Extreme electric fields power catalysis in the active site of ketosteroid isomerase

Article abstract of DOI:10.1126/science.1259802

Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

Full text of DOI:10.1126/science.1259802

RESEARCH
| REPORTS
magnetosphere can attain when the IMF points
northward.
BIOPHYSICS
REFERENCES AND NOTES
Extreme electric fields power
catalysis in the active site of
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Stephen D. Fried,* Sayan Bagchi,† Steven G. Boxer‡
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Enzymes use protein architecture to impose specific electrostatic fields onto their bound
substrates, but the magnitude and catalytic effect of these electric fields have proven
difficult to quantify with standard experimental approaches. Using vibrational Stark
effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase
8.
L. A. Frank et al., J. Geophys. Res. 91, 3177–3224 (1986).
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(KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes
1
1
a charge rearrangement in KSI’s rate-determining step. Moreover, we found that the
magnitude of the electric field exerted by the active site strongly correlates with the
enzyme’s catalytic rate enhancement, enabling us to quantify the fraction of the catalytic
effect that is electrostatic in origin. The measurements described here may help explain
the role of electrostatics in many other enzymes and biomolecular systems.
1
3. N. Østgaard, S. B. Mende, H. U. Frey, L. A. Frank,
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etosteroid isomerase (KSI) is a small, pro-
ficient enzyme with one of the highest
known unimolecular rate constants in
biochemistry (1, 2), which has prompted
extensive study of its mechanism and the
cycle (10). In this study, we have focused on the
C=O group of the inhibitor 19-nortestosterone
(19-NT) (Fig. 1C), because when 19-NT binds, the
C=O group is loaded directly into the catalytic
machinery (11, 12). In this way, 19-NT’s C=O vibra-
tional (infrared) frequency shift probes the electro-
static environment that the substrate’s C=O bond
would experience in the active site, except 19-NT
cannot react due to the position of the C=C bond.
To calibrate the sensitivity of 19-NT’s C=O vi-
brational frequency to an electric field, we used
two complementary approaches. In Stark spec-
troscopy (Fig. 2, A and B), an external electric
field of known magnitude is applied to a frozen
glass containing 19-NT, and the accompanying
effect on the vibrational spectrum is recorded (7).
By fitting the Stark spectrum (Fig. 2B) to deriv-
atives of the absorption spectrum (Fig. 2A), the
vibration’s difference dipole can be extracted:
2
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1
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American Geophysical Union, Washington, DC, 2012), pp. 69–80.
K
catalytic strategies it uses (3–5). In steroid biosyn-
thesis and degradation, KSI alters the position of a
C=C double bond (Fig. 1A) by first abstracting a
nearby a proton (E•S ⇌ E•I), forming a charged
enolate intermediate (E•I), and then reinserting
the proton onto the steroid two carbons away
(E•I ⇌ E•P). The removal of a proton in the first
step initiates a rehybridization that converts the
adjacent ketone group to a charged enolate, an
unstable species that is normally high in free en-
ergy and so slow to form. The reaction is therefore
2
0. R. C. Fear, S. E. Milan, J. Geophys. Res. 117, A09230 (2012).
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May 1996 (SP 389, European Space Agency, Noordwijk,
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4. C. T. Russell, Cosmic Electrodyn. 2, 184–196 (1971).
5. J. H. King, N. E. Papitashvili, J. Geophys. Res. 110, A02104 (2005).
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Earth’s Space Plasma Environment, H. Laakso, M. Taylor, P. Escoubet,
Eds. (Springer, Dordrecht, Netherlands, 2010), pp. 129–144.
7. H. Rème et al., Ann. Geophys. 19, 1303–1354 (2001).
2
2
2
2
expected to produce an increase in dipole moment
ACKNOWLEDGMENTS
→
at the carbonyl bond (jDm j), suggesting that KSI
rxn
Work in the UK was supported by Science and Technology
Facilities Council (STFC) Ernest Rutherford Fellowship
ST/K004298/1 and STFC grants ST/K001000/1 and
may facilitate this reaction by exerting an electric
→
→
–1
field (F enz) on this bond that stabilizes it in the
intermediate form and the preceding transition
state (Fig. 1B). Using vibrational Stark effects, we
have measured the electric field that KSI exerts on
this C=O bond, providing quantitative experimen-
tal evidence for the connection between electro-
statics and catalytic proficiency.
The frequencies of certain vibrations (such as
the C=O stretch) shift in a linear manner with
the electric field experienced by that vibration
from its environment, a phenomenon known as
the linear vibrational Stark effect (6, 7). Through
this effect, we have shown that vibrations can be
used as probes of local electrostatic fields. The
nitrile group has been widely deployed to mea-
sure electric fields inside enzymes and their re-
lationship to mutation (8), ligand occupancy (9),
or conformational changes over the catalytic
jDm
jƒ = 1.39 T 0.05 cm /(MV/cm), where ƒ is
C¼O
ST/K000977/1. R.M. is supported by the Belgian Science Policy
Office through the Solar-Terrestrial Center of Excellence. French
participation in the Cluster project is funded by the Centre National
d’Etudes Spatiales (CNES). IMAGE satellite work at the University
of California, Berkeley, was supported through a Southwest
Research Institute subcontract under NASA contract NAS5-96020.
We acknowledge support from the International Space Science
Institute through funding of its International Team on Polar Cap
Arcs, and we are grateful for discussions with members of the
team. Cluster data were obtained from the Cluster Active Archive
the local field factor (fig. S1) (6, 7, 13). A vibra-
tion’s difference dipole is its linear Stark tuning
rate; that is, 19-NT’s C=O vibrational frequency
–1
shifts ~1.4/ƒ cm for every MV/cm of electric
field projected onto the C=O bond axis, whether
the source of that field is an external voltage (as
in Stark spectroscopy) or an organized environ-
→
ment created by an enzyme active site (F enz) that
we wish to characterize. Whenever an external
field is applied to a vitreous sample, vibrational
bands will broaden because 19-NT molecules
(and their C=O bonds) are randomly oriented
with respect to the fixed direction of the external
electric field (6, 7). By contrast, a vibrational probe
will have a fixed orientation with respect to a
protein electric field when bound to a protein, and
as such the linear Stark effect then produces spec-
tral shifts instead of broadening. The C=O vibra-
tion’s Stark tuning rate does not appreciably change
when C=O accepts a hydrogen bond (fig. S2), im-
plying that the frequency still responds to fields
linearly even when C=O participates in stronger
interactions, although those interactions themselves
are associated with larger electric fields (14).
(
http://caa.estec.esa.int/caa/), and the IMAGE FUV data were
provided by the NASA Space Science Data Center (http://nssdc.
gsfc.nasa.gov/space/). The OMNI IMF data were obtained through
NASA’s CDAWeb (http://cdaweb.gsfc.nasa.gov/), for which we
acknowledge J. H. King, N. Papatashvilli, and the principal
investigators of the magnetic field and plasma instruments on the
Geotail and Advanced Composition Explorer (ACE) spacecraft. The
DMSP particle detectors were designed by D. Hardy of Air Force
Research Laboratory, and data were obtained from the Johns
Hopkins University Applied Physics Laboratory.
SUPPLEMENTARY MATERIALS
www.sciencemag.org/content/346/6216/1506/suppl/DC1
Supplementary Text
Fig. S1
References (28–38)
Movie S1
Department of Chemistry, Stanford University, Stanford, CA
4305-1052, USA.
Present address: Protein and Nucleic Acid Chemistry Division,
Medical Research Council Laboratory of Molecular Biology, Cambridge
CB2 0QH, UK. †Present address: Physical and Materials Chemistry
Division, National Chemical Laboratory (CSIR), Pune 411008, India.
‡Corresponding author. E-mail: sboxer@stanford.edu
9
*
1
1
2 June 2014; accepted 19 November 2014
0.1126/science.1257377
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1
6
103
We also pursued a second approach to cali-
brate the sensitivity of 19-NT’s C=O frequency
shifts to electric fields and to assign these fre-
quencies to an absolute field scale: Specifically,
we measured 19-NT’s vibrational spectrum in a
series of solvents (table S1) and examined the
correlation of the frequencies with the solvents’
reaction fields, estimated by molecular dynamics
Tyr and Asp
(11, 12), and its vibrational
By exploring a series of structurally conserva-
tive (but catalytically detrimental) mutants (table
S2), we could systematically perturb the catalytic
efficacy of KSI and quantitatively evaluate its
relationship to the electric field probed by the
C=O vibration. In all cases, the assignment of the
vibrational bands to 19-NT was confirmed with
frequency reflects the electric field at a primary
site of charge rearrangement during KSI’s cata-
lytic cycle. Notably, the C=O vibration red-shifts to
–1
–1
1588.3 cm (Fig. 3A), 46 cm further to the red
from the peak frequency in water, implying an
extremely large electrostatic field. Attributing the
frequency shift to the Stark effect, the linear field-
frequency relationship of Fig. 2D maps this fre-
quency value to an ensemble-average electric field
of –144 T 6 MV/cm. Although this highly red-
shifted frequency lies outside the known linear
range from solvatochromism, additional lines of
evidence suggest that the C=O vibrational fre-
quency maintains an approximately linear rela-
tionship with the field in this regime; neglect of
higher-order terms is expected to result in over-
estimates of the electric field, but by no more than
10% (fig. S4 and text S2). Not only is the C=O band
extremely red-shifted in KSI, it is also extremely
narrow (Fig. 3A), suggesting a rather rigid envi-
ronment (15) that greatly reduces the dispersion
in the electric field. This is very different from what
is observed in H-bonding solvents like water that
exert large, but also highly inhomogeneous, elec-
tric fields because solvent H-bonds can assume a
broad distribution of conformations (dashed
traces in Fig. 3A and fig. S3, B and C) (14). Further-
more, the position of the C=O band in wild-type
KSI is situated at the reddest (highest field) edge of
the frequencies sampled by the C=O group in
water (see the red and dashed traces in Fig. 3A),
suggesting that the active site achieves this large
field by restricting H-bond conformations to those
that are associated with the largest electric fields.
18
isotope replacement studies using C= O 19-NT
16
(MD) simulations (14). As shown in Fig. 2C, 19-NT’s
(figs. S5 and S6). The H-bond provided by Tyr is
C=O band shifts consistently to the red with
known to be essential for KSI’s catalysis, as the
–1
16
increasing solvent polarity, from 1690.2 cm in
conservative Tyr Phe mutation diminishes KSI’s
–1
4
nonpolar hexane to 1634.0 cm in water, so that
rate by factors of ~10 (11, 16). This single point
–1
–1
the C=O frequency shifts across a 56 cm span
from solvatochromic effects. The large redshift in
water reflects the large electric fields that are
created by water’s hydrogen bonds (H-bonds).
The trend in solvent shifts is strongly correlated
with the average electric field that each solvent
exerts on the C=O bond (Fig. 2D and fig. S3A); that
is, the plot of observed frequency versus computed
mutation induced a blue shift from 1588.3 cm to
–1
1647.5 cm (Fig. 3A), implying a much smaller
average electric field. (This change in field mag-
nitude is comparable to that of the change in
solvent field between hexane and water.) The
16
Tyr Ser mutation (17), although less conserva-
tive than the Phe substitution, is actually less
detrimental. This observation has been explained
2
16
field is linear (R = 0.99) and its slope corroborates
by the suggestion that leaving a cavity in Tyr ’s
the Stark tuning rate measured by Stark spectros-
copy. The ~2-fold difference between the slope of
place allows water to remain in the back pocket
of the active site; these water molecules could
also H-bond to the steroid substrate, thereby
–1
this curve [0.702 cm /(MV/cm)] and the measured
Stark tuning rate is consistent with the current
understanding of the local field effect (ƒ ~ 2), based
on other vibrational probes and electrostatic mod-
els (text S1) (13, 14). The regression line implies
that the frequency variation due to different mo-
lecular environments can be well explained as a
field effect and suggests that we can model 19-NT’s
C=O peak frequency in terms of the average electric
field experienced by the vibration.
16
partially compensating for the loss of Tyr ’s key
H-bond (18). Indeed, the C=O stretching fre-
16
quency in Tyr Ser is not as dramatically blue-
16
103
shifted as it is in Tyr Phe. Asp is the other
primary H-bond donor in KSI’s active site (Fig.
103
1A). In the Asp Asn mutant, the H-bonding
proton is much less acidic, but N–H and O–H
bonds have similar sterics and dipole moments.
Consistent with these considerations, the change
in electric field and the rate impairment this
mutation entails with respect to wild-type are
much smaller compared to the other mutants.
Although it is conventionally accepted that
When 19-NT is bound to wild-type KSI, the
C=O probe engages in short, strong H-bonds with
E
•
S
O
E
•
I
O
•
E P
O
16
103
KSI uses Tyr ’s and Asp ’s H-bonds to stabilize
its transition state (1, 11), these measurements
show that the interaction between these H-bonds
and the C=O group can be described fundamen-
tally in terms of the electric field they produce. In
all the mutants studied, 19-NT’s C=O band re-
mains relatively narrow, suggesting that electro-
static rigidity is conferred by KSI’s scaffold,
rather than by the H-bonding residues. Also,
sizable electric fields persist in the KSI active site
after removing the critical H-bond donated by
C=O
C=O
µS
µI
Tyr16
Tyr16
Tyr16
OH
OH
OH
O
O
O
H
H
O
H
O
H H
O
CO H
2
CO2H
Asp103
CO H
2
O
O
Asp103
O
Asp103
Asp40
Asp40
Asp40
C
O
µTS
no F
16
C=O
µTS
Tyr (–60 T 3 MV/cm), implying that a substan-
tial electrostatic field contribution also arises
from the environment fashioned by the enzyme
with F
•
OH
enz
–Fenz
•
E
19-NT
C O
C
O
16
µ
scaffold (Fig. 3B). Nevertheless, that the Tyr
H-bond alone contributes a static field of 84 T
MV/cm without an accompanying increase in
S
Tyr16
C=O
µI
OH
–
Fenz µS
•
O
7
H
H
CO H
2
‡
C=O
C=O
electric field dispersion makes it quite distinct
from water, which donates close to two H-bonds
to C=O on average, but taken altogether these
H-bonds generate an average field of ~40 MV/cm
that is also highly heterogeneous (13).
∆
∆G = –F
enz•
(µTS – µS
µrxn
Reaction Coordinate, q
)
Asp103
O
O
∆
Asp40
40
Fig. 1. Catalysis by ketosteroid isomerase. (A) The chemical mechanism of ketosteroid isomerase. In
the first step, Asp removes an a proton from the steroid to form an enolate, stabilized by two H-bonds
from Tyr and Asp . This transformation results in an increase of the dipole moment along the C=O
bond. E, enzyme; S, substrate; I, intermediate; P, product. (B) Simple model for electrostatic catalysis,
illustrating the effect of an electric field from the organized environment of an enzyme active site (Fenz) on a
, dipole of substrate’s C=O bond; m TS , dipole of transition state’s
C=O bond. (C) Complex between KSI and the product-like inhibitor, 19-NT, used in this study to probe
The Asp Asn mutant decreases KSI’s cata-
40
6
lytic rate by a factor of ~10 (16) but only blue-
16
103
shifts the C=O vibrational frequency of bound
–1
19-NT by 6 cm relative to wild-type KSI (table
S2). This behavior strongly contrasts with
other mutants studied, which produce blue-
shifts commensurate with their deleterious
effect on catalysis (Fig. 3B). To explain this
→
‡
→C¼O
→C¼O
reaction’s activation barrier (DG ). m
S
electric fields in the KSI active site.
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observation, we posit that, whereas the function
of Tyr and Asp is to stabilize the reaction’s
relation can be explained using the simple model
for electrostatic catalysis in Fig. 1B. The forma-
tion of a transition state generally involves re-
distribution of electron density, resulting in bonds
with larger dipole moments than the analogous
state. A chemical reaction with a larger charge
displacement would consequently be exponen-
tially more accelerated by these electric fields,
suggesting that electric field effects may provide
a natural framework for explaining the catalysis
of more proficient enzymes as well (22).
16
103
transition state by exerting electrostatic fields
40
onto the carbonyl moiety, Asp provides an
orthogonal catalytic function (text S3), by acting
as the general base in the proton-transfer re-
action (Fig. 1A).
→
C¼O
→C¼O
S
bonds in the substrate (e.g., jm
j > jm
j)
TS
(20, 21). An electric field will therefore differentially
By comparing the intercept extrapolated to
→
–1
When nitrile vibrational probes were placed in
other locations around the KSI active site in pre-
vious work, frequencies were found well within
the range demarcated by the solvent series (9). In
other words, the extreme electric field experi-
enced by 19-NT’s C=O in wild-type KSI is specific
to its precise position in the active site where
strong local interactions and the collective effect
of the overall enzyme architecture mutually re-
inforce each other.
stabilize the transition state in proportion to the
jF enzj ¼ 0 from Fig. 3B (18.8 kcal mol ) to wild-
→
→C¼O →C¼O
–1
reaction difference dipole (Dm ¼ m
− m
S
→
),
type KSI’s activation barrier (11.5 kcal mol ), we
rxn
TS
→
altering the activation barrier by F enz ⋅ Dm
estimate that the active site’s electric field con-
rxn
–1
(Fig. 1B). If we make a simplifying assumption
that the electric field experienced by the C=O
bond is the same in the substrate and transition
state, the model can be directly mapped onto the
data in Fig. 3B; the slope of the plot (1 D) corre-
tributes 7.3 T 0.4 kcal mol to KSI’s barrier
5
reduction. This corresponds to an ~10 -fold rate
enhancement and accounts for 70% of KSI’s cat-
alytic speedup relative to an uncatalyzed ref-
erence reaction in solution (Fig. 3C and text S3).
Moreover, the electrostatic stabilization energy is
quite similar to the enthalpic component of KSI’s
→
–1
sponds to jDm j, and the intercept (18.8 kcal mol )
rxn
corresponds to the hypothetical activation bar-
–1
A plot of each mutant’s apparent activation
barrier (calculated from the Michaelis-Menten
rier if KSI contributed no stabilizing electric
barrier reduction (9 kcal mol ), suggesting that
the thermodynamic manifestation of the electric
field effect is enthalpic (23). The active site’s sup-
pression of electric field variability, as evidenced
by the approximately lifetime-limited linewidth
of the C=O vibrational band in all the KSI mutants,
is possibly also an important catalytic feature; for
→
field. The small value for jDm j implies that there
rxn
kcat) (fig. S7A) (16, 18, 19) against its correspond-
is a rather small perturbation in the electrostatic
character of the substrate upon activation, con-
sistent with ab initio density functional theory
calculations (fig. S7C and table S3). Apparently,
the very large field present in the KSI active site
is needed to leverage what small charge displace-
ment is associated with the reaction’s transition
ing ensemble-average active-site electric field
magnitude (derived from the field-frequency cal-
ibration curve in Fig. 2D) reveals a robust linear
trend (Fig. 3B; see also fig. S7B). This relationship
suggests that electric fields in KSI’s active site are
intimately linked to catalysis. Moreover, this cor-
16
example, the Tyr Phe mutant produces a smaller
average electric field than water (Fig. 3A), although
Fig. 2. Sensitivity of the C=O
stretch frequency of 19-NT to
electrostatic field. (A) The
hexanes
dibutylether
chloroform
tetrahydrofuran
dichloromethane
pentanitrile
acetonitrile
methyl sulfoxide
water
1
700 1690 1680 1670 1660 1650 1640
absorption spectrum of 19-NT
50 mM) in glassy 2-methyl
(
1.0
absorption
numerical fit
8
00
tetrahydrofuran at 77 K. (B) The
Stark spectrum of 19-NT at
0
0
0
.8
.6
.4
1
.0 MV/cm, shown as a
difference between the field-on
and field-off spectra. The Stark
tuning rate is related to the
second derivative fitting
600
4
2
00
00
parameter (figs. S1 and S2).
(C) Infrared spectra of 19-NT
dissolved in organic solvents of
various polarities or water; the
small peak at 1615 cm is the
0.2
0
–
1
C=C stretch. (D) Plot of
0
6
4
1
9-NT’s C=O peak frequency,
1
700
1680
1660
1640
11620
1600
nC¼O against the calculated
–
→
Wavenumbers / cm
solvent electric field, jFsolvj, the
C=O group experiences in each
Stark spectrum
numerical fit
of those solvents (13, 14). The
least-squares regression line is
νC=O = 0.702 Fsolv + 1689
1
690
→
nC¼O ¼ 0:702jFsolvj þ 1689
2
1680
1670
(R
= 0.99). Error ranges for
2
0
frequencies are contained
within symbols; for electric
fields, error bars report the
correlation-adjusted error of
the average electric field from
1
1
1
660
650
640
–
2
4
6
2
-ns simulations.
–
–
1630
1
700 1690 1680 1670 1660 1650 1640
-80 -70 -60 -50 -40 -30 -20 -10
Solvent Field / (MV/cm)
0
–
1
Wavenumbers / cm
1
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it is still a modest catalyst relative to solution. How-
ever, the linear infrared experiments described here
cannot directly quantitate this effect.
What is the physical basis for the extreme
with our experiments (text S4 and fig. S8). Better
estimates of active-site electric fields have been
obtained with more sophisticated computational
models, which have provided a theoretical foun-
dation supporting the link between electric fields
and catalysis (10, 21).
electric field detected in KSI’s active site? Large
16
103
electric fields arising from Tyr and Asp are
expected when the carbonyl group of the ligand
closely approaches the OH groups of these two
residues and in a coplanar orientation [as indeed
is seen in crystal (3) and solution (12) structures].
Nevertheless, these static structures cannot pre-
dict or reproduce the electric fields determined
by vibrational Stark effects, likely because struc-
tures represent ensemble averages and because
electric fields depend sensitively on atomic posi-
tions down to resolutions not accessible in most
structural data (text S4). Moreover, electric fields
calculated from classical MD simulations of the
KSI•19-NT complex (text S4) also do not agree
The remaining catalytic effect beyond electro-
statics can likely be attributed to the precise po-
40
sitioning of the general base (Asp ) with respect
to the proton on the steroid to be abstracted,
which is expected to be an entropic effect. From
this discussion, we surmise that in enzymatic
proton abstraction, electrostatic stabilization and
exact positioning of reacting moieties provide the
physical basis to achieve enthalpic and entropic
contributions to catalysis, respectively; however,
contrary to earlier views (24, 25), electrostatic
stabilization can be the more important of the
two (Fig. 3C).
Unusual spectral shifts have been observed
previously in enzyme active sites (including KSI)
(11, 26–28) and have been variously interpreted
as implying strain, distortion, or polarization. In
contrast, the vibrational Stark effect enables a
quantitative connection between spectroscopic
observables and a fundamental physical quantity
(electric field). As we demonstrate here, these
experiments can be applied to measuring the
H-bond electric field strength in proteins in
functional contexts. Moreover, the electric field
description provides a framework to quantify the
contributions of specific H-bonding interactions,
as well as the overall electrostatic environment
with the same units, to give a simple and unified
model for electrostatic catalysis (Fig. 1B). That a
substantial portion of KSI’s catalytic rate enhance-
ment can be explained in terms of its average
electric field suggests that the electric field could
be a useful design criterion in the ongoing efforts
to engineer enzymes with unnatural or enhanced
functions. More generally, we anticipate that elec-
tric field measurements with functionally relevant
vibrational probes will elucidate the physical basis
for a broad spectrum of biomolecular and con-
densed phase interactions and processes.
Increasing kcat
Y16S
hexanes
DMSO
Y16F
water D103L
1
0
0
0
0
.0
.8
.6
.4
.2
0
D103N
WT
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700
1675
1650
1625
1600
1575
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–
1
Wavenumbers / cm
.
.
D. A. Kraut et al., PLOS Biol. 4, e99 (2006).
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solution
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–3.6
–2.4
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1
5
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Y16F
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enz
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Active Site Electric Field / (MV/cm)
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‡
–1
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SCIENCE sciencemag.org
19 DECEMBER 2014 • VOL 346 ISSUE 6216 1513

RESEARCH
| REPORTS
ACKNOWLEDGMENTS
performed experiments and collected data. S.D.F. analyzed the data.
S.D.F. and S.G.B. discussed results and wrote the manuscript.
Supplementary Text S1 to S4
Figs. S1 to S8
Tables S1 to S3
We thank the laboratory of D. Herschlag for providing plasmids
of some of the KSI mutants. S.D.F thanks the NSF predoctoral
fellowship program and the Stanford Bio-X interdisciplinary graduate
fellowship for support. This work was supported in part by a grant
from NIH (grant GM27738). S.D.F. designed the study. S.D.F. and S.B.
SUPPLEMENTARY MATERIALS
References (29–70)
www.sciencemag.org/content/346/6216/1510/suppl/DC1
Materials and Methods
8 August 2014; accepted 17 November 2014
10.1126/science.1259802
PALEOCEANOGRAPHY
The penultimate glacial period [marine iso-
tope stage (MIS) 6] at Site 1094 is characterized
by low export production (Fig. 2B), typical of
the Antarctic South Atlantic during ice ages
A stagnation event in the deep
South Atlantic during the last
interglacial period
(9). Although substantial sediment focusing oc-
curred during MIS 6 (Y = 4, Fig. 2C), these gla-
cial age sediments are low in biogenic opal, our
proxy of organic carbon export. Given the absence
of an enhanced supply of organic carbon to the
sea floor (either vertically or laterally), the aU
enrichment during glacial MIS 6 (Fig. 2E) must
be due to low bottom water oxygen concentra-
tions, consistent with sluggish ventilation of the
Southern Ocean during glacial periods (10). This
scenario is corroborated by glacial-age aU enrich-
ments observed in other sediment records from
the Atlantic (9, 11) and Indian (12) sectors of the
Southern Ocean.
1,2
3
3
4
Christopher T. Hayes, * Alfredo Martínez-García, Adam P. Hasenfratz, Samuel L. Jaccard,
5
6
3
2
David A. Hodell, Daniel M. Sigman, Gerald H. Haug, Robert F. Anderson
During the last interglacial period, global temperatures were ~2°C warmer than at present
and sea level was 6 to 8 meters higher. Southern Ocean sediments reveal a spike in
authigenic uranium 127,000 years ago, within the last interglacial, reflecting decreased
oxygenation of deep water by Antarctic Bottom Water (AABW). Unlike ice age reductions in
AABW, the interglacial stagnation event appears decoupled from open ocean conditions
and may have resulted from coastal freshening due to mass loss from the Antarctic ice
sheet. AABW reduction coincided with increased North Atlantic Deep Water (NADW)
formation, and the subsequent reinvigoration in AABW coincided with reduced NADW
formation. Thus, alternation of deep water formation between the Antarctic and the North
Atlantic, believed to characterize ice ages, apparently also occurs in warm climates.
During the deglacial period (T-II), export pro-
230
duction increased rapidly, with Th-normalized
opal fluxes increasing roughly 10-fold (Fig. 2B).
Accounting for concurrent changes in sediment
focusing, although aU concentrations decreased
(Fig. 2E), the mass accumulation rate (MAR) of
aU increased from MIS 6 into the earliest part
of T-II (Fig. 2D), consistent with an increased load
of organic matter to the sea floor enhancing aU
deposition. During the course of T-II, export pro-
duction remained high or even increased, whereas
aU MAR gradually decreased (Fig. 2D). We there-
fore infer that the gradual deglacial decline in aU
MAR was driven by increased oxygenation in deep
water as a consequence of increased Southern
Ocean overturning during T-II.
he circulation and biological productivity
of the Southern Ocean may help regulate
surements with controls on the supply of or-
ganic matter to the sediments. In this core, the
2
30
atmospheric CO
2
over millennial to glacial-
Th-normalized biogenic opal flux records
interglacial time scales (1, 2). Evidence
changes in the vertical rain of organic matter
(2, 6). Lateral supply of organic matter by se-
diment redistribution, or focusing, is assessed
by calculating the focusing factor, Y (7). For age
control during the period surrounding Termina-
tion II (T-II), a new sea-surface temperature
record from Site 1094, based on the marine lipid
T
underpinning this view comes largely from
study of climate oscillations during ice ages.
Here, we report evidence for rapid changes in
Southern Ocean circulation during the last inter-
glacial period that is particularly relevant to
today’s warming climate. Authigenic uranium
Against this backdrop of MIS 6–to–T-II changes
that are consistent with previous findings, we
observed an entirely unexpected feature in the
L
(
aU), a redox-sensitive trace-element proxy for
index, TEX86 , was correlated to the Vostok ice
core dD (7, 8) (Fig. 2A, fig. S1, and table S1).
the oxygen content in sediment pore waters (3),
reveals a millennial-scale reduction in the ven-
tilation of the deep Southern Ocean around
Salinity
Oxygen [ml/l]
1
27,000 years ago, when global temperatures
0
8
were ~2°C warmer than they are today (4, 5).
Most sediment pore water oxygen concen-
trations are controlled primarily by the balance
between diffusive supply from bottom water and
consumption by respiration of sedimentary or-
ganic matter. We find evidence in samples from
Ocean Drilling Program (ODP) Site 1094 (53.2°S
1
7
2
CDW
NADW
6
3
AABW
5.1°E, 2807 m water depth, Fig. 1) for changes in
3
4.65
4
ODP Site 1094
5
4
deep water oxygen supply by combining aU mea-
60˚W 20˚W 20˚E
1
5
Department of Earth, Atmospheric and Planetary Sciences,
Massachusetts Institute of Technology (MIT), Cambridge,
2
40˚S
45˚S
50˚S
60˚S
MA 02142, USA. Lamont-Doherty Earth Observatory of
3
Columbia University, Palisades, NY 10964, USA. Geological
4
Institute, ETH Zürich, 8092 Zürich, Switzerland. Institute of
Geological Sciences and Oeschger Centre for Climate Change
Research, University of Bern, 3012 Bern, Switzerland.
Fig. 1. Vertical section along 5°E in the South Atlantic putting ODP Site 1094 into hydrographic
context. Color-mapped oxygen concentrations, with salinity contours overlain, as drawn from the 2009
World Ocean Atlas (30, 31). ODP Site 1094 is marked with a white circle. The inset map indicates the plotted
section (red line) and the locations of other cores mentioned in the text: MD03-2664, white triangle; ODP
Site 1089, white diamond; and PS2561-2, white square. Figure created with Ocean Data View (odv.awi.de).
5
Department of Earth Sciences, University of Cambridge,
6
Cambridge CB2 3EQ, UK. Department of Geosciences,
Princeton University, Princeton, NJ 08544, USA.
*Corresponding author. E-mail: cthayes@mit.edu
1
514 19 DECEMBER 2014 • VOL 346 ISSUE 6216
sciencemag.org SCIENCE

Extreme electric fields power catalysis in the active site of
ketosteroid isomerase
Stephen D. Fried et al.
Science 346, 1510 (2014);
DOI: 10.1126/science.1259802
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