of 25
28
Supporting Information: Yeast Require Redox Switching in DNA Primase
O’Brien, E.
; Salay, L.E.
, et al
.
Supplementary Materials and Methods
Figures S1 to S13
Tables S1 to S3
www.pnas.org/cgi/doi/10.1073/pnas.1810715115
29
Supplementary
Materials and Methods
Protein Expression and Purification.
Yeast p58C (Pri2) residues 316
-
512 were cloned into
pGB100 using the BamHI and NotI restriction sites. This plasmid was transformed into Rosetta
DE3 cells. The cells were grown to an OD
600
of 1.0
at 37
°C
. They were then induced with
1
mM
isopropyl β
-
D
-
1
-
thiogalactopyranoside (IPTG) and transferred to 20
°C
for 16 hr. The cells
were harvested and stored at
-
80
°C
.
For purification, the cells pellets were thawed and resuspended in 50
mM
NaH
2
PO
4
(pH
7.8),
500
mM
NaCl, 10
mM
imidazole. Dnasin and lysozyme were added at 2 mg/g of
cells. Protease
inhibitor tablets (Roche) were added to the suspension. The suspension was homogenized using
a Dounce homogenizer and then sonicated on ice for 10 min. The
solution was spun at 50,000
rcf for 20 min at 4
°C
. The supernatant was filtered and passed over a Ni
-
NTA column (GE
Healthcare). The column was washed with 3 column volumes of
50
mM
NaH
2
PO
4
(pH 7.8), 500
mM
NaCl, 20
mM
imidazole. The protein was eluted
with 50
mM
NaH
2
PO
4
(pH 7.8), 500
mM
NaCl, 250
mM
imidazole. The eluent was dialyzed overnight at 4
°C
into 50
mM
NaH
2
PO
4
(pH
7.8), 500
mM
NaCl, 10
mM
imidazole and the
6xHis tag was cleaved using H3C protease. The
dialyzed protein was repassed over the Ni
-
NTA column as in the previous step. The flow
-
through and wash fractions were collected. These fractions were dialyzed into 20
mM
HEPES
(pH 7.2), 50
mM
NaCl, 2
mM
dithiothreitol (
DTT) (Heparin Buffer A), then
was filtered and
passed over a 5 ml Hi
-
Trap HP h
eparin column (GE Healthcare) that had been pre
-
equilibrated
with Heparin Buffer A.
The protein was eluted in
Heparin Buffer B
(
20
mM
HEPES (pH 7.2), 1
M NaCl, 2
mM
DTT
)
using a linear gradient of 0 to 100% over 40 column volumes. The protein
typically elu
ted
at
~20% Heparin Buffer B (250
mM
NaCl). The protein
was
dialyzed into 20
mM
HEPES (pH 6.8),
2
mM
DTT,
and
200
mM
NaCl,
then
flash frozen in N
2
, and stored at
-
80
°C
.
Site Directed Mutagenesis.
Site directed mutagenesis for single
-
site and
multi
-
site variants was
performed using a Q5 mutagenesis kit (New England Biolabs). The primers to create the
mutations are listed below. The same reverse primer was used for all mutations. The annealing
temperature for amplification was 58
°C
.
Site
directed mutagenesis primers
Mutation
Primer
Y395F
5’GAGAAGTTCAATAAAGAATTCCGTTACAGCTTCAGGC
Y397L
5’GAGAAGTTCAATAAAGAACTGCGTTACAGCTTCAGGC
Y397F
5’GAGAAGTTCAATAAAGAATACCGTTTCAGCTTCAGGC
Y397L
5’GAGAAGTTCAATAAAGAATACCGTCTGAGCTTCAGGC
Reverse
primer
5’CATTGTCATGTTCCCATTTCTTGTAAATGC
30
Oligonucleotide preparation.
All standard or modified phosphoramidites
and DNA synthesis
reagents were purchased from Glen Research. Unmodified DNA oligonucleotides for
electrochemical experiments were purchased from Integrated DNA Technologies, Inc. DNA
sequences for electrochemistry assays are shown in
Table S1
. Thiol
-
modi
fied DNA strands for
electrochemistry were made on an Applied Biosystems 3400 DNA synthesizer, with a C6 S
-
S
phosphoramidite incorporated at the 5’
-
terminus. Single
-
stranded DNA was purified using
standard procedures as described previously (1). High pres
sure liquid chromatography (HPLC)
using a reverse
-
phase PLRP
-
S column (Agilent) was used, and oligonucleotide mass confirmed
using MALDI
-
TOF Mass Spectrometry. Thiol
-
modified strands were reduced after the initial
HPLC purification with 100
mM
dithiothreit
ol (Sigma) for 2
-
3 h in 50
mM
Tris
-
HCl, pH 8.4, 50
mM
NaCl. Reduced thiol
-
modified DNA was purified by size exclusion chromatography (Nap5
Sephadex G
-
25, GE Healthcare) and subsequent reverse
-
phase HPLC. Single
-
stranded
oligonucleotides were then desalted
using ethanol precipitation and stored in low salt
buffer
(5
mM
Phosphate, pH 7.0, 50
mM
NaCl). Duplex DNA for electrochemistry was prepared by
quantification of the complementary single
-
stranded oligonucleotides by UV
-
Visible
spectrosco
py, followed by annealing at 90
°C
. A mixture of equimolar complementary single
-
stranded DNA (50
μM
) was pr
epared in low salt buffer. Thiol
-
modified duplex DNA substrates
were then deoxygenated by bubbling argon gas through the solution for 90
-
180
s. Duplex DNA
was annealed on a thermocycler (Beckman Instruments) by initial heating to 90
°C
, followed by
slow co
oling to 4
°C
over 90 minutes. DNA was quantified using absorbance at 260 nm, with
extinction coefficients at 260 nm for DNA obtained using Integrated DNA Technologies online
OligoAnalyzer tool. Single
-
stranded DNA substrates were quantified using UV
-
Visib
le
spectroscopy and stored in low salt buffer at a stock concentration for activity assays.
Multiplexed Chip Fabrication
. DNA was prepared as described in the
SI Text.
Multiplexed
electrode platforms were prepared using standard photolithography technique
s, adapted from
established protocols (1
-
3). Nine 1 in. by 1 in. chips were patterned on 525 um thick silicon
wafers (SiliconQuest). A thermal oxide layer roughly 4000 Å thick was grown on the silicon
wafers using a Tytan tube furnace (Tystar). S1813 photo
resist (2 μm layer) was deposited onto
the wafers for patterning of the chips before metal deposition. Electron beam evaporation (CHA
Industries) was then used to deposit a 3
nm titanium adhesion layer followed by a 100nm gold
layer, without breaking vacuu
m between depositions. Metal lift
-
off using Remover PG
(MicroChem) was performed overnight (10
-
12 h) at room temperature. Wafers were
subsequently dried with a nit
rogen gun and dehydrated at 140
°C
for 10 minutes. A 3
m layer
of insulating SU
-
8 photoresis
t was deposited and patterned onto the wafer as described
previously (1
-
3), with connective wires between contact pads on the edges of the chips and
working electrodes in the center were covered but the contact pads and working electrodes left
exposed. Thi
s ensured a fixed working electrode surface area of 2
mM
2
. SU
-
8 photoresist was
cured (150
°C
, 15 minutes) and wafers cleaved into individual chips using
a Dynatex
Scriber/Breaker or broken manually after scoring with a diamond tip scriber.
DN
A
-
Modified Electrode Assembly/Preparation
. Chips were dried thoroughly using argon
gas and ozone
-
cleaned for 20 minutes at 20 mW using a Uvo brand ozone cleaner. Clean chips
31
were assembled onto polycarbonate holders with acrylic clamp and Buna
-
N rubber gas
ket
according to previous protocols, with four quadrants in the chip separated by fastened gasket and
clamp (1). Duplex DNA substrates, with a thiol modifier at the 5’
-
end, (25
μM
) were deposited
in a 20
μL
volume onto each quadrant
of
the multiplex chip
. Substrates incubated for 18
-
24
hours on the gold surface to allow formation of self
-
assembled DNA monolayer. DNA
monolayers were washed with phosphate buffer (5
mM
phosphate, pH 7.0, 50
mM
NaCl, 5%
glycerol) and subsequently backfilled with 1
mM
6
-
mercap
tohexanol (Sigma) in phosphate
buffer for 45 minutes. Monolayers are then washed 10 times per quadrant with phosphate buffer
and twice per quadrant with TBP buffer (5
mM
phosphate, pH 7.0, 50
mM
NaCl,
4
mM
MgCl
2
,
4
mM
spermidine) to aid in formation of a monolayer with termini accessible for
p58C binding.
Sample preparation for electrochemistry.
Wild type and mutant p58C samples were stored
prior to experiments in p58C storage buffer (20
mM
Tris, pH 7.2, 75
mM
NaCl) or
crystallography buffer (20
mM
HEPES, pH 6.8, 200
mM
NaCl, 2
mM
DTT). All p58C variants
were transferred to HEPES electrochemistry buffer (20
mM
HEPES, pH 7.2, 75
mM
NaCl)
using Amicon ultra centrifugal filters (0.5
mL
, 3MWCO)
(Millipore Sigma). Protein was applied
in a 90
-
140
μL
volume to the filter and centrifuged for 15 minutes at 14000 x g at 4
°C
. After
centrifugation, 400
μL
of HEPES electrochemistry buffer was applied to the filter and
centrifuged at 14000 x g for 20 minu
tes. This was repeated four times to exchange the p58C
protein into HEPES electrochemistry buffer. After buffer exchange and recovery of sample by
centrifugation (2 minutes, 1000 x g), concentrations of [4Fe4S] cluster
-
containing p58C or
mutants were measu
red using UV
-
Visible spectroscopy, by absorbance of the [4Fe4S] cluster at
410 nm (extinction coefficient = 17000 M
-
1
cm
-
1
) (4). Recovered samples (approx. 100
-
150
μL
volume) were deoxygenated for 2
-
3 minutes with argon. Samples were then transferred into
the
anaerobic chamber (Coy Laboratory products). Before deposition onto the gold electrode
surface, p58C/mutant samples were diluted to a molar concentration
of
30
μM
or
57
μM
[4Fe4S]
p58C variant with previously deoxygenated
HEPES
electrochemistry buffer
.
Mutant Selection and Design.
Mutations in the p58C domain of human DNA primase were
designed based on previously determined structural data (5
-
8) bioinformatics (6) compiled for
conserved residues in this domain. Chimera software was used to analyze the
relevant residues
in the 1.54 Å resolution structure of yeast p58C (6) (PDB 3LGB). Distances between tyrosine
residues were measured between centroids.
Circular Dichroism.
Near
-
UV circular dichroism was performed as described in (9).
Fluorescence anisotropy.
Fluorescence anisotropy was performed as described in (3). Yeast
p58C and the related mutants were assayed in a buffer containing 20
mM
HEPES (pH 6.8),
2
mM
DTT, and 75
mM
NaCl.
32
DNA Electrochemistry Measurements of p5
8C Variants
. Multiplex gold electrodes were part
of a three
-
electrode system with an external Ag/AgCl reference electrode (Bioanalytical
Systems) and platinum counter electrode. Initial cyclic
voltammetry
scans of the monolayers in
p58C buffer were perform
ed to ensure monolayer formation on each electrode. All washes were
performed with 20
μL
buffer volumes on each quadrant. Before scanning, a 200
μL
volume was
deposited over the chip surface, a bulk solution well for completion of a three
-
electrode circui
t
with an external reference and counter electrode. Cyclic
voltammetry
scans were performed at
100 mV/s scan rates, over a potential range of +0.412 V to
-
0.288 V vs. NHE or +512
mV to
188
mV vs NHE. Bulk electrolysis on DNA was performed at an applied p
otential of +0.412 V
vs. NHE
or
+512mV vs. NHE for all electrochemical oxidation reactions and
-
0.188 V vs. NHE
for all electrochemical reduction reactions. The oxidizing potential was applied for at least 8.33
minutes for single oxidation reactions on a s
urface, and 6.67 minutes for the iterative oxidation
cycles of p58C variants. The reducing potential
was
applied for 8.33 minutes in all
electrochemical reduction reactions. All bu
lk electrolysis and cyclic
voltammetry
was performed
in previously deoxygena
ted p58C storage buffer (20
mM
HEPES, pH
7.2,
75
mM
NaCl). Charge
transfer (nC) in the cathodic peak of oxidized samples CV scans was assessed using the area
under the current wave of the reduction signal. Variants of p58C were compared for charge
transfer
proficiency using at least three trials of oxidation at +0.412 V vs.
NHE or +512
mV vs.
NHE, after at least 8.33 minutes of oxidation at the applied
potential. Charge transfer was
measured for oxidized samples using CHI software, assessing the area under
the reductive peak
in CV after electrochemical oxidation. Yields for bulk electrolysis were assessed by subtracting
the total charge reported in Coulombs from the product of the electrolysis time (s) and the final
current value (A). NTP
-
dependence of elect
rochemical
signals were measured by pipetting a
small volume (1
-
3
μL
) of 0.1
M ATP stock solution into each quadrant of the multiplexed chip
setup. These stocks should be added by quadrant, rather than into the bulk solution. Divide the
NTP stock into quar
ters and pipet ¼ of the total solution onto a quadrant. Samples were added
by quadrant, as physical barriers in the setup prevent diffusion of NTPs between electrode
quadrants. After the volume of ATP stock was deposited onto the electrode quadrant, result
ing in
a 2.5
mM
or 5
mM
concentration of ATP in the quadrant, CV scans were measured at a 100
m
V/s scan rate. Square wave
voltammetry
in both reductive and oxidative sweeps was measured
using a sy
mm
etric square wave form (25
mV amplitude),
superimposed on
a staircase (E
step
= 4
mV). Scans were performed at 15 Hz frequency, 60
mV/s
scan rate. Charge transfer was
assessed using CHI software; charge values were determined by calculation of the area under the
reductive and oxidative peak curves.
Midpoint potentials of NTP
-
dependent redox signals were
assessed using the peak selection function in CHI software.
X
-
ray Crystallography.
Crystals were grown at 16
°C
by mixing 1:1 yeast p58C variants (~5
mg/ml) and mother liquor (100
mM
TRIS (pH 8.5), 4
0
-
65% MPD) using hanging drop vapor
diffusion. Crystals formed after 2 days. They were then looped, transferred to a cryo
-
protectant
containing the mother liquor and 20% glycerol.
Data collection for yeast p58C Y397F was performed at 100 K using an in
-
hous
e
Bruker Microstar rotating anode x
-
ray generator with a Bruker Proteum PT135 CCD area
detector at a wavelength of 1.54 Å. Sulfur single anomalous dispersion (SAD) was used to phase
this data set with the program suite HKL2MAP (10). The model was built usi
ng arp/Warp in the
CCP4 suite of programs (11) followed by iterative rounds of refinement using Phenix (12)
and
Coot
(13)
.
33
Crystallographic data for the remaining variants were collected at the Advanced Photon
Source (Argonne National Laboratory, Chicago, IL) on Life Sciences Collaborative Access
Team beamlines. Data were indexed and scaled using HKL2000 (14). Molecular replac
ement
using Phaser (15) with the model of the yeast p58C variant Y397F was used to solve the phases
of the remaining mutants. The models were refined iteratively using Phenix (12) and
Coot
(13).
Access to these programs was provided by SBGrid (16)
.
Yeast
Strain Construction.
Integration plasmids were constructed by subcloning a
PRI2
C
-
terminal domain (CTD) fragment of plasmid PRI2
-
3HA (
17
,
18
) into the pRS406 vector (
19
) and
mutations were created using overlap
-
extension PCR (
20
). Silent nucleotide changes
were
included to create restriction site polymorphisms tightly linked to each mutation. Plasmids
linearized with
Age
I
-
HF (NEB) were transformed into YKF201 and Ura+ transformants were
subsequently plated on media containing 5
-
fluoroorotic acid. Chromosoma
l integration of all
mutations was verified by restriction digestion and sequencing (cells losing the
URA3
marker by
recombination also lost the 3HA tag). To test for viability of the
pri2
-
Y395L
allele, strain
YKF201 was transformed with the complementing
plasmid pRS416
-
PRI2,
containing a 3.1kb
fragment amplified from yeast genomic DNA inserted at the
Bam
HI and
Eco
RI sites of pRS416.
PRI2
-
3HA plasmids (either WT or containing the Y397F or Y497L alleles) were linearized with
Age
I
-
HF (NEB) and integrated at t
he
PRI2
locus with selection on media lacking uracil and
tryptophan. Individual transformants were verified by PCR and sequencing, then restreaked on
media lacking uracil and tryptophan to confirm growth and on media lacking tryptophan and
containing 5
-
flu
oroorotic acid (5
-
FOA) to select for loss of the complementing plasmid.
For inducible overexpression of PRI2 alleles, the open reading frame of
PRI2
was
cloned into plasmid p416
-
Gal1 at the
Bam
HI and
Xba
I sites (
21
). The Y397L mutation was
incorporated by
subcloning from the integration plasmid described above. All plasmids were
verified by sequencing. Overexpression plasmids were transformed into haploid strain
YKF1675 (
MAT
a
::DEL.HOcs::HisG ura3 trp1 leu2::KAN hml::hisG HMRa
-
stk
; isogenic to
strain
CL11
-
7) (
22
) with selection on media lacking uracil. Individual transformants were
restreaked on media lacking uracil and containing 2% glucose or galactose. Sequences of
oligonucleotides utilized for cloning and mutagenesis are available by request.
34
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36
Figure S1
. P58C does not produce a redox signal
on ss/dsDNA in the absence of
electrochemical alteration. CV performed on 30
μM
[4Fe4S] p58C in Tris storage buffer
(light grey trace) and 40
μM
[4Fe4S] p58C in HEPES storage buffer (dark grey trace) using a
Ag/AgCl reference electrode is shown. Buffer con
ditions were 20
mM
Tris buffer, pH 7.2, 75
mM
NaCl or 20
mM
HEPES, pH 7.2, 75
mM
NaCl. CV scanning was performed in anaerobic
conditions, at a 100
mV/s scan rate, for both samples.
37
0
5
10
15
20
25
30
0
-
200
-
400
-
600
Time Elapsed (minutes)
Figure S2
. Bulk oxidation of WT p58C (
S. cerevisiae
), shown in red, compared to a buffer
blank control (grey). The yield for oxidation is
relatively low, but the technique is effective in
converting the protein from the [4Fe4S]
2+
form to the [4Fe4S]
3+
form. Electrochemical
oxidation was performed in anaerobic conditions at an applied potential of 512mV vs. NHE,
on 10
mM
[4Fe4S] p58C in 20
mM
MES, pH 7.2, 75
mM
NaCl, 10% glycerol.
Buffer
10uM Yeast p58C
Current (nA)
38
Figure S3.
WT and mutant yeast p58C biophysical characterization. left) Circular dichroism
(CD) spectroscopy of WT and mutant p58C indicate the mutations do not perturb any of the
elements of
secondary structure. All spectra normalized to WT at 222 nm. right) UV
-
Visible
spectroscopy of WT and mutant p58C shows similar 280 nm/410 nm absorbance ratios,
indicating similar degrees of [4Fe4S] cluster cofactor loading in all variants. All spectra
nor
malized to WT at 410 nm.
39
Figure S4
. Fluorescence anisotropy measuring DNA binding of p58C variants. WT and
tyrosine mutants bind DNA with similar affinity, suggesting that differences in
electrochemical signals are due to
differences in redox proficiency.
40
Figure S5.
CV scans of yeast p58C Y395F. a) Electrochemically unaltered p58C Y395F
mutant displays no electrochemical signal on DNA. b) p58C Y395F tyrosine mutant displays
a cathodic peak after oxida
tion at an applied potential of +412
mV vs. NHE, but the peak is
significantly smaller than that observed for wild type. c) p58C Y395F displays no redox
signal
on DNA after electrochemical reduction at an applied potential of
-
188
mV vs. NHE. All
scans per
formed on 30
μM
[4Fe4S] p58C Y395F, in
20
mM
Tris, pH 7.2, 75
mM
NaCl, at a
100mV/s scan rate.
41
Figure S6
. Cyclic
voltammetry
of electrochemically unaltered WT p58C (top left, grey trace)
and p58C Y397L (top right, grey trace) and elect
rochemically reduced WT p58C (bottom left,
red trace) and p58C Y397L (bottom right, red trace). Electrochemically unaltered and
electrochemically reduced variants under identical conditions display no redox activity,
suggesting that the unaltered samples i
s predominantly in the redox
-
inert, [4Fe4S]
2+
form. All
CV scans were performed in anaerobic conditions, with 57
μM
[4Fe4S] WT p58C or 40
μM
[4Fe4S] Y397L in 20
mM
HEPES, pH 7.2, 75
mM
NaCl. Scans were performed at 100
mV/s
scan rate.
42
Figure S7.
CV scans of yeast p58C Y395L. a) Electrochemically unaltered p58C Y395L
mutant displays no electrochemical signal on DNA. b) p58C Y395L tyrosine mutant displays
a cathodic peak after oxidation at an applied potential of +512
mV vs. NHE, but th
e peak is
significantly smaller than that observed for wild type. c) p58C Y395L displays no redox
signal on DNA after electrochemical reduction at
an
applied potential
of
-
188
mV vs. NHE.
All scans performed on 57
μM
[4Fe4S] p58C variant,
in
20
mM
HEPES,
pH 7.2, 75
mM
NaCl, at a 100
mV/s scan rate.
43
Figure S8.
Electrochemistry of yeast p58C Y395L in the presence of 1.25
mM
ATP
(orange)
and 2.5
mM
ATP (red)
CV scans
(above)
show no
measurable
redox signaling activity for
p58C Y395L in the presence of DNA and NTPs. below) SWV of yeast p58C Y395L in the
presence of 1.25
mM
ATP. Reductive (left) and oxidative (right) scans show no measurable
redox signaling activity in this variant.
P58C Y395L i
s CT
-
deficient in the presence of DNA
and NTPs.
All scans performed in anaerobic conditions on 57
μM
[4Fe4S] p58C Y395L and
1.25
mM
ATP, in 20
mM
HEPES, pH 7.2, 75
mM
NaCl, at a 100
mV/s scan rate for CV or a
15 Hz frequency, 25
mV amplitude for SWV.
-80
-40
0
40
80
-0.4
-0.2
0
0.2
0.4
0.6
Current (nA)
Potential (V vs. NHE)
44
Figure S9.
Electrochemistry of yeast p58C Y395F in the presence of 2.5
mM
ATP. above) CV
scans show some redox signaling activity in the presence of DNA and NTPs. Even in the
presence of a higher concentration of ATP, this variant displ
ays diminished redox signaling
relative to WT. below) SWV of yeast p58C Y395F in the presence of 2.5
mM
ATP. Reductive
(left) and oxidative (right) scans show
only a small amount of
redox signaling activity in this
variant.
All scans performed in anaerobi
c conditions on 40
μM
[4Fe4S] p58C Y395F and 2.5
mM
ATP, in 20
mM
HEPES, pH 7.2, 75
mM
NaCl, at a 100
mV/s scan rate for CV or a 15 Hz
frequency, 25
mV amplitude for SWV. Arrows indicate reductive and oxidative signals.
45
Figure S10.
Electrochemistry of yeast p58C Y397F in the presence of 2.5
mM
ATP. left) CV
scans show a small peak in the reductive wave near
-
80
mV vs. NHE, which is likely a
[3Fe4S]
+
oxidative degradation product. right) SWV of yeast p58C Y397F in the
presence of
2.5
mM
ATP. All scans performed in anaerobic conditions on 57
μM
[4Fe4S] p58C Y397F and
2.5
mM
ATP, in 20
mM
HEPES, pH 7.2, 75
mM
NaCl, at a 100
mV/s scan rate for CV or a 15
Hz frequency, 25
mV amplitude for SWV. Arrows indicate [3Fe4S]
+/0
red
uctive peak.
46
Figure S11.
Electrochemistry of yeast p58C Y397L in the presence of 2.5
mM
ATP. left) CV
scans show a small reversible peak centered at 140 ± 17 mV vs. NHE. right) SWV of yeast
p58C Y397L in the presence of 2.5
mM
ATP. This variant shows a small amount of
measurable redox signaling activity, though it is decreased from the redox signaling activity
observed in WT yeast p58C. All scans performed in anaerobic conditions on 40
μM
[4Fe4S]
p58C Y397L and 2.5
mM
ATP, i
n 20
mM
HEPES, pH 7.2, 75
mM
NaCl, at a 100
mV/s scan
rate for CV or a 15 Hz frequency, 25
mV amplitude for SWV.
-40
-30
-20
-10
0
10
20
30
40
-0.4
-0.2
0
0.2
0.4
0.6
Current (nA)
Potential (V vs. NHE)
0
5
10
15
20
25
30
35
40
-0.4
-0.2
0
0.2
0.4
0.6
Current (nA)
Potential (V vs. NHE)
47
Figure S12
.
Electrochemistry of WT yeast p58C in the presence of 5
mM
ATP. left) CV scans
show a small peak in the reductive wave near
-
80
mV vs. NHE, likely a [3Fe4S]
+
oxidative
degradation product. right) SWV of yeast p58C in the presence of 5
mM
ATP. All scans
performed in anaerobic conditions on 40
μM
[4Fe4S] p58C and 5
mM
ATP, in 20
mM
HEPES,
pH 7.2, 75
mM
NaCl, at a 100
mV/s scan rate for CV or a 15 Hz frequency, 25
mV amplitude
for SWV.