, 451 (2010);
et al. Monika Swierczek,
1 Cytochrome bc
An Electronic Bus Bar Lies in the Core of
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on July 24, 2010
among the prime candidates to harbor proton-
translocation sites, distances for energy-transfer
reactions of more than 100 Å have to be en-
visaged.Acontinuouselectrondensity most likely
with the membrane arm embedded in the trans-
membrane segments near the matrix-facing surface
the distal domain (Fig.2A). At the proximal end, it
is connected by continuous electron density to
one of the transmembrane segments in a nearly or-
thogonal orientation. Strikingly, on the PDdomain
resembling a discontinuous helix arrangement,
suggesting the presence of a proton-translocation
unit (Fig. 3B) (22). We propose that this observed
connection between the P domains is a critical
transmission element within the proton-pumping
machinery of complex I.
The modular architecture of complex I as re-
vealed by crystallographic analysis of the complete
bipartite functional organization, conformational
energy is generated by the redox chemistry occur-
ring in the N and Q modules of the peripheral arm
and is transmitted to the two proton-pumping
modules of the membrane arm, which are con-
nected by a helical transmission element.
References and Notes
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33. Excellent technical assistance by K. Siegmund and technical
supportbyA. Duchene,F.Streb,andB.Wenzel aregratefully
acknowledged. We thank S. Kerscher for his essential
contributions regarding all aspects of Y. lipolytica genetics
and B. Wrzesniewska for testing the impact of antibody
fragments to improve crystallization. We are indebted to
H. Michel for valuable advice during the initial stages of
the project and for his continuous support. We thank the
European Synchrotron Radiation Facility and the Swiss
Light Source (SLS) for granting beam time, many scientists
especially at SLS beamlines X06SA and X10SA for constantly
supporting our work, and the staff of the Helmholtz
Zentrum für Infektionsforschung (HZI)/Gesellschaft für
Biotechnologische Forschung, Braunschweig for large-scale
fermentation of Y. lipolytica. Funding by the Deutsche
Forschungsgemeinschaft (SFB 472 Projects P2 and P17) is
gratefully acknowledged. This study was supported by the
Excellence Initiative of the German Federal and State
Governments (EXC 115 and EXC 294). Electron density maps
are available on request.
Supporting Online Material
Materials and Methods
Tables S1 and S2
16 April 2010; accepted 10 June 2010
Published online 1 July 2010;
Include this information when citing this paper.
An Electronic Bus Bar Lies in the
Core of Cytochrome bc1
Monika Świerczek,1Ewelina Cieluch,1Marcin Sarewicz,1Arkadiusz Borek,1
Christopher C. Moser,2P. Leslie Dutton,2Artur Osyczka1*
The ubiquinol–cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are
homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are
functionally important. This has resulted in a proliferation of contradictory models. Here, we duplicated
and fused cytochrome b subunits, and then broke symmetry by introducing independent mutations into
each monomer. Electrons moved freely within and between monomers, crossing an electron-transfer
bridge between two hemes in the core of the dimer. This revealed an H-shaped electron-transfer system
that distributes electrons between four quinone oxidation-reduction terminals at the corners of the
dimer within the millisecond time scale of enzymatic turnover. Free and unregulated distribution of
electrons acts like a molecular-scale bus bar, a design often exploited in electronics.
igure 1 shows a bacterial ubiquinol–
cytochrome c oxidoreductase (1), often
called cytochrome bc1, displaying homodi-
meric core subunit structure typical of respiratory
and photosynthetic electron transfer systems (2, 3).
It is well established that adjacent cofactors in
each monomer serve to separate electronic charge
across the membrane in the catalytically relevant
microsecond-to-millisecond electron transfer pro-
and electrochemical symmetry between the mono-
mers of the dimer has confounded efforts to
determine whether a functional electron-transfer
connection exists between monomers. At the dis-
tances displayed in Fig. 1, calculations show that
electron-tunneling times between cofactors in dif-
ferent monomers are much slower than the physi-
ologically relevant time scale, except for tunneling
between the two bLhemes. Electron tunneling
across the 13.9 Å separating these two hemes is
slightly faster than the measured 0.5- to 5.0-ms
physiological turnover time. However, electron-
tunneling theory itself (6, 7) provides only an up-
per limit for the rate of electron transfer between
redox cofactors. Many electron transfers in oxido-
reductases are limited not by electron tunneling but
by slower coupled events of chemistry, con-
formational change, or motion (8, 9). Indeed,
many models have been proposed for ubiquinol–
cytochrome c oxidoreductases that include just
such regulation of electron transfer within or be-
tween monomers (10–15) or even strict electronic
isolation of monomers (16). Given the inherent
tunneling speed, a relatively small amount of
coupling of thiselectrontunneling to chemical or
conformational events could effectively regulate
interaction between monomers or even isolate
To resolve the underlying dimer engineering,
we broke the symmetry of the cytochrome bc1
and Biotechnology, Jagiellonian University, Kraków, Poland.
and Biophysics, University of Pennsylvania, PA 19104, USA.
*To whom correspondence should be addressed. E-mail:
VOL 32923 JULY 2010
on July 24, 2010
meric cytochrome b subunits, analogous to the
subunits accommodate the core cofactors in the
electron-transfer chain and the putative bridge
between monomers (Figs. 1 and 2). The N and C
termini of the eight-transmembrane–a-helical
chain of the monomeric cytochrome b protrude at
the cytoplasmic side of the membrane. We joined
these termini by extending the gene encoding
cytochrome b with the linker peptide sequence
followed by the second copy of the same gene
containing Strep-tag at its C terminus (figs. S2
and S3) (18). The other two genes of the operon,
encoding the subunits containing the FeS cluster
fused cytochrome bc1are designated BB and
B-B, respectively. Electrophoresis verified that
the subunits of B-B had the correct molecular
mass (fig. S4), and ultraviolet (UV)–visible and
electron paramagnetic resonance spectroscopy
(EPR) demonstrated normal cofactor assembly
(Fig. 3 and figs. S5 and S6). Measurements of
the fused protein remained functional.
To uncover dimer-specific operation and test
the putative H-shaped electron transfer system,
we need only two asymmetrically positioned
point mutations in B-B. We chose two sites that
have been extensively characterized in BB. The
mutation H212N (symmetricalNBBNin Fig. 2A)
cofactors in the cytochrome bc1structure (5). This
heme bHknockout markedly cuts short electron
transfer in both upper H branches and, because
linked electron transfer into the lower branch. The
second site, G158W (symmetricalWBBWin Fig.
(Qosite knockout), again without affecting the
function of the other cofactors, and effectively
inactivates dual electron transfer from quinol into
both lower and upper branches (4). We achieved
expression and assembly of mutants with asym-
metrically placed copies of H212N or G158W in
B-B, either separately or together in various com-
binations [table S1 and supporting online material
(SOM) text]. Figure S4 confirms the proper size of
double-mutant forms, and Fig. 3 and figs. S5 and
S6 demonstrate that levels of expressed heme bH
(reported by the EPR spectrum of the FeS cluster)
As depicted in Fig. 2B, permutations of these
two strategically positioned mutations unambig-
uously expose all possible electron-transfer paths
through the individual branches and bridge of
this H-shaped electron transfer system. Figure 4
shows two types of kinetic assays, flash-induced
on the left and steady-state on the right. After a
flash of light activates the photosynthetic reaction
Fig. 1. Cofactorsanddis-
tances in homodimer of
cytochrome bc1 [Protein
Data Bank ID: 1ZRT (1)].
Each monomer comprises
cytochrome b (yellow), cy-
tochrome c1 (magenta),
and FeS subunit (green).
Functional distances (blue
lines) and nonfunctional
between cofactors (black)
are in angstroms. Qosite
quinone is approximated
from the crystallographic
and Qisite quinone posi-
tion is adopted from (28).
FeS head domain move-
ment (29) is indicated by
the dashed arrow.
Fig. 2. Symmetric and
asymmetric knockout pat-
terns. Distribution of the
structed with unfused na-
fused gene coding (B). BB,
upper branches removed;
removed;WNBBNW, all four
branches removed. B-B,
per branch removed;WB-B,
WNB-B, two branches on
the same side removed;
removed. N and W refer to
H212N and G158W point
mutations in cytochrome b
(G, Gly; H, His; N, Asn; W,
Trp). Black arrows, func-
ble arrow, electron entry
point at the Qosite. Brown
overlay: intraprotein elec-
tronic bus bar.
23 JULY 2010VOL 329
on July 24, 2010
center to oxidize cytochrome c, the re-reduction
of cytochrome c provides a sensitive indicator of
electron-transfer activity and quinol oxidation catal-
ysis in cytochrome bc1. Cytochrome c oxidation-
reduction in B-B (Fig. 4A, top trace) is similar to
that of wild-type BB (not shown). Flash-activated
a prompt downward change. In the ensuing milli-
seconds, the upward trending cytochrome c trace
lower branches of the H from oxidations of quinol
in the Qosite (Fig. 4A, top trace, black).
The critical involvement of upper and lower
branches in quinol oxidation is demonstrated by
inhibition by antimycin. It inactivates both Qisites
and prevents movement of electrons through and
out of the upper branches, which in turn restricts
branch to cytochrome c; thus cytochrome c reduc-
tion is greatly impeded (Fig. 4A, top trace, red).
(NBBN) trims the upper branch at the point before
unlike the symmetricNBBN, the single asymmetric
heme bHknockout (NB-B) that inactivates one
of the two upper branches has cytochrome c re-
free B-B (Fig. 4A, third trace). Parallel results are
Table 1. Enzymatic activity supported by the complete H-shaped electron transfer system and its
Without inhibitor With antimycin‡
*Letter code corresponds to schemes of Fig. 2. Obtainable second versions of some forms (table S1) and their activities are
shown in parentheses.†Measured for cytochrome bc1in membranes.
antimycin abolished activity to almost zero in all forms.
‡Myxothiazol or stigmatellin in place of
Fig. 4. Testing functional branch connection in
the H-shaped electron transfer system. (A) Light-
induced oxidation and re-reduction of cytochrome
c at 550 minus 540 nm in membranes containing
complete knockout variations described in Fig. 2.
Black, uninhibited; red, inhibited with antimycin.
B-BWand B-BNWdisplayed kinetics similar to that of
WB-B andWNB-B, respectively (not shown). (B) Cor-
responding steady-state enzymatic reduction of cy-
tochrome c at 550 nm. Rates are listed in Table 1.
(C) Light-induced heme bHkinetics inWB-BNin the
presence of antimycin abolishing Qiaction (red) or
blocked with antimycin (ant), the only route to
reduce heme bH(red) must involve the heme bLto
bLelectron transfer. stg, stigmatellin.
Fig. 3. Spectroscopic proof of structural asymmetry imposed by mutations in B-B. (A) X-band continuous-
wave EPR spectra of the FeS cluster in membranes.Left: B-Bwith native gxtransition at 1.804 (intact Qosite);
of single Gaussian curve to the shape of gxin B-B (red) andWBBW(green) with equal contribution of each
one head domain of the FeS subunit, as expected for an assembly of one fusion protein per set of two FeS
subunits. (B) Optical redox difference spectra of hemes in membranes: B-B with native-like spectrum with
hemesC (peak at 550nm) and B(peak at 560 nm) components;NBBNwithdiminishedamplitudeat 560nm
reflecting absence of both hemes bHin dimer;NB-B shows decreased peak at 560 nm with the amplitude in
betweenthatofthespectrumofB-BandNBBN,asexpected foralossofonly oneheme bHinNB-B.Solid and
dashed lines, dithionite minus ferricyanide and ascorbate minus ferricyanide spectra, respectively.
VOL 329 23 JULY 2010
on July 24, 2010
found for the Qosite mutants. Cytochrome c re- Download full-text
out (WBBW) is impeded (Fig. 4A, fourth trace),
whereas the inactivation of only one of the two
chrome c re-reduction kinetics only slightly slower
than in knockout-free B-B (Fig. 4A, fifth trace).
Moreover, knocking out both upper and lower
branches of electron transfer in the same monomer
(WNB-B or B-BNW) causes a similar minor slowing
bottom). These results demonstrate that the activ-
ity of one intact monomer is independent of the
functional status of the other monomer.
The mutant combinationWB-BN(Fig. 2B and
mer electron transfer. The result is unambiguous.
After light-flash–induced oxidation of cytochrome
c, re-reduction follows the same general pattern
observed with the unmutated B-B and mutants
the millisecond time scale.
cytochrome bc1 in respiratory systems. These
analyses confirm the results from flash activation
in showing that forNB-B,WB-B or B-BW,WNB-
B or B-BNW, andWB-BNthe observed steady-
state rates are never less than half that of B-B.
Figure 4C reveals approximately millisecond
electron transfer across the bridge inWB-BNmore
ly matches enzymatic turnover and demonstrates
that intermonomer electron transfer is a physiolog-
ically relevant event. This time is also within the
uncertainty of the calculated pure tunneling time,
coupled to electron tunneling between hemes bL
electron transfer between the hemes bLdoes not
monomer or any upper or lower monomer branch
time scale electron transfer throughout the dimer,
there is serious doubt that intermonomer or inter-
branch conformational interactions play an impor-
tant role in regulating energy coupling or function
of cytochrome bc1(10–15).
In the absence of intricate regulation, the nat-
dimeric cytochrome bc1appears relatively simple
and robust. Dimerization of proteins is common
and proceeds for any number of different reasons.
But merely by permitting two of the core redox
cofactors on either side of a dimer interface to
approach to within a 14 Å electron-tunneling dis-
verted to an H-shaped electron transfer system
opposite sides of the membrane to be enzymati-
cally competent. This simple electronic distribution
molecular version of a conducting bus bar familiar
bar offers several advantages for respiration and
photosynthesis, especially under stress. Multiple
unpaired electrons produced at the Qosite (19, 20)
and implicated in the production of reactive oxy-
gen species (ROS) (21–24) can be collected and
neutralized (5, 25). The bus bar also builds in re-
dundancy to allow physiological function of the
protein even after operational damage of one part,
perhaps from ROS. Finally, this design increases
in the overcrowded bioenergetic membrane (27).
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material on Science Online.
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Acad. Sci. U.S.A. 104, 7887 (2007).
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A. N. Tikhonov, E. K. Ruuge, FEBS Lett. 155, 19 (1983).
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30. We are grateful to F. Daldal (University of Pennsylvania,
Philadelphia, USA) for the genetic system for cytochrome
bc1in R. capsulatus. This work was supported by The
Wellcome Trust International Senior Research Fellowship
(A.O.) and by the United States National Institutes of
Supporting Online Material
Materials and Methods
Figs. S1 to S6
14 April 2010; accepted 9 June 2010
Sfrp5 Is an Anti-Inflammatory
Adipokine That Modulates Metabolic
Dysfunction in Obesity
Noriyuki Ouchi,1* Akiko Higuchi,1Koji Ohashi,1Yuichi Oshima,1Noyan Gokce,2
Rei Shibata,3Yuichi Akasaki,1Akihiko Shimono,4Kenneth Walsh1*
Adipose tissue secretes proteins referred to as adipokines, many of which promote inflammation and
disrupt glucose homeostasis. Here we show that secreted frizzled-related protein 5 (Sfrp5), a protein
previously linked to the Wnt signaling pathway, is an anti-inflammatory adipokine whose expression is
severe glucose intolerance and hepatic steatosis, and their adipose tissue showed an accumulation of
activated macrophages that was associated with activation of the c-Jun N-terminal kinase signaling
pathway. Adenovirus-mediated delivery of Sfrp5 to mouse models of obesity ameliorated glucose
intolerance and hepatic steatosis. Thus, in the setting of obesity, Sfrp5 secretion by adipocytes exerts
salutary effects on metabolic dysfunction by controlling inflammatory cells within adipose tissue.
grade inflammatory state in adipose tissue. Adi-
to as adipokines (1–3). Most adipokines—such as
tumor necrosis factor a (TNFa), interleukin-6
(IL-6), and leptin—are proinflammatory. One
besity is a predisposing factor for meta-
bolic disorders, such as type 2 diabetes,
which are often associated with a low-
prominent exception is adiponectin (APN), an
anti-inflammatory adipokine that promotes insu-
lin sensitization and protects cardiovascular
tissue from ischemic injury (2, 4).
Because adipokine dysregulation can contrib-
we sought to identify new adipokines by compar-
ing the gene expression profile of adipose tissue
23 JULY 2010VOL 329
on July 24, 2010