxiliary electrodes were the same used for DNA-modified gold electro-chemistry
experiments described above. All potentials were converted to the NHE scale.
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Electrophoretic Mobility Shift Assays.
A non-thiol-functionalized analogue of the oligonucleotide used for DNA-modified
electrochemistry (5
GTG CTG CAA CGT GTC TGC GC 3
) was purchased from IDT,
purified by HPLC and ethanol precipitation, and radiolabeled through 5
phosphorylation
using [
γ
32
P] ATP (NEG035C005MC, 6000 Ci/mmol, 150 mCi/mL, PerkinElmer) and T4
polynucleotide kinase (New England BioLabs) at 37 °C for 30 min followed by heat
inactivation at 65 °C for 20 min. Purification of
32
P-labeled oligonucleotide was
accomplished by size-exclusion purification through a Micro Bio-Spin 6 column (Bio-Rad)
followed by a Monarch PCR & DNA Cleanup Kit (New England BioLabs) with a modified
protocol. Briefly, to 50
μ
L of filtrate from size exclusion were added 100
μ
L Monarch DNA
Cleanup Binding Buffer and 300
μ
L ethanol. The resulting solution was loaded into a
Monarch DNA Cleanup Column and spun at 16,000 ×
g
for 1 min. After discarding the
flow-through, 500
μ
L of a 1:4 solution of Monarch DNA Cleanup Binding Buffer and
ethanol were added to the column and spun at 16,000 ×
g
for 1 min. This washing procedure
was repeated once more and flow-through discarded each time. The column was spun at
16,000 ×
g
for 1 min to remove residual ethanol, transferred to a clean 1.5 mL tube, and 25
μ
L of Monarch Elution Buffer was carefully added to the center of the column matrix. After
1 min, the column was spun at 16,000 ×
g
for 1 min to collect the purified, radiolabeled
oligonucleotide, which was then annealed to its non-radiolabeled complement (10
μ
M
oligonucleotides in buffer containing 5 mM sodium phosphates, 50 mM NaCl, pH 7.0) by
heating at 90 °C for 10 min followed by cooling to ambient temperature over 3 h. Complete
annealing was verified by native 10% polyacrylamide gel electrophoresis at 4 °C (50 V, 0.5×
Tris/borate/EDTA buffer) using single-stranded controls. Gels were imaged by dark
exposure to BAS storage phosphor screens followed by imaging on a Typhoon FLA 9000
Imager (GE Healthcare Life Sciences). Radiolabeled dsDNA was incubated with native or
nitrosylated EndoIII for 1 h at 4 °C prior to native 10% polyacrylamide gel electrophoresis
at 4 °C (50 V for 3 h in 1× TGX buffer). Gel exposure time was calculated by the
radioactivity of a 10
μ
L aliquot where a scintillation reading of 300,000 counts per minute
corresponded to 1 h exposure on storage phosphor screens. Free dsDNA band intensity was
measured using ImageQuant 5.2, and normalized band intensities were used to calculate
dissociation constants by the Hill function in OriginPro 2017.
RESULTS AND DISCUSSION
Characterization of Nitrosylated EndoIII by UV−vis Spectroscopy and the Ferene Assay.
We sought to characterize the products of EndoIII nitrosylation to enable a rational
interpretation of our biophysical observations. At the outset of our work, the reaction
between NO and the [Fe
4
S
4
]
2+
cluster of EndoIII was reported with a focus on the titration
of EndoIII with NO, enzyme activity, and the reversibility of [Fe
4
S
4
]
2+
cluster nitrosylation.
27
Additionally, the dinitrosyl iron complex, [(Cys)
2
Fe(NO)
2
]
, was the only protein-bound
iron nitrosyl species observed.
27
In the absorbance spectrum of native EndoIII, we observe
absorbances at 280 and 410 nm consistent with the presence of a protein-bound
[Fe
4
S
4
]
2+
cluster.
33
The [Fe
4
S
4
]
2+
cluster of native EndoIII is relatively stable to handling in
air, but NO degrades the cluster within a 10 min reaction period. The absorbance spectrum
of nitrosylated EndoIII is similar to native EndoIII at 280 nm but differs at longer
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wavelengths primarily through a 46 nm hypsochromic shift of the cluster absorbance
maximum to a new feature at 364 nm (Figure 2). This nitrosylation reaction proceeds
similarly in the presence of excess dsDNA (EndoIII/dsDNA ratio of 1:10) as evidenced by a
nearly identical UV− vis absorbance spectrum (Figure S1). The new absorbance centered at
364 nm is consistent with a Roussin’s red ester, [(
μ
-Cys)
2
Fe
2
(NO)
4
], which exhibits a highly
conserved absorbance maximum in the 360 ± 10 nm range in proteins, peptides, and
synthetic model complexes.
22
,
24
,
42
45
Additional support for the presence of a multinuclear iron nitrosyl complex stems from
ferene colorimetric assays, which were used to quantify protein-bound Fe of native and
nitrosylated EndoIII. The concentration of protein-bound [Fe
4
S
4
] cluster was determined
using the reported extinction coefficient of 17.0 × 10
3
M
−1
cm
−1
at 410 nm,
33
and the
concentration of Fe per cluster was measured using the ferene colorimetric assay where
ferene-chelated Fe(II) is detected at 593 nm (Figure 2). Native EndoIII was found to contain
4.00 ± 0.09 Fe per cluster, and this value decreased to 3.05 ± 0.01 Fe for nitrosylated
EndoIII prepared from a 10 min exposure to NO. If [(Cys)
2
Fe(NO)
2
]− is the only iron
nitrosyl species to form in the tetracysteine cluster-binding pocket, then nitrosylated EndoIII
should retain only 1 or 2 Fe per nitrosylated cluster. Accordingly, the retention of 3 Fe per
nitrosylated cluster strongly indicates that an iron nitrosyl species with more than 1 equiv of
Fe is present, which is likely the [(
μ
-Cys)
2
Fe
2
(NO)
4
] Roussin’s red ester based on UV−vis
observations described above. The retention of 3 Fe in nitrosylated EndoIII necessitates the
release of 1 Fe equivalent in a labile form which is subsequently removed from our reaction
solutions by size-exclusion purification. The release of labile Fe upon reaction with NO is
consistent with cluster degradation.
17
Characterization of Nitrosylated EndoIII by CW EPR Spectroscopy.
To gain further insight into the products of EndoIII nitrosylation, we performed CW and
pulse EPR experiments using native and nitrosylated EndoIII in the absence of DNA. The
CW EPR spectrum of native EndoIII is devoid of signal and consistent with a diamagnetic
sample (Figure S2), while the corresponding spectrum of nitrosylated EndoIII contains a
slightly rhombic signal with
g
= [2.042, 2.036, 2.015], consistent with a mononuclear
[(Cys)
2
Fe(NO)
2
]
dinitrosyl iron complex (Figure 2).
22
,
42
When nitrosylated EndoIII is treated with sodium dithionite (protein/dithionite ratio of
1:25), a new axial signal with
g
= 2.011 and
g
= 1.972 emerges consistent with the
binuclear [(
μ
-Cys) Fe
2
(NO)
4
]
complex referred to as the reduced Roussin’s red ester.
22
,
42
A ratiometric comparison of the double-integration spin intensities indicates that
[(Cys)
2
Fe(NO)
2
]
and [(
μ
-Cys)
2
Fe(NO)
4
]
form in a 1:1 ratio (see Figures S3 and S4 for
comparison of single-integration signals and simulations of CW EPR spectra, respectively).
To simplify electron counting and avoid assigning formal oxidation states in iron nitrosyl
complexes, the Enemark− Feltham notation, {M(NO)
x
}
n
, is used where M is the metal
center,
x
is the number of metal-bound nitrosyl ligands, and n is the total number of
electrons in the metal d and NO
π
* orbitals.
46
The CW EPR spectrum of native EndoIII is
devoid of any observable signal, consistent with a diamagnetic [Fe
4
S
4
]
2+
cluster
47
, while
nitrosylated EndoIII exhibits a rhombic signal centered at
g
= 2.029 consistent with the
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presence of an {Fe(NO)
2
}
9
dinitrosyl iron complex.
22
,
42
Interestingly, when nitrosylated
EndoIII is reduced with dithionite, the {Fe(NO)
2
}
9
profile diminishes, and a new axial
signal is observed with
g
= 2.011 and
g
= 1.972. The {Fe(NO)
2
}
9
dinitrosyl iron complex
is the only species observed by EPR directly after cluster nitrosylation because it is the only
paramagnetic (
S
= 1/2) species initially formed, while the {Fe(NO)
2
}
9
−{Fe(NO)
2
}
9
Roussin’s red ester, a dimerized analogue of the dinitrosyl iron complex, contains two
antiferromagnetically coupled Fe centers and is therefore EPR silent as synthesized. Upon
reduction by dithionite, the {Fe(NO)
2
}
9
dinitrosyl iron complex is reduced into an EPR-
silent {Fe(NO)
2
}
10
complex, while the EPR-silent {Fe-(NO)
2
}
9
−{Fe(NO)
2
}
9
Roussin’s red
ester is reduced into the EPR-active {Fe(NO)
2
}
9
−{Fe(NO)
2
}
10
(
S
= 1/2) species referred to
as the reduced Roussin’s red ester (Scheme 1).
22
,
43
Our assignment of the reduced Roussin’s
red ester, [(
μ
-Cys)
2
Fe
2
(NO)
4
]
, is supported by similar EPR resonances observed for
synthetic and peptide-bound model complexes.
23
,
42
,
48
It should be noted that the reduced Roussin’s red ester signal with principal g-values of
g
=
2.011 and
g
= 1.972 was originally assigned as a d
9
dinitrosyl iron complex having been
reduced from a d
7
form.
19
However, compelling spectroscopic data of structurally well-
characterized model complexes disagree with the initial assignment and instead support the
assignment of a reduced Roussin’s red ester.
42
,
45
The spectroscopic agreement between
reduced Roussin’s red ester model complexes and other protein-bound iron nitrosyl species,
once considered d
9
dinitrosyl iron complexes, has been noted previously.
23
It therefore
appears the protein-bound Roussin’s red ester, once overlooked due to its EPR-silent nature
in the neutral form, is indeed more common than previously thought.
22
26
Collectively, these CW EPR data support the observation that nitrosylated EndoIII contains
both the dinitrosyl iron complex and Roussin’s red ester. The reason that the Roussin’s red
ester has not been explicitly reported for EndoIII is likely because it is EPR silent. To
measure the ratio of dinitrosyl iron complex to reduced Roussin’s red ester, the CW EPR
signals were compared. The doubly integrated spectral intensities of {Fe(NO)
2
}
9
and
Fe(NO)
2
}
9
−{Fe(NO)
2
}
10
reveal they form in a 1:1 ratio, indicating EndoIII retains 1 Fe
equivalent from [(Cys)
2
Fe(NO)
2
]
and 2 Fe equivalents from [(
μ
-Cys)
2
Fe
2
(NO)
4
] for a total
retention of 3 Fe per nitrosylated cluster. This observation is consistent with the ferene assay
results where nitrosylated EndoIII retains an average of 3.05 ±0.01 Fe per nitrosylated
cluster. Considering the [Fe
4
S
4
]
2+
cluster is bound by four cysteine residues, and that
dinitrosyl iron complex and Roussin’s red ester each require only two cysteine residues, it is
likely that the cysteine residues originally bound to the [Fe
4
S
4
] cluster are responsible for
binding the iron nitrosyl species after nitrosylation.
Characterization of Nitrosylated EndoIII by Pulse-EPR Spectroscopy with
14/15
NO.
To gain further insight into the electronic structure of protein-bound iron nitrosyl complexes
through measurement of the nitrosyl
14/15
N hyperfine interactions, we performed pulse EPR
experiments. Initial characterization by X-band HYSCORE spectroscopy of
[(Cys)
2
Fe(NO)
2
]
and [(
μ
-Cys)
2
Fe
2
(NO)
4
]
prepared using natural abundance (
14
N =
99.63%) NO reveals features consistent with two distinct classes of hyperfine couplings
from
14
N nuclei of the nitrosyl ligands (Figure 3), with one relatively weakly coupled
14
N
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exhibiting signals in the (+,+) quadrant and a more strongly coupled
14
N exhibiting features
in the (−,+) quadrant (Figure 3).
In order to further confirm these assignments and constrain the hyperfine coupling and
nuclear quadrupole interaction parameters independently, Q-band (34 GHz) HYSCORE
spectroscopy was performed on samples generated with isotopically enriched
15
N (I = 1/2),
generated from
15
NO
2
and quantified using deoxymyoglobin (see Figures S5 and S6 for
representative UV−vis spectra), and natural abundance NO (Figure 4). The Q-band
HYSCORE spectra of the
15
NO isotopologues of both the of [(Cys)
2
Fe(NO)
2
]
and [(
μ
-
Cys)
2
Fe
2
(NO)
4
]− differ markedly from their natural abundance variants, confirming that
these features indeed arise from hyperfine couplings to the NO nitrogens. Elimination of
nuclear quadrupole interactions in the
15
NO samples allowed for the 15N hyperfine
couplings to be determined via simulation (see Figures S7−S9 for Q-band HYSCORE
spectra and simulations of
14
N/
15
N hyperfine couplings). Accounting for the ratio of
15/14
N
nuclear g-values (
15
N
g
n
/
14
N
g
n
=−1.403), the
14
N hyperfine couplings were then fixed
(Table 1), and the nuclear quadrupole coupling constant (
e
2
qQ/h
) and electric field gradient
asymmetry (
η
) determined for each
14
N nucleus via simulation of the data.
Simulations of the combined multifrequency HYSCORE data set for the EndoIII
[(Cys)
2
Fe(NO)
2
]
dinitrosyl iron complex indicate nitrogen hyperfine couplings to two
distinct classes of NO, one more strongly coupled with
14
N
a
iso
= 9.5 MHz and the other
much more weakly coupled with
14
N
a
iso
= 0.4 MHz. The non-equivalence the two NO
ligands is expected because dinitrosyl iron species exhibit two distinct NO stretching
frequencies.
35
This non-equivalence is also predicted by density functional theory.
49
These
relatively small 14N hyperfine couplings are consistent with the assignment of an
{Fe(NO)
2
}
9
dinitrosyl iron complex with spin density largely localized on the Fe center as
opposed to an {Fe(NO)}
7
species, which exhibit much larger
14
N hyperfine couplings (
a
iso
30 MHz, up to 220 MHz) due to unpaired spin being primarily localized on the NO moiety.
35
Similarly, the multifrequency HYSCORE data set for the EndoIII [(
μ
-Cys)
2
Fe
2
(NO)
4
]
reduced Roussin’s red ester also indicates nitrogen hyperfine couplings to two distinct
classes of NO, one more strongly coupled with
14
N
a
iso
= 3.9 MHz and the other more
weakly coupled with
14
N
a
iso
= 1.9 MHz. Because this species consists of a mixed-valence,
antiferromagnetically coupled {Fe(NO)
2
}
9
−{Fe(NO)
2
}
10
(
S
= 1/2) dimer, these two classes
of NO
14
N hyperfine couplings likely represent two non-equivalent NO ligands on the
{Fe(NO)
2
}
9
fragment (analogous to the dinitrosyl iron complex) with no significant
contribution from the NO ligands on the diamagnetic {Fe(NO)
2
}
10
fragment.
Collectively, the relatively low magnitude of the
14
N nuclear quadrupole coupling constants
observed for all of the NO nitrogens, e
2
qQ/h = 0.8–1.4 MHz combined with the low
estimation for the electric field gradient asymmetry (
η
) indicate axial symmetry of charge
about the NO nitrogen with significant population of charge density in the NO
π
orbitals
orthogonal to the NO
σ
orbital, as expected for the multiply bonded NO moiety (see Table 1
for specific simulation parameters).
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In sum, these HYSCORE data further demonstrate that the dinitrosyl iron complex and
Roussin’s red ester are both products of [Fe
4
S
4
]
2+
cluster nitrosylation in EndoIII.
Mass Spectrometry of Native and Nitrosylated EndoIII
Having identified and characterized iron nitrosyl complexes through UV−vis and EPR
spectroscopies, we turned to mass spectrometry for further characterization of protein-bound
iron nitrosyl species. Whole protein was analyzed by diluting native and nitrosylated EndoIII
in formic acid (0.2% v/v) followed by HPLC−MS. Native EndoIII has a lone mass at 33,264
Da corresponding to apoprotein where the cysteines originally binding the cluster are instead
protonated (Figure 4). This observation indicates the EndoIII [Fe
4
S
4
] is acid labile.
Nitrosylated EndoIII yields multiple masses of relatively high abundance that provide
insight into nitrosylation reaction products. First, masses at 33,260 and 33,292 Da
correspond to apoprotein with two cysteine disulfides (2 Cys-SS-Cys) and apoprotein with
one cysteine disulfide and one cysteine trisulfide (Cys-SS-Cys + Cys-SSS-Cys),
respectively. Disulfide formation and the retention of cluster sulfides as polysulfides of
cysteine are common features of [Fe
4
S
4
] cluster degradation by both NO and O
2
.
17
In
addition to apoprotein masses, we observe a mass at 33,494 Da corresponding to protein-
bound [(
μ
-Cys)
2
Fe
2
(NO)
4
] present with a cysteine disulfide (33,264 −2 + 232). To test
whether the mass at 33,494 Da arises from protein-bound [(
μ
-Cys)
2
Fe
2
(NO)
4
], we
performed additional whole-protein mass spectrometry on nitrosylated EndoIII prepared
using
15
NO and observe a new mass at 33,498 Da (Figure 4). This 4 Da mass shift is
consistent with the substitution of four
14
NO ligands for
15
NO and therefore supports the
assignment of 33,494 Da as protein-bound [(
μ
-Cys)
2
Fe
2
(NO)
4
]. The
15
N-enriched sample
displays a 4 Da mass shift because only the Roussin’s red ester is present in EndoIII after
dilution in acid. This can be rationalized considering that the p
K
a
of bridging cysteine
thiolates of the Roussin’s red ester should be lower than the p
K
a
of non-bridging thiolates of
the dinitrosyl iron complex, rendering the dinitrosyl iron complex more acid labile than the
Roussin’s red ester. These observations are similar to a recent report on the mass
spectrometric detection of protein-bound Roussin’s red ester of the transcription factor
NsrR.
50
We next turned to mass spectrometry of trypsin-digested protein to test for specific off-target
modifications to EndoIII caused by NO exposure. By itself, NO has selective reactivity for
metal centers and thiols, but does not react directly with other amino acids. However, direct
amino acid modification, commonly in the form of tyrosine nitration, can occur in the
presence of NO
2
formed from the reaction between NO and O
2
.
51
For this reason, the
nitrosylation reactions performed in this work were performed under anaerobic conditions.
However, we still tested whether tyrosine nitration of EndoIII occurs specifically from NO
treatment from the presence of residual O
2
in reaction solutions. To test for tyrosine nitration
specific to NO treatment, we digested native and nitrosylated EndoIII with trypsin, and the
resulting peptides were analyzed by electron-transfer dissociation mass spectrometry. While
we detect peptides modified by tyrosine nitration, the modification does not appear specific
to NO exposure (Figure S10) thereby indicating minimal off-target effects.
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Cluster Nitrosylation Abates DNA-Bound Redox Activity of EndoIII.
DNA-bound redox activity is a hallmark of DNA-processing proteins that participate in
DNA CT. The DNA-bound redox activity of [Fe
4
S
4
] cluster proteins is typically evaluated
using DNA-modified gold electrodes, where the [Fe
4
S
4
]
2+/3+
redox couple of the DNA-
bound protein is observed near a midpoint potential of 80 mV vs NHE.
29
32
Because the
current at 80 mV vs NHE reflects redox activity of the DNA-bound [Fe
4
S
4
] cluster protein,
signal intensity at 80 mV vs NHE reflects the ability of a protein to participate in DNA CT.
We observe DNA-bound redox activity of native EndoIII at 80 mV vs NHE purified by size-
exclusion filtration (Figure 5). However, no redox activity above the buffer background is
observed on the same DNA-modified electrode for nitrosylated EndoIII purified by size-
exclusion filtration.
52
The absence of DNA-bound redox activity can be explained by either
of the following two factors: (1) The DNA-binding affinity of nitrosylated EndoIII is lower
than that of native EndoIII, or (2) the reduction potential of nitrosylated EndoIII is no longer
at 80 mV vs NHE and is shifted out of the potential window for the DNA-modified gold
electrode. In both of these scenarios, the redox activity at 80 mV vs NHE would decrease for
nitrosylated EndoIII relative to the native enzyme. To examine these hypotheses in more
detail, we turned to DNA-binding affinity measurements.
Native and Nitrosylated EndoIII Have Different Conformations but Similar DNA-Binding
Affinities.
One explanation for the lack of observable redox activity with nitrosylated EndoIII on DNA-
modified gold electrodes is that nitrosylated EndoIII binds DNA with a lower affinity than
native EndoIII. We therefore examined changes to EndoIII DNA-binding affinity caused by
[Fe
4
S
4
] cluster nitrosylation. To test if nitrosylated EndoIII has a lower DNA-binding
affinity than native EndoIII, we performed electrophoretic mobility shift assays. The assays
were performed by incubating native and nitrosylated EndoIII with radiolabeled dsDNA
followed by native gel electrophoresis. The percentage of EndoIII-bound dsDNA was
visualized by phosphoimagery (see representative gels in Figure S11), quantified by
measuring band intensities, and used to calculate dissociation constants with Hill function
fitting (Figure 5). Through these assays, native and nitrosylated EndoIII show similar DNA
dissociation constants of 3.7 ± 0.4 and 2.2 ± 0.1
μ
M, respectively. If anything, the DNA
affinity for the nitrosylated protein is higher, as would be expected given the more positive
charge on the cluster site.
31
Interestingly, circular dichroism spectra indicate that [Fe
4
S
4
]
cluster nitrosylation decreases the
ε
222
/
ε
208
ellipticity ratio from 1.199 ± 0.005 to 0.90
± 0.04 from native to nitrosylated EndoIII (Figure 5), which is consistent with disordering of
α
helices.
53
Despite this disordering, [Fe
4
S
4
] cluster nitrosylation does not appear to lower
the DNA-binding affinity. Accordingly, the absence of DNA-bound redox activity of
nitrosylated EndoIII cannot be explained by nitrosylated EndoIII having a significantly
lower DNA-binding affinity than native EndoIII. Since the DNA-binding affinity of
nitrosylated EndoIII is not lower than native EndoIII, we suspected that the reduction
potential of nitrosylated EndoIII is no longer at 80 mV vs NHE.
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Cluster Nitrosylation Shifts the EndoIII Reduction Potential by 800 mV in the Negative
Direction.
Since redox activity of nitrosylated EndoIII is not observed on DNA-modified gold
electrodes despite native and nitrosylated EndoIII having similar DNA-binding affinities, we
turned to direct electrochemistry experiments in the absence of DNA on a graphite electrode
with a wider potential window. Using differential pulse voltammetry, we observe a reduction
potential of 127 ± 6 mV vs NHE in the voltammogram of native EndoIII (Figure 5). This
value is consistent with previous measurements of EndoIII in the absence of DNA and is
assigned as the [Fe
4
S
4
]
2+/3+
reduction potential.
41
However, the differential pulse
voltammogram of nitrosylated EndoIII reveals a new reductive peak at −674 ± 8 mV vs
NHE. The new reduction signal is specific to nitrosylated EndoIII and corresponds to an
approximately 800 mV negative shift in reduction potential relative to native EndoIII. This
shift places the reduction potential of nitrosylated EndoIII within a reasonable range to be
reduced by dithionite (reduction potential range of −650 to −752 mV vs NHE from pH 6.9–
7.9)
54
and also in the range of reduction potentials (−500 to −1000 mV vs NHE) measured
for dinitrosyl iron and Roussin’s red ester model complexes.
55
59
We are unable to assign
the reduction potential of −674 mV vs NHE to either iron nitrosyl species specifically.
However, considering that by EPR we observe the reduction of both the dinitrosyl iron
complex and Roussin’s red ester by dithionite, it is reasonable that the reductive wave at
−674 mV vs NHE reflects the reduction of both iron nitrosyl species. Moreover, this
reduction potential is outside the potential window of DNA-modified gold electrodes; the
thiolated DNA and mercaptohexanol monolayer is compromised when sweeping potentials
below −300 mV vs NHE (Figure S12). This shift in potential furthermore explains the lack
of DNA-bound redox activity of nitrosylated EndoIII despite having a similar DNA-binding
affinity to native EndoIII (Figure 5).
EndoIII Nitrosylation and Implications for DNA CT and NO Signaling.
Collectively, these data demonstrate that [Fe
4
S
4
] cluster nitrosylation of the bacterial DNA
repair enzyme EndoIII results in two distinct protein-bound iron nitrosyl complexes, the
dinitrosyl iron complex and Roussin’s red ester. The reaction of NO with EndoIII is similar
in the absence and presence of DNA and leads to rapid and direct changes to the cluster with
seemingly inconsequential changes in protein conformation. It is noteworthy that, despite
this NO reactivity, the cluster in EndoIII is relatively stable to oxygen.
Importantly, unlike native EndoIII with an intact [Fe
4
S
4
] cluster, nitrosylated EndoIII does
not exhibit DNA-bound redox activity on DNA-modified gold electrodes. While there are
conformational changes between native and nitrosylated EndoIII, nitrosylated EndoIII does
not have a lower affinity for duplex DNA than native EndoIII. This observation is in contrast
to NO-sensitive transcription factors in which [Fe
4
S
4
] cluster nitrosylation modulates DNA-
binding affinity as a result of a conformational change. Instead of changes to DNA-binding
affinity, we observe an 800 mV decrease in reduction potential between native and
nitrosylated EndoIII using direct electrochemical measurements. It therefore appears that the
primary consequence of EndoIII [Fe
4
S
4
] cluster nitrosylation is electrochemical in nature.
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The results of this study have critical implications for the DNA CT model based on two
primary observations: (1) native and nitrosylated EndoIII bind dsDNA to a similar extent
and (2) nitrosylated EndoIII is more difficult to reduce than native EndoIII by 800 mV.
These observations suggest that [Fe
4
S
4
] cluster nitrosylation might affect DNA CT between
nitrosylated and native DNA repair enzymes. Moreover, we observe that the presence of
excess dsDNA does not inhibit cluster nitrosylation under our reaction conditions indicating
that both free and DNA-bound EndoIII are susceptible to NO. Within the context of NO
signaling, this study expands the known bioinorganic effects of NO. In contrast to
transcription factor cluster nitrosylation for modulating DNA binding and gene expression,
we observe that [Fe
4
S
4
] cluster nitrosylation of a DNA repair enzyme results in a minimal
change to DNA binding but a substantial modulation of redox activity.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
ACKNOWLEDGMENTS
This work was supported by GM126904 from the National Institutes of Health (to J.K.B.). The Caltech EPR facility
is supported by the National Science Foundation (NSF-1531940) and the Dow Next Generation Educator Fund. The
Proteome Exploration Laboratory is supported by the Beckman Institute and the National Institutes of Health
(1S10OD02001301). This research benefited from the use of the Autoflex MALDI TOF mass spectrometer in the
Caltech CCE Multiuser Mass Spectrometry Laboratory, acquired with funds from the Dow Corporation. The
authors thank Andy Zhou for assistance with protein overexpression and purification.
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