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S K-edge XAS Studies of the Effect of DNA Binding on the
[Fe
4
S
4
] Site in EndoIII and MutY
Yang Ha
†,‡
,
Anna R. Arnold
§
,
Nicole N. Nuñez
||
,
Phillip L. Bartels
§
,
Andy Zhou
§
,
Sheila S.
David
||,*
,
Jacqueline K. Barton
§,*
,
Britt Hedman
‡,*
,
Keith O. Hodgson
†,‡,*
, and
Edward I.
Solomon
†,‡,*
Department of Chemistry, Stanford University, Stanford, California, 94035, United States
Stanford Synchrotron Radiation Lightsource, SLAC, Stanford University, Menlo Park, California,
94025, United States
§
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena,
California, 91125, United States
||
Department of Chemistry, University of California Davis, Davis, California, 95616, United States
Abstract
S K-edge X-ray Absorption Spectroscopy (XAS) was used to study the [Fe
4
S
4
] clusters in the
DNA repair glycosylases EndoIII and MutY to evaluate the effects of DNA binding and solvation
on Fe-S bond covalencies (ie. the amount of S 3p character mixed into the Fe 3d valence orbitals).
Increased covalencies in both iron-thiolate and iron-sulfide bonds would stabilize the oxidized
state of the [Fe
4
S
4
] clusters. The results are compared to those on previously studied [Fe
4
S
4
]
model complexes, ferredoxin (Fd), and to new data on high potential iron-sulfur protein (HiPIP).
A limited decrease in covalency is observed upon removal of solvent water from EndoIII and
MutY, opposite to the significant increase observed for Fd where the [Fe
4
S
4
] cluster is solvent
exposed. Importantly, in EndoIII and MutY, a large increase in covalency is observed upon DNA
binding which is due to the effect of its negative charge on the iron-sulfur bonds. In EndoIII, this
change in covalency can be quantified and makes a significant contribution to the observed
decrease in reduction potential found experimentally in DNA repair proteins, enabling their HiPIP
like redox behavior.
Graphical Abstract
Corresponding Authors: david@chem.ucdavis.edu, jkbarton@caltech.edu, hedman@ssrl.slac.stanford.edu,
hodgson@ssrl.slac.stanford.edu, solomone@stanford.edu.
Associated Content
Computational results and analysis and addition figures for XAS spectra and protein structure comparison are available free of charge
on the ACS Publications website
http://pubs.acs.org
HHS Public Access
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J Am Chem Soc
. Author manuscript; available in PMC 2017 August 24.
Published in final edited form as:
J Am Chem Soc
. 2017 August 23; 139(33): 11434–11442. doi:10.1021/jacs.7b03966.
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Introduction
Iron-sulfur clusters occur in a wide range of proteins with roles in electron transfer,
catalysis, and regulation.
1
Those involved in electron transfer are the rubredoxins and
ferredoxins. Rubredoxin contains a mononuclear iron center with thiolate ligands, while
ferredoxins contain multinuclear iron-sulfide clusters which includes [Fe
2
S
2
], [Fe
3
S
4
] and
[Fe
4
S
4
] sites.
2
The [Fe
4
S
4
] proteins can be further divided into two categories, the low-
potential ferredoxins (Fd), which utilize the [Fe
4
S
4
]
2+/1+
redox couple with a potential as
low as −600 mV, and high potential iron proteins (HiPIP), which have an [Fe
4
S
4
]
3+/2+
redox
couple with a reduction potential as high as +350 mV.
1
From X-ray crystallography
3
,
Extended X-Ray Absorption Fine Structure (EXAFS)
4
and resonance Raman
5
spectroscopic
results, the [Fe
4
S
4
]
2+
sites in the Fds and HiPIPs are almost identical. They have similar
bond distances and angles, as well as similar vibrational modes. The major difference is in
their protein environments. The [Fe
4
S
4
] site in Fd is solvent exposed while this cluster in
HiPIP is buried.
6
Lyophilization of Fd and unfolding of HiPIP lead to significant changes in
their S K-edge XAS spectra, showing that the H-bonds from solvent water change the Fe-S
bond covalency.
7
The higher covalency in HiPIP stabilizes the oxidized state over the
reduced state, and this significantly contributes to their [Fe
4
S
4
] clusters utilizing different
redox couples from Fd.
1
The [Fe
4
S
4
]
2+
in Fd is less covalent and activated toward reduction,
while the cluster in HiPIP is more covalent, thus tuned toward oxidation.
Endonuclease III (EndoIII) and MutY are DNA glycosylases present in bacteria to humans
that excise oxidized bases or their mispaired base partners as initiating events in the base
excision repair (BER) pathway. Specifically, EndoIII removes oxidized pyrimidines while
MutY removes adenine from A:oxoG mispairs (where oxoG = 8-oxo-7,8-
dihydroguanine).
8
9
The importance of these enzymes is highlighted by the correlation of
inherited defects in human MutY (MUTYH) and early onset colorectal cancer, referred to as
MUTYH-associated polyposis (MAP).
10
EndoIII and MutY both contain an [Fe
4
S
4
]
2+
cluster, which is redox inactive in solution in the absence of DNA.
11
The [Fe
4
S
4
]
2+
cluster in
these enzymes has been shown to be essential for substrate binding and catalysis, but it is
not required for the global structural integrity of the enzyme.
9
However, electrochemical
experiments carried out with EndoIII and MutY on DNA-modified gold electrodes showed
reversible redox signals at potentials ranging from 60–95 mV versus NHE, suggesting that
DNA binding was able to activate these proteins for redox activity. A role for DNA in
activating the cluster for redox activity was confirmed by electrochemistry on highly
oriented pyrolytic graphite (HOPG) electrodes in the presence and absence of DNA; these
experiments revealed that upon binding to DNA, the reduction potential of the [Fe
4
S
4
]
3+/2+
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couple in EndoIII shifted ~ −200 mV into the physiological range corresponding to a
significantly increased DNA binding affinity of the oxidized form of the protein.
12
13
From
electrochemical and
in vivo
experiments, A model has been proposed in which long-distance
DNA-mediated electron transfer between two [Fe
4
S
4
] proteins with similar DNA-bound
redox potentials facilitates the search for damage across a vast genome.
8
,
14
15
If there is no
DNA damage between the two binding sites, the inter-protein charge transfer (CT) can
proceed efficiently, and one of the DNA-bound proteins is reduced, thus its affinity for DNA
is decreased. This protein can then dissociate and diffuse to another region of the genome.
However, if there is a mismatch or lesion that disrupts the
π
-stacking of DNA, charge
transfer will be attenuated and both proteins would remain bound to the DNA in the vicinity
of the lesion. While the CT signaling model has been strongly supported in numerous
studies, the origin of the large DNA-induced potential shift that makes such a damage search
possible has remained puzzling. Electrostatic effects remain the most likely explanation,
given that no significant structural changes are apparent between the free and DNA-bound
forms of EndoIII and MutY. Unfortunately, the electrochemical techniques used in earlier
work cannot reveal the fundamental molecular-scale changes involved, making an alternative
approach necessary to elucidate the origin of this DNA induced potential shift.
S K-edge X-ray Absorption Spectroscopy (XAS) has been developed and applied to
experimentally determine the covalency (
α
2
) of sulfur-metal bonds (i.e. the amount of S 3p
character mixed into the metal 3d antibonding (
Ψ
*
) valence orbitals:
.
16
The S 1s orbital is localized on the S atom and the S 1s
S 3p
transition is electric dipole allowed, thus the intensity of the 1s
→Ψ
*
pre-edge transition
reflects the covalency of this sulfur-metal bond. The energy of the pre-edge transition
reflects the energy of the unoccupied or partially occupied Fe d-orbitals, which depends on
the effective nuclear charge (Z
eff
) of the metal and the nature of the ligand field.
16
The pre-
edge transition energy also depends on the charge of the S; sulfide has a lower Z
eff
than
thiolate, thus the sulfide donor orbitals are at higher energy than those of the thiolate, and
their associated S 1s
→Ψ
*
pre-edge transitions are at lower energy.
16
The intensity of a pre-edge peak
D
0
is given by
(Equation 1)
where
c
is a constant,
r
is the dipole operator,
α
2
is the bond covalency (i.e. sulfur p
character mixed into the metal d orbitals),
N
is the total number of sulfurs bound to the
metal,
h
is the total number of d electron holes, and
I
s
is the S 1s
3p electric dipole
integral, which is also dependent on the Z
eff
of the S ligand and has been experimentally
determined for thiolate and sulfide ligands in previous work
17
. Our previous XAS studies on
[Fe
4
S
4
] sites showed that the contributions of thiolate and sulfide can be distinguished at the
pre-edge, with the μ
3
-sulfide pre-edge ~0.7 eV lower than the thiolate.
6
,
18
Quantitatively,
for the [(RS)
4
Fe
4
S
4
]
2+
clusters, there are 9
α
and 9
β
holes (ie. unoccupied valence orbitals),
which have mainly Fe 3d and S 3p character
6
, thus the maximum covalency value possible is
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1800%. In particular, 1 unit of sulfide intensity corresponds to 30.6% S character per Fe-S
bond, while 1 unit of thiolate corresponds to 70.8% S character in unoccupied valence
orbitals. This difference reflects the different number of bonds to each Fe (3 from sulfides
and 1 from thiolate) and the difference in dipole integral due to the Z
eff
of sulfide relative to
thiolate S (6.54 vs 8.47).
17
Note that only the sulfur atoms bound to the Fe contribute to the
pre-edge feature in the XAS spectra, while all sulfur atoms including the free Cys and Met
residues contribute to the edge. Thus the pre-edge intensity directly reflects the covalency of
the thiolate-Fe and bridging sulfide-Fe bonds, but the edge normalization must be corrected
for the total number of sulfurs in the protein.
The total pre-edge intensity, and therefore S covalency, in HiPIP is significantly higher than
in Fd. The pre-edge intensity in a relevant alkyl thiolate [Fe
4
S
4
] model complex is even
slightly higher than in HiPIP.
7
Importantly, there is a direct correlation between the total S
covalency and the redox potential of the [Fe
4
S
4
] clusters, with an increase of 1% of total S
covalency corresponding to ~3.3 mV decrease in redox potential
7
which reflects the fact that
higher covalency stabilizes the oxidized over the reduced state of the [Fe
4
S
4
] cluster. With
respect to DNA repair proteins, the power of S K-edge XAS to monitor changes in [Fe
4
S
4
]
cluster covalency in different environments and to correlate these changes to redox potential
results in an ideal method to elucidate the origin of the DNA-induced potential shift at a
molecular level.
In the present study, S K-edge XAS is applied to experimentally measure the Fe-S bond
covalency of the [Fe
4
S
4
] clusters in EndoIII and MutY, both in the absence of and bound to
DNA, and with and without solvent water. The effect of DNA binding on the S K-edge
intensity and hence covalency is correlated to the reduction potential of the cluster according
to a relationship defined in previous XAS studies on [Fe
4
S
4
] clusters
7
, and is in agreement
with the electrochemically observed reduction potential decrease upon DNA binding.
12
The
solvent effect is compared to those previously observed for Fd
7
and for the HiPIP proteins in
this study. This work provides direct molecular evidence for the proposal that the negative
charge of bound DNA tunes the potential of [Fe
4
S
4
] clusters and shows that this involves a
change in the covalency of the cluster that enables the [Fe
4
S
4
]
3+/2+
redox couple when
EndoIII (and MutY) are bound to DNA.
Experimental
Expression and purification of EndoIII
WT
E. coli
EndoIII was overexpressed in BL21star-(DE3)pLysS cells containing a pET11-
ubiquitin-His
6
-
nth
construct and purified as detailed previously
19
, with the exception that the
final buffer contained 10% glycerol, rather than 20% glycerol (20 mM sodium phosphate,
pH 7.5, 0.5 mM EDTA, 150 mM NaCl, 10% glycerol, Buffer A). EndoIII is less stable in the
absence of glycerol; therefore glycerol was not removed until the day of sample preparation.
Glycerol was removed from half the volume of protein solution using HiPrep 26/10
desalting column (GE Healthcare) equilibrated with Buffer A lacking glycerol. Next, the
protein solutions either containing 10% glycerol or no glycerol were separately concentrated
first with 10,000 MWCO (molecular weight cutoff) Amicon Ultra 15 mL centrifugation
filter units (Millipore) and then with 10,000 MWCO Amicon Ultra 0.5 mL centrifugation
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filter units (Millipore) until the protein solutions were very dark colored, to approximately
300 μL each if using an entire protein preparation from 6 L of bacterial culture.
Expression and purification of MutY
WT MutY was expressed as an N-terminal fusion with MBP (Maltose Binding Protein) to
increase the solubility at the concentrations needed for XAS experiments. Of note, the MBP
tag has additional Met residues that can complicate XAS data analysis. The MBP-MutY
protein was overexpressed in BL21 DE3 competent cells and purified as detailed
previously
13
, with the following modification. Pelleted cells from overexpression were re-
suspended in resuspension buffer (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 1mM
EDTA, 10 % glycerol, 1 mM PMSF), sonicated on ice and centrifuged at 12,000 RPM for 15
minutes. The supernatant was saved in a conical tube on ice, and the pellet was re-suspended
and underwent a repeat of sonification and centrifugation. The supernatant was batch bound
to amylose resin (New England BioLands) for one hour, poured over a PD10 column and
washed with amylose wash buffer (20 mM sodium phosphate, pH 7.5, 200 mM NaCl, 1mM
EDTA) and eluted in amylose elutant buffer; amylose wash buffer with the addition of 10
mM maltose). The elutant was concentrated using an ultrafiltration cell with a 10,000
MWCO filter with stirring at 4°C. Protein was then diluted 10-fold in heparin buffer A (20
mM sodium phosphate, pH 7.5, 1 mM EDTA, 5% glycerol). The sample was applied to a
Pharmacia Hi-trap heparin column on an AKTApurifier FPLC system, and eluted using a
10% linear gradient in heparin buffer A to 100% heparin buffer B and buffer A with the
addition of 1 M NaCl). Fractions corresponding to MBP-MutY were combined and
concentrated using an ultrafiltration cell with a 10,000 MWCO filter with stirring at 4 °C, to
approximately 10mL. Purity of MBP-MutY samples were confirmed via 12 % SDS page
stained with SYPRO orange and 7.5 % acetic acid.
DNA preparation
DNA strands for EndoIII studies were purchased from Integrated DNA Technologies (a 20-
mer mixed sequence strand: 5
-GTGAGCTAACGTGTCAGTAC-3
and its complement).
DNA strands (5 μmol) were resuspended in MilliQ water (200 μL), and purified by ethanol
precipitation. The purified strands were resuspended in EndoIII Buffer A or Buffer A
lacking glycerol and quantified based on calculated
ε
260
values for the strands (Integrated
DNA Technologies) of 197,800 M
−1
cm
−1
for the 20-mer strand and 190,200 M
−1
cm
−1
for
its complement. Annealing of the strands in either Buffer A or Buffer A without glycerol
was accomplished by combining equimolar amounts of the single-stranded DNAs, heating at
90°C for 5 minutes, and slowly cooling to room temperature.
DNA strands for MutY studies containing oxoG or FA (2
-fluoro-adenine) were synthesized
at the University of Utah DNA and Peptide Synthesis Core Facility and unmodified strands
were purchased from Integrated DNA Technologies. Two lengthed DNA duplexes were
used, a 15-mer (5
-GGAGCCAXGAGCTCC-3
and its compliment 5
-
GGAGCTCYTGGCTCC -3
), and a 30-mer (5
-
CGATCATGGAGCCACXAGCTCCCGTTACAG-3
and its compliment 5
-
CTGTAACGGGAGCTYGTGGCTCCATGATCG-3
) where X = G or oxoG and Y = C or
FA. Oligonucleotides containing the central oxoG or FA were deprotected and cleaved from
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the solid support by incubation in NH
4
OH, with the addition of 2-mercaptoethanol to oxoG
samples to prevent further oxidation. The cleaved DNA substrates were dissolved in H
2
O,
filtered with a 0.2 μm filter, and HPLC purified using a Beckman Gold Nouveau system with
a Waters AP1DEAE 8HR column with a 10–100% gradient of 90:10 H
2
O/acetonitrile with 2
M NH
4
Ac. Isolated fractions were lyophilized and de-salted with a SEP-PAK C18 column,
and DNA integrity was confirmed using MALDI-MS. The purified strands were
resuspended in MBP-MutY concentration buffer with or without glycerol and quantified
based on calculated
ε
260
values for the strands (Integrated DNA Technologies) of 145,800
M
−1
cm
−1
for the 15-mer strand with central G and 131,700 M
−1
cm
−1
for its complement,
136,000 M
−1
cm
−1
for the 15-mer strand with central oxoG and 137,900 M
−1
cm
−1
for its
complement, 285,200 M
−1
cm
−1
for the 30-mer strand with central G and 280,800 M
−1
cm
−1
for its complement, and lastly 279,600 M
−1
cm
−1
for the 30-mer strand with central oxoG
and 287,000 M
−1
cm
−1
for its complement. Annealing of the strands in either MBP-MutY
concentration buffer with or without glycerol was accomplished by combining equimolar
amounts of the single-stranded DNAs, heating at 90°C for 5 minutes, and allowed to slowly
anneal overnight to 4°C.
XAS sample preparation
To prepare the EndoIII XAS samples, concentrated solutions of EndoIII protein were mixed
with the DNA duplex at a ratio of 1 mol EndoIII: 20 mol base-pairs DNA. An equivalent
volume of buffer was added to EndoIII alone solutions so that identical EndoIII
concentrations were obtained both with and without DNA. Mixtures were allowed to
incubate on wet ice for 30 minutes to allow for binding before freezing in liquid nitrogen.
The no glycerol samples were lyophilized. Both samples were placed on dry ice and sent to
Stanford University for measurement. The EndoIII [4Fe4S] cluster loading was calculated
using the protein concentration determined by UV-visible absorbance at 410 nm that is
specific to [4Fe4S] clusters (
ε
410
= 17,000 M
−1
cm
−1
) relative to the protein concentration
determined by Braford Assay; samples were typically 70–75% loaded with cluster.
To prepare the MutY XAS samples, purified MBP-MutY samples were buffer exchanged in
concentration buffer, with the final buffer consisting of 20 mM sodium phosphate, pH 7.5,
150 mM NaCl, 1mM EDTA, 10% glycerol for samples to remain in buffer, or concentration
buffer lacking glycerol for samples to be lyophilized for XAS. The protein sample was
concentrated to a final concentration of 1.2mM. 125 uL of the purified MBP-MutY was
incubated with 25uL of the 6mM annealed DNA duplex at 25 °C for 30 minutes to afford a
final concentration of 1mM for both protein and DNA. Following lyophilization or
immediately for samples in buffer, samples were snap frozen in liquid nitrogen, stored at
−80 °C or on dry ice, and were thawed on wet ice just prior to XAS experiments. The MutY
[4Fe4S] cluster loading was determined using the UV-visible absorbance at 410 nm (
ε
410 =
17,000 M
−1
cm
−1
) and at 280 nm (
ε
280 = 143,240 M
−1
cm
−1
); samples were typically 65–
75% loaded with cluster.
S K-edge XAS
Sulfur K-edge XAS data were measured at the Stanford Synchrotron Radiation Lightsource
on the unfocussed 20-pole, 2.0-Tesla wiggler Beam Line 4–3, under SPEAR3 storage ring
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parameters of 3 GeV and 500 mA. A Ni-coated, flat, bent pre-monochromator mirror was
used for harmonic rejection and vertical collimation. A Si(111) double crystal
monochromator was used for energy selection. The energy calibration, data reduction and
error analysis follow the methods described in reference.
20
Solid samples were ground into a
fine powder and dispersed as thinly as possible on Kapton tape to minimize potential self-
absorption effects. The solution samples were loaded into 50μL teflon cells, with Kapton
tape as the back window, and 6 μm-thick, sulfur-free polypropylene film as front window. A
shutter was inserted automatically during each monochromator move to minimize
photoreduction. The photon energy was calibrated to the maximum of the first pre-edge
feature of Na
2
S
2
O
3
·5H
2
O at 2472.02 eV. At least three scans were measured for each
sample to ensure reproducibility. Raw data were calibrated and averaged using MAVE in the
EXAFSPAK package.
21
Using the PySpline program
22
, the background was removed from
all spectra by fitting a second-order polynomial to the pre-edge region and subtracting this
from the entire spectrum. Normalization of the data was accomplished by fitting a straight
line to the post-edge region and normalizing the edge jump to 1.0 at 2490.0 eV. The error
from background subtraction and normalization is less than 3%. Intensities of the pre-edge
features were quantified by fitting the data with pseudo-Voigt line shapes with a fixed
Lorentzian to Gaussian ratio of 1:1, using the EDG_FIT program.
21
Pre-edge energies and
widths of single peaks were locked based on the previously published results on [Fe
4
S
4
]
models and proteins (Supporting Info)
6
. The error from the fitting procedure is less than 3%.
The fitted sulfide and thiolate intensities were converted to %S 3p character according to
reference
17
.
Note that all the perturbations (lyophilization, mixing with DNA, and the combination) on
the EndoIII and MutY were done on aliquots of the same protein sample. This means that all
the data on each protein presented in this study have the same loading ratio, thus the
differences observed upon DNA binding and lyophylization are independent of the loading.
DFT calculations
DFT calculations with broken symmetry spin polarization were performed using Gaussian
09
23
, with the pure functional BP86, and with 6–311G(d) basis sets on Fe and S, and 6–
31G(d) basis sets on C and H. This functional and basis set were chosen to be consistent
with previous studies.
7
The
α
-carbons of the 4 Cys ligands were fixed to their crystal
structure positions
24
during geometry optimizations. To qualitatively evaluate the
electrostatic effect of DNA binding, a point charge was placed at 5 Å from either a sulfide or
a thiolate S atom in the [Fe
4
S
4
] cluster, and the electronic structure was reoptimized.
Results and Analysis
1) EndoIII without and with DNA
Sulfur K-edge XAS spectra of EndoIII in the absence and presence of a 20-mer mixed
sequence DNA duplex (5
-GTGAGCTAACGTGTCAGTAC-3
and its complement) at a
ratio of 1 mol protein to 20 mol base-pairs were measured in both solution and lyophilized
forms. Protein concentrations at 1 mM or greater were used for high quality XAS data.
Equal concentrations of EndoIII were compared without and with the DNA 20-mer. The data
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