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Metastable radical state, nonreactive with oxygen, isinherent to
catalysis by respiratory and photosyntheticcytochromes
bc1/b6fMarcin Sarewicza,1, Łukasz Bujnowicza,1, Satarupa Bhadurib,
Sandeep K. Singhb, William A. Cramerb,and Artur Osyczkaa,2
aDepartment of Molecular Biophysics, Faculty of Biochemistry,
Biophysics and Biotechnology, Jagiellonian University, 30-387
Krakow, Poland; andbDepartment of Biological Sciences, Purdue
University, West Lafayette, IN 47907
Edited by Harry B. Gray, California Institute of Technology,
Pasadena, CA, and approved December 23, 2016 (received for review
November 15, 2016)
Oxygenic respiration and photosynthesis based on quinone
redoxreactions face a danger of wasteful energy dissipation by
diversionof the productive electron transfer pathway through the
genera-tion of reactive oxygen species (ROS). Nevertheless, the
widespreadquinone oxido-reductases from the cytochrome bc family
limit theamounts of released ROS to a low, perhaps just signaling,
levelthrough an as-yet-unknown mechanism. Here, we propose that
ametastable radical state, nonreactive with oxygen, safely
holdselectrons at a local energetic minimum during the oxidation
ofplastohydroquinone catalyzed by the chloroplast cytochrome
b6f.This intermediate state is formed by interaction of a radical
with ametal cofactor of a catalytic site. Modulation of its energy
level onthe energy landscape in photosynthetic vs. respiratory
enzymesprovides a possible mechanism to adjust electron transfer
ratesfor efficient catalysis under different oxygen tensions.
cytochrome b6f | reactive oxygen species | semiquinone |
electronparamagnetic resonance | electron transport
Photosynthetic and respiratory cytochromes bc1/b6f (Fig.
1A)generate a proton-motive force (pmf) that powers
cellularmetabolism by using the Gibbs free energy difference (ΔG)
be-tween hydroquinone (QH2) derivatives (Fig. 1B) and
oxidizedsoluble electron transfer proteins (e.g., cytochrome c or
plasto-cyanin) (1, 2). To increase the efficiency of this process,
which iscritical for the yield of the generated pmf, one part of
the enzymerecirculates electrons to the quinone pool in the
membrane(Q pool), whereas the second part steers the electrons to
thecytochrome c pool, powering the electron recirculation (Fig.
1C).This mechanism, which is best established for the cytochrome
bc1(cyt bc1) (3, 4), with supporting data for the cytochrome b6f
(cytb6f) (5), discussed in ref. 2, is based on bifurcation of the
routefor two electrons released upon oxidation of QH2 at one of
thecatalytic sites—the Qp site, (Qp), (Fig. 1D) (3–5). A model
forthe energetics of this reaction assumes that one electron
derivedfrom the two-electron QH2 donor is transferred, through
thehigh-potential cofactor chain (“steering part” in Fig. 1C)
toplastocyanin or cytochrome c, whereas the second electron
istransferred across the membrane through low-potential
cofactors(“recirculation” part in Fig. 1C).The electronic
bifurcation process requires formation of a
short-lived and reducing redox intermediate—ubisemiquinone(USQ)
or plastosemiquinone (PSQ) (4, 6, 7). However, such anintermediate
in an oxygenic environment would readily reduceoxygen to form
superoxide radical, (O2
−), compromising theefficiency of energy conservation (8). Even
in cyt b6f where thelevel of superoxide production through this
pathway is at least anorder of magnitude greater than that from
yeast cyt bc1, thebranching ratio for electron transfer to O2
forming O2
− is only 1–2% of the total flux (6). The low absolute level of
O2
− productionin native proteins implies the existence of a
mechanism that isnot understood. In fact, contemporary models are
based on at-tempts to decrease stability of semiquinone (SQ) as a
means to
decrease the stationary level of SQ to avoid reactive
oxygenspecies (ROS) (9–12). This destabilization of SQ, in turn,
in-evitably leads to an increase in the rate constant for the
reactionof SQ with oxygen, which might have deleterious
consequences,especially for enzymes exposed to the relatively high
local oxygenconcentrations associated with oxygenic photosynthesis
(8, 13).Here, it is shown that both cyt b6f and cyt bc1 generate
a
metastable radical state, nonreactive with oxygen, under
steady-state turnover. This result sheds light on thermodynamic
prop-erties of intermediates of electronic bifurcation at the Qp,
imply-ing a mechanism that explains how cyt b6f/bc1 maintain a
balancebetween energy-conserving reactions and ROS production to
se-cure the enzymatic reactions at physiologically competent
rates.This mechanism provides a thermodynamic basis for the
signifi-cant difference in the O2
− generation in the two enzymes, andadvances our understanding
of the molecular mechanism of con-trol of electron flow through the
photosynthetic and respiratorychains and its contribution to ROS
generation, which are postulatedto function as signaling mediators
released from bioenergeticorganelles.
Materials and MethodsReagents. Equine cytochrome c,
decylubiquinone (DB), decylplastoquinone(PQ), antimycin A,
dibromothymoquinone (DBMIB), sodium borohydride,sodium dithionite,
potassium ferricyanide (PFC), and other reagents were
Significance
Photosynthesis and respiration are crucial
energy-conservingprocesses of living organisms. These processes
rely on redoxreactions that often involve unstable radical
intermediates. Inan oxygenic atmosphere, such intermediates present
a dangerof becoming a source of electrons for generation of
reactiveoxygen species. Here, we discover that cytochrome b6f, a
keycomponent of oxygenic photosynthesis, generates a meta-stable
state nonreactive with oxygen during enzymatic turn-over. In this
state, a radical intermediate of a catalytic cycleinteracts with a
metal cofactor of a catalytic site via spin-spinexchange. We
propose that this state is a candidate for regu-lation of cyclic
vs. noncyclic photosynthesis and also allowsphotosynthetic and
respiratory cytochrome bc complexes tofunction safely in the
presence of oxygen.
Author contributions: M.S., Ł.B., and A.O. designed research;
M.S., Ł.B., S.B., and S.K.S.performed research; M.S., Ł.B., W.A.C.,
and A.O. analyzed data; and M.S., W.A.C., andA.O. wrote the
paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Freely available online through the PNAS open access
option.1M.S. and Ł.B. contributed equally to this work.2To whom
correspondence should be addressed. Email:
[email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1618840114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1618840114 PNAS | February 7,
2017 | vol. 114 | no. 6 | 1323–1328
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purchased from Sigma-Aldrich. Dodecyl-maltoside detergent was
purchasedfrom Anatrace. PQ and DB were suspended in ethanol and
DMSO, re-spectively, and reduced to hydroquinone form with H2 gas
released fromacidic water solution of sodium borohydride in the
presence of platinum.Ethanolic stock of reduced PQ was mixed with
DMSO in 1:2 (vol/vol) ratio todecrease the rate of spontaneous
oxidation of plastohydroquinone (PQH2).Both substrates were kept at
−80 °C until used.
Enzymes. Cyt bc1 was isolated according to the procedure
described in ref. 14from wild-type Rhodobacter capsulatus grown
under semiaerobic condi-tions. Thylakoid membranes were isolated
from spinach as described in ref.15. Cyt b6f was isolated according
to the procedure described in SI Materialsand Methods.
Electron Paramagnetic Resonance Spectroscopy. Electron
paramagnetic reso-nance (EPR) spectra were measured by the
continuous wave method at 20 Kon a Bruker Elexsys E580 equipped
with an Oxford Instruments liquid heliumtemperature controller. The
X-band spectra were measured as described inref. 16. For Q-band
measurements, a ER5106QT/W resonator inserted intoCF935O cryostat
was used and calibrated at 20 K by using a trityl radicalsignal.
Parameters for EPR measurements are described in SI Materials
andMethods. Spectra were analyzed and processed by using the Eleana
com-puter program (larida.pl/eleana).
Sample Preparation for EPR Spectroscopy. Samples for
measurements shownin Fig. 2A were prepared by manual injection of
PQH2 to the EPR tubecontaining a mixture of cyt b6f, plastocyanin
(PC), and PFC. After the addi-tion of the substrate to the reaction
mixture, the solution was rapidly frozenin an ethanol bath cooled
to 200 K after 2 s or 5 min of incubation. Finalconcentrations of
cyt b6f, PC, PFC, and PQH2 were 140 μM, 25 μM, 680 μM,and 910 μM,
respectively. Samples for measurements shown in Fig. 2B (Xband)
were prepared by the freeze-quench method as described in ref.
17with the exception that glycerol was not present in the buffer.
Final con-centrations of cyt bc1, cytochrome c, antimycin, and DBH2
were 20, 190, 130,
and 190 μM, respectively. Samples with cyt bc1 for Q-band
measurements(Fig. 2B) were obtained similarly as for cyt b6f, as
shown in Fig. 2A. Finalconcentrations of cyt bc1, antimycin, PFC,
and DBH2 were 300 μM, 500 μM,2.5 mM, and 4 mM, respectively.
Spectra in Fig. 3 A and B were obtained forsamples containing 90 μM
cyt b6f or 130 μM cyt bc1 supplemented with2 mM DBMIB.
Preparation of Samples Under Anoxic Conditions. Samples
containing cyt b6fand cyt bc1 in aerobic and anaerobic conditions
were prepared similarly asthose for X-band experiments presented in
Fig. 2 A and B, respectively, with
Fig. 1. Structural and functional elements of cyt b6f/bc1
catalysis. (A) Cofactors and catalytic sites (red circles) overlaid
on the protein surfaces (based oncrystal structures of cyt b6f and
cyt bc1 (PDB ID codes: 1VF5 and 1ZRT, respectively). (B) Chemical
structures of hydroxyquinones with estimated average Emvalues of
Q/QH2 couples at pH 7. Red, redox-active groups. (C) General scheme
showing the “steering” and “recirculation” parts. (D) Simplified
sequentialscheme of enzymatic reaction. Yellow hexagons, quinones
bound to the catalytic sites. Cofactors common for cyt b6f and cyt
bc1: heme bp, bn (rhombuses),and FeS (circles) in oxidized (white)
or reduced (red) forms. Gray rhombus, heme cn exclusively present
in cyt b6f.
Fig. 2. EPR spectra of FeS of cyt b6f (A) and cyt bc1 (B) at two
resonancefrequencies (X and Q). Blue, spectra measured for samples
obtained by rapidfreezing of the reaction solution 2 s after mixing
with respective QH2. Black,spectra of the same samples measured
after reaching the equilibrium (5 minafter mixing). Vertical dashed
lines, rounded g values of major EPR transi-tions (see Fig. S1 for
details).
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the exception that the final concentration of cyt b6f was 50 μM.
To obtainanaerobic conditions, glucose oxidase (final activity 1
U/mL) and glucose(final concentration 15 mM) were added to the
reaction mixture. Reagentsafter glucose addition were incubated for
30 min and mixed under atmo-sphere of sulfur hexafluoride gas.
ResultsDetection of PSQ Spin-Coupled to Reduced FeS in
Noninhibited cyt b6fUnder Nonequilibrium Conditions. Fig. 2A shows
that noninhibitedcyt b6f, exposed to its substrates, PQH2 and
oxidized plastocya-nin, generates an intermediate of Qp detected by
EPR at acharacteristic spectral line position defined by an
approximate gvalue ∼1.95 (see Fig. S1 for exact g values). The g
value of thistransition strongly depends on the resonance
frequency. Theshift in the g value indicates that the transition
must be a result ofmagnetic interactions between at least two
paramagnetic centers(18, 19) and not an effect of structural
changes that lead tomodifications of g values of [2Fe-2S] Rieske
cluster (FeS). Fur-thermore, it closely resembles a transition
(also frequency-dependent) found earlier in cyt bc1 (g ∼ 1.94 in
Fig. 2B) andassigned as USQ magnetically coupled to reduced FeS via
spin–spin exchange interaction (designated as USQ-FeS) (17).
Byanalogy to cyt bc1, we propose that the signal in cyt b6f
originatesfrom PSQ that undergoes electron spin–spin exchange
interac-tion with reduced FeS (PSQ-FeS). The occupancy of
SQ-FeScenter in cyt b6f and cyt bc1 (Fig. 2 A and B, blue) was
estimatedas 13% and 42% of the total Qp sites, respectively (see
details inSI Materials and Methods).To record SQ-FeS (this term
corresponds to either USQ-FeS
or PSQ-FeS) in cyt b6f, as in cyt bc1, the steady-state
enzymaticreaction must be interrupted, and the reaction mixture
frozenbefore equilibrium between substrates (PQH2 and
oxidizedplastocyanin) and products (PQ and reduced plastocyanin)
arereached. When the enzymes used all substrates (at
equilibrium),the signals were no longer present (Fig. 2 A and B,
black).Nevertheless, the SQ-FeS in both enzymes, despite being
farfrom the global energy minimum, is relatively long-lived
incomparison with a putative unstable SQ that is commonly
de-scribed (9, 10, 20, 21). A series of control experiments (Fig.
S2)verified that the detected signal did not result from
nonspecificand nonenzymatic reactions between substrates and/or
bufferconstituents. These measurements confirmed that generation
ofthe g ∼ 1.95 transition is possible only when cyt b6f catalyzes
netelectron transfer from PQH2 to plastocyanin.Until now, all
reports of detection of intermediates of Qp,
including USQ-FeS in cyt bc1, were obtained under conditionswhen
the inhibitor antimycin blocked the recirculation pathway,i.e.,
blocked electron flow from the low-potential path to the Qpool (10,
17, 21). Such inhibition severely slows the turnover ofQp, because
electrons entering the low-potential path must findan alternate
route that restores oxidizing equivalents in Qpnecessary to support
turnover. However, PSQ-FeS in cyt b6f wasdetected in the
noninhibited enzyme (Fig. 2A), which shows thatthis state can be
formed in the absence of inhibitors. This
observation implies that the probability of formation of
SQ-FeSin noninhibited enzymes is greater in cyt b6f than in cyt
bc1.
Detection of the Spin-Coupled State Between High-Potential
QuinoneAnalog (DBMIB) and Reduced FeS in cyt b6f/bc1 Under
Equilibrium. Asimilar paramagnetic state can be generated in cyt
b6f and cyt bc1under equilibrium in the presence of the halogenated
quinonederivative, DBMIB (Fig. 3 A and B, respectively), possessing
arelatively high average redox mid-point potential (Em)
(seecomparison of Ems of quinone/hydroquinone couples for
ubi-quinone (UQ), PQ, and DBMIB in Fig. 1B). In fact, this
signalwas observed in spectra of DBMIB-inhibited cyt b6f or cyt
bc1(22–25). However, the nature of the g transitions in the
presenceof DBMIB, as we now propose, was misinterpreted as an
alter-ation of protein structure around FeS. If DBMIB changed the
gtensor of FeS, the g values of the spectra for DBMIB-altered
FeSshould have the same value regardless of the spectrometer
fre-quency used for detection. The clear frequency dependence ofthe
g values measured for samples in the presence of DBMIB(Fig. 3 A and
B) indicates that it binds to reduced FeS as asemiquinone, and
these two centers are subject to similar spin–spin exchange
interaction as USQ-FeS (17) or PSQ-FeS coupledcenters in cyt bc1
and cyt b6f, respectively.
Redox State of Hemes b Under Conditions Favoring Generation of
SQ-FeS. Fig. 4 examines the redox state of the hemes b in cyt
bc1complex under three different conditions relevant to
experi-ments described above. Before mixing the enzyme with
ubihy-droquinone (UQH2) b-type hemes located at n and p side
ofmembrane (bn and bp, respectively) are oxidized (Fig. 4A,
green).However, after 2 s required to generate a relatively large
USQ-FeS signal (Fig. 2B) heme bn undergoes complete
reduction,whereas heme bp is still fully oxidized (Fig. 4A, blue).
This resultappears unexpected because it means that semiquinone
that is inspin–spin exchange interaction with reduced FeS is unable
toreduce heme bp. Even less reducing power is observed
whensemiquinone form of DBMIB creates the spin-spin coupled
statewith FeS. In this case, both hemes remain oxidized (Fig.
4B)because they are unable to take electrons from the
high-potentialsynthetic semiquinone at the Qp.
Testing the Reactivity of PSQ-FeS and USQ-FeS with
MolecularOxygen. Because the experiments revealing SQ-FeS (shown
inFig. 2) were performed in the presence of oxygen, we infer
thatSQ-FeS cannot be highly reactive with oxygen. It follows that
theyield of generated SQ-FeS should not be significantly sensitive
tothe presence or absence of O2. Indeed, in cyt b6f, the amount
ofPSQ-FeS generated under oxygenic and anoxygenic conditions
issimilar (Fig. 5A). However, in the case of cyt bc1, the amount
ofUSQ-FeS is almost always higher when the reaction is carriedout
in the presence of oxygen (see example in Fig. 5B). Thereasons why
USQ-FeS is more efficiently formed in the presenceof oxygen is not
understood. However, one may speculate that aportion of O2
− reduces UQ that stays hydrogen-bonded to
Fig. 3. EPR spectra of FeS measured for samples of cyt b6f (A)
and cyt bc1(B) after 5 min incubation of the enzymes with DBMIB.
Vertical dashed lines,rounded g values of major EPR
transitions.
Fig. 4. EPR gz transitions of oxidized hemes bp and bn of cyt
bc1. (A) Anti-mycin-inhibited enzyme before (green) and 2 s after
initiation of reaction byaddition of UQH2 (blue). (B) Enzyme
incubated with DBMIB in the absence ofantimycin.
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reduced FeS. As a result, O2− is converted to oxygen and UQ
to
USQ that strongly interacts with FeS. In other words, the
portionof USQ-FeS is a product of O2
− scavenging by UQ that is boundto FeS. Such the effect is
clearly visible in cyt bc1, but not in cyt b6f,as only in cyt bc1
do the conditions, antimycin-inhibited cyt bc1 vs.2% in
noninhibited cyt b6f (6, 26, 27), used for detection of USQ-FeS,
represent the conditions in which the enzyme produces sig-nificant
amount of O2
− per single oxidized QH2 (10–18%).
DiscussionOne of the important issues associated with the
understanding offundamentals of oxidative metabolism concerns
elucidation ofthe mechanism by which enzymes catalyzing reactions
that pro-ceed through highly unstable radical intermediates have
adaptedto safely function in the presence of molecular oxygen (8).
Inaddressing this issue for the cyt bc1/b6f family, a concept
emergesfrom the unexpected discovery of the PSQ-FeS state in Qp
ofnoninhibited cyt b6f (Fig. 2A) and subsequent comparativeanalysis
of the conditions favoring appearance of this state in cytb6f and
cyt bc1 under oxygenic and anoxygenic environments(Figs. 2–5).
Based on the results and analysis presented in thiswork, it is
concluded that the generation of SQ-FeS in cyt bc1 orcyt b6f is an
inherent part of enzymatic catalysis that can bedescribed as a
metastable state nonreactive with oxygen. Theconcept of
stabilization of SQ in the Qp site by interaction withreduced FeS
of cyt bc1 was proposed by Link (28), who consid-ered
antiferromagnetic coupling of high energy between thesetwo centers
as a possible explanation for the failure to detect anSQ
intermediate in this site. However, such a strong interactionshould
result in disappearance of the EPR signal of FeS, whichwas not
observed. Furthermore, this concept was in opposition toa more
popular view explaining the lack of SQ detection by ahigh
instability of SQ at Qp (9, 10, 12, 20). Our results indicatethat
coupling between reduced FeS and SQ exists in both cyt bc1and cyt
b6f, but its energy is small enough that, regardless of
theantiferromagnetic or ferromagnetic character of coupling, it
pro-duces detectable EPR transitions of SQ-FeS (with a
characteristicg ∼ 1.94) at temperatures higher than a few degrees
Kelvin.Quite importantly, SQ-FeS in cyt bc1 is observed along
with
reduced heme bn and oxidized heme bp (Fig. 4A). On
thermo-dynamic grounds, this observation implies that electron
transferfrom SQ-FeS to heme bp is an uphill step. As a consequence,
ifheme bp is unable to transfer electrons further to heme bn,
theelectron moves back to quinone (Q) at Qp and SQ-FeS is
re-formed. After reaching equilibrium, no net reactions that lead
tosignificant occupation of the metastable state occur and,
conse-quently, this state is no longer detected spectroscopically.
Toobserve it at equilibrium, one must use a high potential
quinoneanalog (DBMIB in our case). However, a large increase in
Emdramatically stabilizes this state to the point that it becomes
in-hibitory, at least for cyt b6f. The electron from DBMIB
semiquinone, because of its relatively oxidizing potential,
cannever reduce hemes bp or bn, leaving them oxidized (Fig.
4B).Considering the results presented here, a mechanism can be
proposed for inclusion of the metastable SQ-FeS into the
ther-modynamic diagram of electronic bifurcation (Fig. 6). This
dia-gram follows the generally accepted scheme of enzymatic
cyclebut adds a new state, state 4, which is a result of an
energeticallydownhill electron transfer from heme bp to Q at Qp
(transitionfrom state 3). This state protects the enzyme against
ROS pro-duction by: (i) the fact that electron transfer from SQ-FeS
tomolecular oxygen (to state 8) is energetically unfavorable
and(ii) blocking Qp for the next QH2 oxidation until electrons
areremoved from the low-potential chain. The metastable state
existsuntil electrons from the low-potential chain are removed
throughthe Qn site (Qn) back to the Q pool through state 3 of
higher ΔG,followed by subsequent downhill reactions through states
5 and 6.Hence, any factor that decreases the rate of electron
release fromthe low-potential chain to the Q pool, in relation to
the rate of thereduction of this chain by Qp, creates conditions
that favor ap-pearance of the SQ-FeS metastable state. Such
conditions mayexist in some physiological states, e.g., in the
presence of a hightransmembrane potential or a high concentration
of QH2 in the Qpool. It is intriguing that a similar g = 1.94
transition of unknownorigin, which might reflect the USQ-FeS state,
was reported toappear under ischemia and disappear under
reperfusion of rathearts (29).The free energy (ΔG) diagram, shown
in Fig. 6, which depends
not only on Em but also on other processes, e.g.,
reconfigurationof H bonds within Qp, provides a possible
explanation for sig-nificant occupation of the energetic state
representing PSQ-FeSin noninhibited cyt b6f. Although the
difference in Em betweenhemes bn and bp in cyt b6f is not well
defined (shown as the widthin the level of state 5 in Fig. 6A), the
average is somewhat morenegative than in hemes b in cyt bc1 (30,
31). This difference,together with the fact that PQ possesses a
more positive Em thanUQ (13) makes the energetic gap between state
4 (with SQ-FeS)and state 5 (with reduced heme bn) smaller in cyt
b6f comparedwith cyt bc1. In other words, in cyt bc1, the electron
transfer fromUSQ-FeS (state 4) to heme bn (state 5) is
energetically muchmore favorable than the electron transfer from
PSQ-FeS toheme bn in cyt b6f. In addition, the existence of
high-spin heme cnis possibly a bottleneck for electron flow from
the low-potentialchain to the Q pool, or reduction of PQ in Qn
requires a co-operative two-electron transfer from hemes bn/cn (32,
33).With the proposed mechanism, it can be appreciated that
energetic states associated with oxidation of QH2 by cyt b6f/cyt
bc1are positioned at levels that allow smooth catalysis while
limitingreleased ROS to perhaps just signaling levels that
carefully reportthe dynamically changing redox state of cofactors
(6, 34). It isproposed that the SQ-FeS metastable state serves as a
“buffer”for electrons that are unable to be relegated from Qp
through thelow-potential chain. Availability of the buffer allows
the enzymeto be held in the state that protects against
energy-wasting reac-tions including the short circuits (two
electrons from QH2 goingto the same cofactor chain) and the leaks
of electrons to mo-lecular oxygen (1, 7, 8). At the molecular
level, the stabilization ofSQ by its spin-coupling to reduced FeS
is likely to occur throughthe creation of an H bond between SQ and
histidine ligatingreduced FeS (transition from 2 to 4 in Fig. 6)
(35).The stabilization of SQ-FeS can occasionally be broken
(depic-
ted in the Fig. 6 as the transition from state 4 to 2),
resulting in theformation of a highly unstable semiquinone (SQ not
spin-coupledto FeS), which is able to reduce molecular oxygen. This
semi-quinone remains the most likely state responsible for limited
su-peroxide release. In the reversible transition between SQ-FeS
andSQ that is not spin-coupled to FeS (states 4 and 2,
respectively),stationary levels of these two states will be
proportional. It followsthat the amount of ROS will correlate with
the amount of the
Fig. 5. Comparison of the amplitudes of g ∼ 1.95/1.94
transition, in oxy-genic and anoxygenic environment for cyt b6f (A)
and cyt bc1 (B), obtainedunder conditions similar to those of Fig.
2.
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detected SQ-FeS, despite its nonreactivity with oxygen.
Indeed,noninhibited cyt b6f produces larger amounts of superoxide
thannoninhibited cyt bc1 (6), which stays in line with our
observationthat in the case of noninhibited enzymes SQ-FeS can only
bedetected in cyt b6f (Fig. 2A). Nevertheless, when semiquinone
ispresent in Qp, the equilibrium is always shifted toward its
sta-bilization by formation of the SQ-FeS spin-coupled
centerwhereby ROS release is limited to the level of ∼2 molecules
ofO2
− per 100 electrons transferred to FeS. Such residual
ROSgeneration detected in cyanobacterial cyt b6f has been
proposedto activate the p side of the chloroplast transmembrane
Stt7 (36)and to carry out longer range signaling in the plant cell
(37).The probability of creation of the SQ-FeS metastable state
may vary in different species depending on the relative Em
valuesof quinones and low-potential cofactors. Perhaps it is
adjusted tothe oxygen tension in the cellular environment (8).
Indeed, cytb6f, which experiences more than an order of magnitude
higherlevel of oxygen in chloroplasts than cyt bc1 in mitochondria,
has agreater tendency to reside in this buffered state. Also, a
possibleconsequence of the existence of PSQ-FeS in noninhibited cyt
b6fis that it may be responsible for regulation of the electron
transferpathways of oxygenic photosynthesis. As proposed, cyt b6f
innative chloroplasts can catalyze PQH2 oxidation according to
twoalternative mechanisms: (i) noncyclic, in which one molecule
ofPQ in Qn undergoes a sequential reduction by two electronsderived
from Qp and (ii) cyclic, in which one electron is deliveredto Qn
from Qp, whereas the second electron comes from reducedferredoxin
(2). We speculate that creation of the metastable statePSQ-FeS may
serve as a factor that changes the efficiency ofcyclic vs.
noncyclic electron transfer between photosystem I andII. This
inference is explained by the fact that transient
stabilization of the PSQ-FeS blocks the oxidation of anotherPQH2
in Qp, and, thus, creates a condition in which the proba-bility of
delivering the second electron needed to complete thereduction of
PQ in Qn by ferredoxin is significantly increased.The phenomenon of
spin–spin exchange interactions between
semiquinones and metal centers has been observed many timesin
different biological systems, including a coupling between
tightlybound semiquinone QA and Fe
2+ in photosynthetic reaction centers(38, 39), between flavin
semiquinones and metal cofactors (18, 40),and between iron-sulfur
cluster N2 and ubisemiquinone in mito-chondrial complex I. However,
the role of metal cofactors in thevicinity of radicals is not
always clear and remains a subject of de-bate, as exemplified by
recent discussion on possible origin on theunusual properties of
the SQ signals in complex I (41). In light of thepresent study, we
envisage that FeS in cyt bc1/b6f has a dual role inelectron
transfer. Its obvious role is to accept electrons from sub-strate
but besides this function, it offers a mean of stabilization
ofpotentially dangerous intermediates that are inherently
associatedwith the stepwise quinone redox reactions. This metal
center behavesas a Lewis acid with electrophilic properties. When
it creates a bondwith SQ, its electron-withdrawing properties
decrease the probabilityof reaction of SQ with oxygen. We propose
that this feature is notrestricted to the iron-sulfur cluster of
cyt bc1/b6f, but may be commonfor other metal cofactors that,
besides having a role in electrontransfer, stabilize potentially
reactive radicals.
ACKNOWLEDGMENTS. This work was supported by The Wellcome
TrustGrant 095078/Z/10/Z (to A.O.). Faculty of Biochemistry,
Biophysics andBiotechnology of Jagiellonian University is a partner
of the Leading NationalResearch Center supported by Ministry of
Science and Higher Education, partof which included Grant
35p/1/2015 (to M.S.) and a scholarship (to Ł.B.).Studies of W.A.C.,
S.B., and S.K.S. were supported by NIH Grant GMS-038323.
Fig. 6. Simplified diagram of relative energy levels at
different stages of reaction catalyzed by cyt b6f (A) and cyt bc1
(B). Black dots represent electrons onthe respective cofactor or
quinone molecule. Yellow squares, Qp empty or occupied by
substrate; purple squares, Qp with bound DBMIB semiquinone.
Greenand red arrows, downhill and uphill transitions between the
states, respectively. Red frame indicates the states that are
inaccessible in antimycin-inhibited cytbc1. State 1 represents the
most populated initial state for QH2 oxidation under steady-state
turnover (in antimycin-inhibited cyt bc1 heme bn is alreadyreduced
after the first turnover that takes place within experimental dead
time). This reaction results in reduction of FeS and heme bp
(transition from 1 to 3involving unstable SQ in 2). From 3 downhill
reactions to 4 or 5 are possible and 5 undergoes further downhill
transition to 6 (oxidation of heme bn) allowingnext turnover. State
4 is metastable state containing SQ spin-coupled to reduced FeS.
Population of 4 increases when transition to 5 is blocked
(antimycin) ortransition from 5 to 6 is slow with respect to the
transition from 1 to 3. The stationary level of 4 depends on
energetic gap between 4 and 5 (gray doublearrow), which differs
within bc family. State 4 lays below energetic level of 8 in which
O2
− is generated. State 7 involves stable DBMIB semiquinone
spin-coupled to FeS. The gray square with a gradient depicts
uncertainty in Em values for hemes b in cyt b6f.
Sarewicz et al. PNAS | February 7, 2017 | vol. 114 | no. 6 |
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BIOCH
EMISTR
Y
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9, 2
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-
1. Sarewicz M, Osyczka A (2015) Electronic connection between
the quinone and cyto-chrome c redox pools and its role in
regulation of mitochondrial electron transportand redox signaling.
Physiol Rev 95(1):219–243.
2. Cramer WA, Hasan SS (2016) Structure-function of the
cytochrome b6f lipoproteincomplex. Cytochrome Complexes: Evolution,
Structures, Energy Transduction, andSignaling, eds Cramer WA,
Kallas T (Springer, Dordrecht), pp 177–207.
3. Mitchell P (1975) The protonmotive Q cycle: A general
formulation. FEBS Lett 59(2):137–139.
4. Crofts AR, Meinhardt SW, Jones KR, Snozzi M (1983) The role
of the quinone pool inthe cyclic electron-transfer chain of
Rhodopseudomonas sphaeroides: A modifiedQ-cycle mechanism. Biochim
Biophys Acta 723(2):202–218.
5. Joliot P, Joliot A (1988) The low-potential electron-transfer
chain in the cytochrome bfcomplex. Biochim Biophys Acta
933(2):319–333.
6. Baniulis D, Hasan SS, Stofleth JT, Cramer WA (2013) Mechanism
of enhanced super-oxide production in the cytochrome b6f complex of
oxygenic photosynthesis.Biochemistry 52(50):8975–8983.
7. Osyczka A, Moser CC, Dutton PL (2005) Fixing the Q cycle.
Trends Biochem Sci 30(4):176–182.
8. Rutherford AW, Osyczka A, Rappaport F (2012) Back-reactions,
short-circuits, leaksand other energy wasteful reactions in
biological electron transfer: Redox tuning tosurvive life in O2.
FEBS Lett 586(5):603–616.
9. Cape JL, Bowman MK, Kramer DM (2006) Understanding the
cytochrome bc com-plexes by what they don’t do. The Q-cycle at 30.
Trends Plant Sci 11(1):46–55.
10. Cape JL, Bowman MK, Kramer DM (2007) A semiquinone
intermediate generated atthe Qo site of the cytochrome bc1 complex:
Importance for the Q-cycle and superoxideproduction. Proc Natl Acad
Sci USA 104(19):7887–7892.
11. Quinlan CL, Gerencser AA, Treberg JR, Brand MD (2011) The
mechanism of superoxideproduction by the antimycin-inhibited
mitochondrial Q-cycle. J Biol Chem 286(36):31361–31372.
12. Bergdoll L, ten Brink F, Nitschke W, Picot D, Baymann F
(2016) From low- to high-potential bioenergetic chains:
Thermodynamic constraints of Q-cycle function.Biochim Biophys Acta
1857(9):1569–1579.
13. Song Y, Buettner GR (2010) Thermodynamic and kinetic
considerations for the re-action of semiquinone radicals to form
superoxide and hydrogen peroxide. Free RadicBiol Med
49(6):919–962.
14. Valkova-Valchanova MB, Saribas AS, Gibney BR, Dutton PL,
Daldal F (1998) Isolationand characterization of a two-subunit
cytochrome b-c1 subcomplex from Rhodo-bacter capsulatus and
reconstitution of its ubihydroquinone oxidation (Qo) site
withpurified Fe-S protein subunit. Biochemistry
37(46):16242–16251.
15. Hurt E, Hauska G (1981) A cytochrome f/b6 complex of five
polypeptides withplastoquinol-plastocyanin-oxidoreductase activity
from spinach chloroplasts. Eur JBiochem 117(3):591–599.
16. Sarewicz M, Dutka M, Pietras R, Borek A, Osyczka A (2015)
Effect of H bond removaland changes in the position of the
iron-sulphur head domain on the spin-lattice re-laxation properties
of the [2Fe-2S]2+ Rieske cluster in cytochrome bc1. Phys ChemChem
Phys 17(38):25297–25308.
17. Sarewicz M, Dutka M, Pintscher S, Osyczka A (2013) Triplet
state of the semiquinone-Rieske cluster as an intermediate of
electronic bifurcation catalyzed by cytochromebc1. Biochemistry
52(37):6388–6395.
18. Fournel A, et al. (1998) Magnetic interactions between a
[4Fe–4S]1+ cluster and aflavin mononucleotide radical in the enzyme
trimethylamine dehydrogenase: A high-field electron paramagnetic
resonance study. J Chem Phys 109(24):10905–10913.
19. Calvo R (2007) EPR measurements of weak exchange
interactions coupling unpairedspins in model compounds. Appl Magn
Reson 31(1–2):271–299.
20. Zhu J, Egawa T, Yeh S-R, Yu L, Yu C-A (2007) Simultaneous
reduction of iron-sulfurprotein and cytochrome bL during ubiquinol
oxidation in cytochrome bc1 complex.Proc Natl Acad Sci USA
104(12):4864–4869.
21. Zhang H, Osyczka A, Dutton PL, Moser CC (2007) Exposing the
complex III Qo semi-quinone radical. Biochim Biophys Acta
1767(7):883–887.
22. Malkin R (1981) Redox properties of the DBMIB-Rieske
iron-sulfur complex in spinachchloroplast membranes. FEBS Lett
131(1):169–172.
23. Schoepp B, Brugna M, Riedel A, Nitschke W, Kramer DM (1999)
The Qo-site inhibitorDBMIB favours the proximal position of the
chloroplast Rieske protein and induces apK-shift of the
redox-linked proton. FEBS Lett 450(3):245–250.
24. Roberts AG, Bowman MK, Kramer DM (2002) Certain metal ions
are inhibitors ofcytochrome b6f complex ‘Rieske’ iron-sulfur
protein domain movements. Biochemistry41(12):4070–4079.
25. Roberts AG, Bowman MK, Kramer DM (2004) The inhibitor DBMIB
provides insightinto the functional architecture of the Qo site in
the cytochrome b6f complex.Biochemistry 43(24):7707–7716.
26. Borek A, Sarewicz M, Osyczka A (2008) Movement of the
iron-sulfur head domain ofcytochrome bc1 transiently opens the
catalytic Qo site for reaction with oxygen.Biochemistry
47(47):12365–12370.
27. Sarewicz M, Borek A, Cieluch E, �Swierczek M, Osyczka A
(2010) Discrimination be-tween two possible reaction sequences that
create potential risk of generation ofdeleterious radicals by
cytochrome bc1. Implications for the mechanism of
superoxideproduction. Biochim Biophys Acta 1797(11):1820–1827.
28. Link TA (1997) The role of the ‘Rieske’ iron sulfur protein
in the hydroquinone oxi-dation (Qp) site of the cytochrome bc1
complex. The ‘proton-gated affinity change’mechanism. FEBS Lett
412(2):257–264.
29. Baker JE, Felix CC, Olinger GN, Kalyanaraman B (1988)
Myocardial ischemia and re-perfusion: Direct evidence for free
radical generation by electron spin resonancespectroscopy. Proc
Natl Acad Sci USA 85(8):2786–2789.
30. Cramer WA, Hasan SS, Yamashita E (2011) The Q cycle of
cytochrome bc complexes: Astructure perspective. Biochim Biophys
Acta 1807(7):788–802.
31. Zhang H, et al. (2008) Quinone and non-quinone redox couples
in Complex III.J Bioenerg Biomembr 40(5):493–499.
32. Zhang H, et al. (2004) Characterization of the high-spin
heme x in the cytochrome b6fcomplex of oxygenic photosynthesis.
Biochemistry 43(51):16329–16336.
33. Baymann F, Giusti F, Picot D, Nitschke W (2007) The ci/bH
moiety in the b6f complexstudied by EPR: A pair of strongly
interacting hemes. Proc Natl Acad Sci USA 104(2):519–524.
34. Bleier L, Dröse S (2013) Superoxide generation by complex
III: From mechanistic ra-tionales to functional consequences.
Biochim Biophys Acta 1827(11-12):1320–1331.
35. Pietras R, Sarewicz M, Osyczka A (2016) Distinct properties
of semiquinone speciesdetected at the ubiquinol oxidation Qo site
of cytochrome bc1 and their mechanisticimplications. J R Soc
Interface 13(118):20160133.
36. Singh SK, et al. (2016) Trans-membrane signaling in
photosynthetic state transitions:Redox- and structure-dependent
interaction in vitro between Stt7 kinase and thecytochrome b6f
complex. J Biol Chem 291(41):21740–21750.
37. Baniulis D, Hasan SS, Miliute I, Cramer WA (2016) Mechanisms
of superoxide gener-ation and signaling in cytochrome bc complexes.
Cytochrome Complexes: Evolution,Structures, Energy Transduction,
and Signaling, eds Cramer W, Kallas T (Springer,Dordrecht), pp
397–417.
38. Calvo R, Passeggi MC, Isaacson RA, Okamura MY, Feher G
(1990) Electron para-magnetic resonance investigation of
photosynthetic reaction centers from Rhodo-bacter sphaeroides R-26
in which Fe2+ was replaced by Cu2+. Determination ofhyperfine
interactions and exchange and dipole-dipole interactions between
Cu2+
and QA-. Biophys J 58(1):149–165.
39. Sedoud A, et al. (2011) Effects of formate binding on the
quinone-iron electron ac-ceptor complex of photosystem II. Biochim
Biophys Acta 1807(2):216–226.
40. Ohnishi T (1998) Iron-sulfur clusters/semiquinones in
complex I. Biochim Biophys Acta1364(2):186–206.
41. Hirst J, Roessler MM (2016) Energy conversion, redox
catalysis and generation of re-active oxygen species by respiratory
complex I. Biochim Biophys Acta 1857(7):872–883.
42. Liu J, et al. (2014) Metalloproteins containing cytochrome,
iron-sulfur, or copper re-dox centers. Chem Rev
114(8):4366–4469.
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