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Significance Photosynthesis and respiration are crucial energy-conserving processes of living organisms. These processes rely on redox reactions that often involve unstable radical intermediates. In an oxygenic atmosphere, such intermediates present a danger of becoming a source of electrons for generation of reactive oxygen species. Here, we discover that cytochrome b 6 f , a key component of oxygenic photosynthesis, generates a metastable state nonreactive with oxygen during enzymatic turnover. In this state, a radical intermediate of a catalytic cycle interacts with a metal cofactor of a catalytic site via spin-spin exchange. We propose that this state is a candidate for regulation of cyclic vs . noncyclic photosynthesis and also allows photosynthetic and respiratory cytochrome bc complexes to function safely in the presence of oxygen.
Simplified diagram of relative energy levels at different stages of reaction catalyzed by cyt b 6 f (A) and cyt bc 1 (B). Black dots represent electrons on the respective cofactor or quinone molecule. Yellow squares, Q p empty or occupied by substrate; purple squares, Q p with bound DBMIB semiquinone. Green and red arrows, downhill and uphill transitions between the states, respectively. Red frame indicates the states that are inaccessible in antimycin-inhibited cyt bc 1 . State 1 represents the most populated initial state for QH 2 oxidation under steady-state turnover (in antimycin-inhibited cyt bc 1 heme b n is already reduced after the first turnover that takes place within experimental dead time). This reaction results in reduction of FeS and heme b p (transition from 1 to 3 involving 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 b n ) allowing next turnover. State 4 is metastable state containing SQ spin-coupled to reduced FeS. Population of 4 increases when transition to 5 is blocked (antimycin) or transition 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 double arrow), which differs within bc family. State 4 lays below energetic level of 8 in which O 2 − is generated. State 7 involves stable DBMIB semiquinone spincoupled to FeS. The gray square with a gradient depicts uncertainty in E m values for hemes b in cyt b 6 f.
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Metastable radical state, nonreactive with oxygen, is
inherent to catalysis by respiratory and photosynthetic
cytochromes bc
1
/b
6
f
Marcin Sarewicz
a,1
,Łukasz Bujnowicz
a,1
, Satarupa Bhaduri
b
, Sandeep K. Singh
b
, William A. Cramer
b
,
and Artur Osyczka
a,2
a
Department of Molecular Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30-387 Krakow, Poland; and
b
Department 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 redox
reactions face a danger of wasteful energy dissipation by diversion
of the productive electron transfer pathway through the genera-
tion of reactive oxygen species (ROS). Nevertheless, the widespread
quinone oxido-reductases from the cytochrome bc family limit the
amounts of released ROS to a low, perhaps just signaling, level
through an as-yet-unknown mechanism. Here, we propose that a
metastable radical state, nonreactive with oxygen, safely holds
electrons at a local energetic minimum during the oxidation of
plastohydroquinone catalyzed by the chloroplast cytochrome b
6
f.
This intermediate state is formed by interaction of a radical with a
metal cofactor of a catalytic site. Modulation of its energy level on
the energy landscape in photosynthetic vs. respiratory enzymes
provides a possible mechanism to adjust electron transfer rates
for efficient catalysis under different oxygen tensions.
cytochrome b
6
f
|
reactive oxygen species
|
semiquinone
|
electron
paramagnetic resonance
|
electron transport
Photosynthetic and respiratory cytochromes bc
1
/b
6
f(Fig. 1A)
generate a proton-motive force (pmf) that powers cellular
metabolism by using the Gibbs free energy difference (ΔG) be-
tween hydroquinone (QH
2
) derivatives (Fig. 1B) and oxidized
soluble electron transfer proteins (e.g., cytochrome cor plasto-
cyanin) (1, 2). To increase the efficiency of this process, which is
critical for the yield of the generated pmf, one part of the enzyme
recirculates electrons to the quinone pool in the membrane
(Q pool), whereas the second part steers the electrons to the
cytochrome cpool, powering the electron recirculation (Fig. 1C).
This mechanism, which is best established for the cytochrome bc
1
(cyt bc
1
) (3, 4), with supporting data for the cytochrome b
6
f(cyt
b
6
f) (5), discussed in ref. 2, is based on bifurcation of the route
for two electrons released upon oxidation of QH
2
at one of the
catalytic sitesthe Q
p
site, (Q
p
), (Fig. 1D)(35). A model for
the energetics of this reaction assumes that one electron derived
from the two-electron QH
2
donor is transferred, through the
high-potential cofactor chain (steering partin Fig. 1C)to
plastocyanin or cytochrome c, whereas the second electron is
transferred across the membrane through low-potential cofactors
(recirculationpart in Fig. 1C).
The electronic bifurcation process requires formation of a
short-lived and reducing redox intermediateubisemiquinone
(USQ) or plastosemiquinone (PSQ) (4, 6, 7). However, such an
intermediate in an oxygenic environment would readily reduce
oxygen to form superoxide radical, (O
2
), compromising the
efficiency of energy conservation (8). Even in cyt b
6
fwhere the
level of superoxide production through this pathway is at least an
order of magnitude greater than that from yeast cyt bc
1
, the
branching ratio for electron transfer to O
2
forming O
2
is only 1
2% of the total flux (6). The low absolute level of O
2
production
in native proteins implies the existence of a mechanism that is
not 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 oxygen
species (ROS) (912). This destabilization of SQ, in turn, in-
evitably leads to an increase in the rate constant for the reaction
of SQ with oxygen, which might have deleterious consequences,
especially for enzymes exposed to the relatively high local oxygen
concentrations associated with oxygenic photosynthesis (8, 13).
Here, it is shown that both cyt b
6
fand cyt bc
1
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 Q
p
,imply-
ing a mechanism that explains how cyt b
6
f/bc
1
maintain a balance
between 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 O
2
generation in the two enzymes, and
advances our understanding of the molecular mechanism of con-
trol of electron flow through the photosynthetic and respiratory
chains and its contribution to ROS generation, which are postulated
to function as signaling mediators released from bioenergetic
organelles.
Materials and Methods
Reagents. 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-conserving
processes of living organisms. These processes rely on redox
reactions that often involve unstable radical intermediates. In
an oxygenic atmosphere, such intermediates present a danger
of becoming a source of electrons for generation of reactive
oxygen species. Here, we discover that cytochrome b
6
f, a key
component of oxygenic photosynthesis, generates a meta-
stable state nonreactive with oxygen during enzymatic turn-
over. In this state, a radical intermediate of a catalytic cycle
interacts with a metal cofactor of a catalytic site via spin-spin
exchange. We propose that this state is a candidate for regu-
lation of cyclic vs.noncyclic photosynthesis and also allows
photosynthetic and respiratory cytochrome bc complexes to
function 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., and
A.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.
1
M.S. and Ł.B. contributed equally to this work.
2
To whom correspondence should be addressed. Email: artur.osyczka@uj.edu.pl.
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
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BIOCHEMISTRY
purchased from Sigma-Aldrich. Dodecyl-maltoside detergent was purchased
from Anatrace. PQ and DB were suspended in ethanol and DMSO, re-
spectively, and reduced to hydroquinone form with H
2
gas released from
acidic 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 to
decrease the rate of spontaneous oxidation of plastohydroquinone (PQH
2
).
Both substrates were kept at 80 °C until used.
Enzymes. Cyt bc
1
was isolated according to the procedure described in ref. 14
from wild-type Rhodobacter capsulatus grown under semiaerobic condi-
tions. Thylakoid membranes were isolated from spinach as described in ref.
15. Cyt b
6
fwas isolated according to the procedure described in SI Materials
and Methods.
Electron Paramagnetic Resonance Spectroscopy. Electron paramagnetic reso-
nance (EPR) spectra were measured by the continuous wave method at 20 K
on a Bruker Elexsys E580 equipped with an Oxford Instruments liquid helium
temperature controller. The X-band spectra were measured as described in
ref. 16. For Q-band measurements, a ER5106QT/W resonator inserted into
CF935O cryostat was used and calibrated at 20 K by using a trityl radical
signal. Parameters for EPR measurements are described in SI Materials and
Methods. Spectra were analyzed and processed by using the Eleana com-
puter program (larida.pl/eleana).
Sample Preparation for EPR Spectroscopy. Samples for measurements shown
in Fig. 2Awere prepared by manual injection of PQH
2
to the EPR tube
containing a mixture of cyt b
6
f, plastocyanin (PC), and PFC. After the addi-
tion of the substrate to the reaction mixture, the solution was rapidly frozen
in an ethanol bath cooled to 200 K after 2 s or 5 min of incubation. Final
concentrations of cyt b
6
f, PC, PFC, and PQH
2
were 140 μM, 25 μM, 680 μM,
and 910 μM, respectively. Samples for measurements shown in Fig. 2B(X
band) were prepared by the freeze-quench method as described in ref. 17
with the exception that glycerol was not present in the buffer. Final con-
centrations of cyt bc
1
, cytochrome c, antimycin, and DBH
2
were 20, 190, 130,
and 190 μM, respectively. Samples with cyt bc
1
for Q-band measurements
(Fig. 2B) were obtained similarly as for cyt b
6
f, as shown in Fig. 2A. Final
concentrations of cyt bc
1
, antimycin, PFC, and DBH
2
were 300 μM, 500 μM,
2.5 mM, and 4 mM, respectively. Spectra in Fig. 3 Aand Bwere obtained for
samples containing 90 μM cyt b
6
for 130 μMcytbc
1
supplemented with
2 mM DBMIB.
Preparation of Samples Under Anoxic Conditions. Samples containing cyt b
6
f
and cyt bc
1
in aerobic and anaerobic conditions were prepared similarly as
those for X-band experiments presented in Fig. 2 Aand B, respectively, with
Fig. 1. Structural and functional elements of cyt b
6
f/bc
1
catalysis. (A) Cofactors and catalytic sites (red circles) overlaid on the protein surfaces (based on
crystal structures of cyt b
6
fand cyt bc
1
(PDB ID codes: 1VF5 and 1ZRT, respectively). (B) Chemical structures of hydroxyquinones with estimated average E
m
values of Q/QH
2
couples at pH 7. Red, redox-active groups. (C) General scheme showing the steeringand recirculationparts. (D) Simplified sequential
scheme of enzymatic reaction. Yellow hexagons, quinones bound to the catalytic sites. Cofactors common for cyt b
6
fand cyt bc
1
: heme b
p
,b
n
(rhombuses),
and FeS (circles) in oxidized (white) or reduced (red) forms. Gray rhombus, heme c
n
exclusively present in cyt b
6
f.
Fig. 2. EPR spectra of FeS of cyt b
6
f(A) and cyt bc
1
(B) at two resonance
frequencies (X and Q). Blue, spectra measured for samples obtained by rapid
freezing of the reaction solution 2 s after mixing with respective QH
2
. Black,
spectra of the same samples measured after reaching the equilibrium (5 min
after mixing). Vertical dashed lines, rounded gvalues of major EPR transi-
tions (see Fig. S1 for details).
1324
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www.pnas.org/cgi/doi/10.1073/pnas.1618840114 Sarewicz et al.
the exception that the final concentration of cyt b
6
fwas 50 μM. To obtain
anaerobic conditions, glucose oxidase (final activity 1 U/mL) and glucose
(final concentration 15 mM) were added to the reaction mixture. Reagents
after glucose addition were incubated for 30 min and mixed under atmo-
sphere of sulfur hexafluoride gas.
Results
Detection of PSQ Spin-Coupled to Reduced FeS in Noninhibited cyt b
6
f
Under Nonequilibrium Conditions. Fig. 2Ashows that noninhibited
cyt b
6
f, exposed to its substrates, PQH
2
and oxidized plastocya-
nin, generates an intermediate of Q
p
detected by EPR at a
characteristic spectral line position defined by an approximate g
value 1.95 (see Fig. S1 for exact gvalues). The gvalue of this
transition strongly depends on the resonance frequency. The
shift in the gvalue indicates that the transition must be a result of
magnetic interactions between at least two paramagnetic centers
(18, 19) and not an effect of structural changes that lead to
modifications of gvalues of [2Fe-2S] Rieske cluster (FeS). Fur-
thermore, it closely resembles a transition (also frequency-
dependent) found earlier in cyt bc
1
(g1.94 in Fig. 2B) and
assigned as USQ magnetically coupled to reduced FeS via spin
spin exchange interaction (designated as USQ-FeS) (17). By
analogy to cyt bc
1
, we propose that the signal in cyt b
6
foriginates
from PSQ that undergoes electron spinspin exchange interac-
tion with reduced FeS (PSQ-FeS). The occupancy of SQ-FeS
center in cyt b
6
fand cyt bc
1
(Fig. 2 Aand B, blue) was estimated
as 13% and 42% of the total Q
p
sites, respectively (see details in
SI Materials and Methods).
To record SQ-FeS (this term corresponds to either USQ-FeS
or PSQ-FeS) in cyt b
6
f, as in cyt bc
1
, the steady-state enzymatic
reaction must be interrupted, and the reaction mixture frozen
before equilibrium between substrates (PQH
2
and oxidized
plastocyanin) and products (PQ and reduced plastocyanin) are
reached. When the enzymes used all substrates (at equilibrium),
the signals were no longer present (Fig. 2 Aand B, black).
Nevertheless, the SQ-FeS in both enzymes, despite being far
from the global energy minimum, is relatively long-lived in
comparison 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 nonspecific
and nonenzymatic reactions between substrates and/or buffer
constituents. These measurements confirmed that generation of
the g1.95 transition is possible only when cyt b
6
fcatalyzes net
electron transfer from PQH
2
to plastocyanin.
Until now, all reports of detection of intermediates of Q
p
,
including USQ-FeS in cyt bc
1
, were obtained under conditions
when the inhibitor antimycin blocked the recirculation pathway,
i.e., blocked electron flow from the low-potential path to the Q
pool (10, 17, 21). Such inhibition severely slows the turnover of
Q
p
, because electrons entering the low-potential path must find
an alternate route that restores oxidizing equivalents in Q
p
necessary to support turnover. However, PSQ-FeS in cyt b
6
fwas
detected in the noninhibited enzyme (Fig. 2A), which shows that
this state can be formed in the absence of inhibitors. This
observation implies that the probability of formation of SQ-FeS
in noninhibited enzymes is greater in cyt b
6
fthan in cyt bc
1
.
Detection of the Spin-Coupled State Between High-Potential Quinone
Analog (DBMIB) and Reduced FeS in cyt b
6
f/bc
1
Under Equilibrium. A
similar paramagnetic state can be generated in cyt b
6
fand cyt bc
1
under equilibrium in the presence of the halogenated quinone
derivative, DBMIB (Fig. 3 Aand B, respectively), possessing a
relatively high average redox mid-point potential (E
m
) (see
comparison of E
m
s of quinone/hydroquinone couples for ubi-
quinone (UQ), PQ, and DBMIB in Fig. 1B). In fact, this signal
was observed in spectra of DBMIB-inhibited cyt b
6
for cyt bc
1
(2225). However, the nature of the gtransitions in the presence
of DBMIB, as we now propose, was misinterpreted as an alter-
ation of protein structure around FeS. If DBMIB changed the g
tensor of FeS, the gvalues of the spectra for DBMIB-altered FeS
should have the same value regardless of the spectrometer fre-
quency used for detection. The clear frequency dependence of
the gvalues measured for samples in the presence of DBMIB
(Fig. 3 Aand B) indicates that it binds to reduced FeS as a
semiquinone, and these two centers are subject to similar spin
spin exchange interaction as USQ-FeS (17) or PSQ-FeS coupled
centers in cyt bc
1
and cyt b
6
f, respectively.
Redox State of Hemes bUnder Conditions Favoring Generation of SQ-
FeS. Fig. 4 examines the redox state of the hemes bin cyt bc
1
complex under three different conditions relevant to experi-
ments described above. Before mixing the enzyme with ubihy-
droquinone (UQH
2
)b-type hemes located at n and p side of
membrane (b
n
and b
p
, respectively) are oxidized (Fig. 4A, green).
However, after 2 s required to generate a relatively large USQ-
FeS signal (Fig. 2B) heme b
n
undergoes complete reduction,
whereas heme b
p
is still fully oxidized (Fig. 4A, blue). This result
appears unexpected because it means that semiquinone that is in
spinspin exchange interaction with reduced FeS is unable to
reduce heme b
p
. Even less reducing power is observed when
semiquinone form of DBMIB creates the spin-spin coupled state
with FeS. In this case, both hemes remain oxidized (Fig. 4B)
because they are unable to take electrons from the high-potential
synthetic semiquinone at the Q
p
.
Testing the Reactivity of PSQ-FeS and USQ-FeS with Molecular
Oxygen. Because the experiments revealing SQ-FeS (shown in
Fig. 2) were performed in the presence of oxygen, we infer that
SQ-FeS cannot be highly reactive with oxygen. It follows that the
yield of generated SQ-FeS should not be significantly sensitive to
the presence or absence of O
2
. Indeed, in cyt b
6
f, the amount of
PSQ-FeS generated under oxygenic and anoxygenic conditions is
similar (Fig. 5A). However, in the case of cyt bc
1
, the amount of
USQ-FeS is almost always higher when the reaction is carried
out in the presence of oxygen (see example in Fig. 5B). The
reasons why USQ-FeS is more efficiently formed in the presence
of oxygen is not understood. However, one may speculate that a
portion of O
2
reduces UQ that stays hydrogen-bonded to
Fig. 3. EPR spectra of FeS measured for samples of cyt b
6
f(A) and cyt bc
1
(B) after 5 min incubation of the enzymes with DBMIB. Vertical dashed lines,
rounded gvalues of major EPR transitions.
Fig. 4. EPR g
z
transitions of oxidized hemes b
p
and b
n
of cyt bc
1
.(A) Anti-
mycin-inhibited enzyme before (green) and 2 s after initiation of reaction by
addition of UQH
2
(blue). (B) Enzyme incubated with DBMIB in the absence of
antimycin.
Sarewicz et al. PNAS
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BIOCHEMISTRY
reduced FeS. As a result, O
2
is converted to oxygen and UQ to
USQ that strongly interacts with FeS. In other words, the portion
of USQ-FeS is a product of O
2
scavenging by UQ that is bound
to FeS. Such the effect is clearly visible in cyt bc
1
, but not in cyt b
6
f,
as only in cyt bc
1
do the conditions, antimycin-inhibited cyt bc
1
vs.
2% in noninhibited cyt b
6
f(6, 26, 27), used for detection of USQ-
FeS, represent the conditions in which the enzyme produces sig-
nificant amount of O
2
per single oxidized QH
2
(1018%).
Discussion
One of the important issues associated with the understanding of
fundamentals of oxidative metabolism concerns elucidation of
the mechanism by which enzymes catalyzing reactions that pro-
ceed through highly unstable radical intermediates have adapted
to safely function in the presence of molecular oxygen (8). In
addressing this issue for the cyt bc
1
/b
6
ffamily, a concept emerges
from the unexpected discovery of the PSQ-FeS state in Q
p
of
noninhibited cyt b
6
f(Fig. 2A) and subsequent comparative
analysis of the conditions favoring appearance of this state in cyt
b
6
fand cyt bc
1
under oxygenic and anoxygenic environments
(Figs. 25). Based on the results and analysis presented in this
work, it is concluded that the generation of SQ-FeS in cyt bc
1
or
cyt b
6
fis an inherent part of enzymatic catalysis that can be
described as a metastable state nonreactive with oxygen. The
concept of stabilization of SQ in the Q
p
site by interaction with
reduced FeS of cyt bc
1
was proposed by Link (28), who consid-
ered antiferromagnetic coupling of high energy between these
two centers as a possible explanation for the failure to detect an
SQ intermediate in this site. However, such a strong interaction
should result in disappearance of the EPR signal of FeS, which
was not observed. Furthermore, this concept was in opposition to
a more popular view explaining the lack of SQ detection by a
high instability of SQ at Q
p
(9, 10, 12, 20). Our results indicate
that coupling between reduced FeS and SQ exists in both cyt bc
1
and cyt b
6
f, but its energy is small enough that, regardless of the
antiferromagnetic or ferromagnetic character of coupling, it pro-
duces detectable EPR transitions of SQ-FeS (with a characteristic
g1.94) at temperatures higher than a few degrees Kelvin.
Quite importantly, SQ-FeS in cyt bc
1
is observed along with
reduced heme b
n
and oxidized heme b
p
(Fig. 4A). On thermo-
dynamic grounds, this observation implies that electron transfer
from SQ-FeS to heme b
p
is an uphill step. As a consequence, if
heme b
p
is unable to transfer electrons further to heme b
n
, the
electron moves back to quinone (Q) at Q
p
and SQ-FeS is re-
formed. After reaching equilibrium, no net reactions that lead to
significant occupation of the metastable state occur and, conse-
quently, this state is no longer detected spectroscopically. To
observe it at equilibrium, one must use a high potential quinone
analog (DBMIB in our case). However, a large increase in E
m
dramatically stabilizes this state to the point that it becomes in-
hibitory, at least for cyt b
6
f. The electron from DBMIB
semiquinone, because of its relatively oxidizing potential, can
never reduce hemes b
p
or b
n
, 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 cycle
but adds a new state, state 4, which is a result of an energetically
downhill electron transfer from heme b
p
to Q at Q
p
(transition
from state 3). This state protects the enzyme against ROS pro-
duction by: (i) the fact that electron transfer from SQ-FeS to
molecular oxygen (to state 8) is energetically unfavorable and
(ii) blocking Q
p
for the next QH
2
oxidation until electrons are
removed from the low-potential chain. The metastable state exists
until electrons from the low-potential chain are removed through
the Q
n
site (Q
n
) 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 from
the low-potential chain to the Q pool, in relation to the rate of the
reduction of this chain by Q
p
, creates conditions that favor ap-
pearance of the SQ-FeS metastable state. Such conditions may
exist in some physiological states, e.g., in the presence of a high
transmembrane potential or a high concentration of QH
2
in the Q
pool. It is intriguing that a similar g=1.94 transition of unknown
origin, which might reflect the USQ-FeS state, was reported to
appear under ischemia and disappear under reperfusion of rat
hearts (29).
The free energy (ΔG) diagram, shown in Fig. 6, which depends
not only on E
m
but also on other processes, e.g., reconfiguration
of H bonds within Q
p
, provides a possible explanation for sig-
nificant occupation of the energetic state representing PSQ-FeS
in noninhibited cyt b
6
f. Although the difference in E
m
between
hemes b
n
and b
p
in cyt b
6
fis not well defined (shown as the width
in the level of state 5 in Fig. 6A), the average is somewhat more
negative than in hemes bin cyt bc
1
(30, 31). This difference,
together with the fact that PQ possesses a more positive E
m
than
UQ (13) makes the energetic gap between state 4 (with SQ-FeS)
and state 5 (with reduced heme b
n
) smaller in cyt b
6
fcompared
with cyt bc
1
. In other words, in cyt bc
1
, the electron transfer from
USQ-FeS (state 4) to heme b
n
(state 5) is energetically much
more favorable than the electron transfer from PSQ-FeS to
heme b
n
in cyt b
6
f. In addition, the existence of high-spin heme c
n
is possibly a bottleneck for electron flow from the low-potential
chain to the Q pool, or reduction of PQ in Q
n
requires a co-
operative two-electron transfer from hemes b
n
/c
n
(32, 33).
With the proposed mechanism, it can be appreciated that
energetic states associated with oxidation of QH
2
by cyt b
6
f/cyt bc
1
are positioned at levels that allow smooth catalysis while limiting
released ROS to perhaps just signaling levels that carefully report
the dynamically changing redox state of cofactors (6, 34). It is
proposed that the SQ-FeS metastable state serves as a buffer
for electrons that are unable to be relegated from Q
p
through the
low-potential chain. Availability of the buffer allows the enzyme
to be held in the state that protects against energy-wasting reac-
tions including the short circuits (two electrons from QH
2
going
to the same cofactor chain) and the leaks of electrons to mo-
lecular oxygen (1, 7, 8). At the molecular level, the stabilization of
SQ by its spin-coupling to reduced FeS is likely to occur through
the creation of an H bond between SQ and histidine ligating
reduced 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 the
formation of a highly unstable semiquinone (SQ not spin-coupled
to 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 and
SQ that is not spin-coupled to FeS (states 4 and 2, respectively),
stationary levels of these two states will be proportional. It follows
that the amount of ROS will correlate with the amount of the
Fig. 5. Comparison of the amplitudes of g1.95/1.94 transition, in oxy-
genic and anoxygenic environment for cyt b
6
f(A) and cyt bc
1
(B), obtained
under conditions similar to those of Fig. 2.
1326
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www.pnas.org/cgi/doi/10.1073/pnas.1618840114 Sarewicz et al.
detected SQ-FeS, despite its nonreactivity with oxygen. Indeed,
noninhibited cyt b
6
fproduces larger amounts of superoxide than
noninhibited cyt bc
1
(6), which stays in line with our observation
that in the case of noninhibited enzymes SQ-FeS can only be
detected in cyt b
6
f(Fig. 2A). Nevertheless, when semiquinone is
present in Q
p
, the equilibrium is always shifted toward its sta-
bilization by formation of the SQ-FeS spin-coupled center
whereby ROS release is limited to the level of 2 molecules of
O
2
per 100 electrons transferred to FeS. Such residual ROS
generation detected in cyanobacterial cyt b
6
fhas been proposed
to 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 E
m
values
of quinones and low-potential cofactors. Perhaps it is adjusted to
the oxygen tension in the cellular environment (8). Indeed, cyt
b
6
f, which experiences more than an order of magnitude higher
level of oxygen in chloroplasts than cyt bc
1
in mitochondria, has a
greater tendency to reside in this buffered state. Also, a possible
consequence of the existence of PSQ-FeS in noninhibited cyt b
6
f
is that it may be responsible for regulation of the electron transfer
pathways of oxygenic photosynthesis. As proposed, cyt b
6
fin
native chloroplasts can catalyze PQH
2
oxidation according to two
alternative mechanisms: (i) noncyclic, in which one molecule of
PQ in Q
n
undergoes a sequential reduction by two electrons
derived from Q
p
and (ii) cyclic, in which one electron is delivered
to Q
n
from Q
p
, whereas the second electron comes from reduced
ferredoxin (2). We speculate that creation of the metastable state
PSQ-FeS may serve as a factor that changes the efficiency of
cyclic vs. noncyclic electron transfer between photosystem I and
II. This inference is explained by the fact that transient
stabilization of the PSQ-FeS blocks the oxidation of another
PQH
2
in Q
p
, and, thus, creates a condition in which the proba-
bility of delivering the second electron needed to complete the
reduction of PQ in Q
n
by ferredoxin is significantly increased.
The phenomenon of spinspin exchange interactions between
semiquinones and metal centers has been observed many times
in different biological systems, including a coupling between tightly
bound semiquinone Q
A
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 the
vicinity of radicals is not always clear and remains a subject of de-
bate, as exemplified by recent discussion on possible origin on the
unusual properties of the SQ signals in complex I (41). In light of the
present study, we envisage that FeS in cyt bc
1
/b
6
fhas a dual role in
electron transfer. Its obvious role is to accept electrons from sub-
strate but besides this function, it offers a mean of stabilization of
potentially dangerous intermediates that are inherently associated
with the stepwise quinone redox reactions. This metal center behaves
as a Lewis acid with electrophilic properties. When it creates a bond
with SQ, its electron-withdrawing properties decrease the probability
of reaction of SQ with oxygen. We propose that this feature is not
restricted to the iron-sulfur cluster of cyt bc
1
/b
6
f, but may be common
for other metal cofactors that, besides having a role in electron
transfer, stabilize potentially reactive radicals.
ACKNOWLEDGMENTS. This work was supported by The Wellcome Trust
Grant 095078/Z/10/Z (to A.O.). Faculty of Biochemistry, Biophysics and
Biotechnology of Jagiellonian University is a partner of the Leading National
Research Center supported by Ministry of Science and Higher Education, part
of 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 b
6
f(A) and cyt bc
1
(B). Black dots represent electrons on
the respective cofactor or quinone molecule. Yellow squares, Q
p
empty or occupied by substrate; purple squares, Q
p
with bound DBMIB semiquinone. Green
and red arrows, downhill and uphill transitions between the states, respectively. Red frame indicates the states that are inaccessible in antimycin-inhibited cyt
bc
1
. State 1 represents the most populated initial state for QH
2
oxidation under steady-state turnover (in antimycin-inhibited cyt bc
1
heme b
n
is already
reduced after the first turnover that takes place within experimental dead time). This reaction results in reduction of FeS and heme b
p
(transition from 1 to 3
involving 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 b
n
) allowing
next turnover. State 4 is metastable state containing SQ spin-coupled to reduced FeS. Population of 4 increases when transition to 5 is blocked (antimycin) or
transition 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 double
arrow), which differs within bc family. State 4 lays below energetic level of 8 in which O
2
is generated. State 7 involves stable DBMIB semiquinone spin-
coupled to FeS. The gray square with a gradient depicts uncertainty in E
m
values for hemes bin cyt b
6
f.
Sarewicz et al. PNAS
|
February 7, 2017
|
vol. 114
|
no. 6
|
1327
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1328
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www.pnas.org/cgi/doi/10.1073/pnas.1618840114 Sarewicz et al.
... A general agreement has been reached that a superoxide generation by Cytbc 1 results from a reaction of USQ o with molecular oxygen (Boveris and Cadenas, 1975;Ksenzenko et al., 1983;Muller et al., 2002;Borek et al., 2008;Dröse and Brandt, 2008;Sarewicz et al., 2010;Pagacz et al., 2021). However, trapping the intermediate USQ o radical during the UQH 2 oxidation and its detection by electron paramagnetic resonance (EPR) has been proven difficult and obtained results have often been disputable or contradictory (de Vries et al., 1981;Jünemann et al., 1998;Cape et al., 2006;Cape et al., 2007;Zhang et al., 2007;Zhu et al., 2007;Sarewicz et al., 2013;Vennam et al., 2013;Victoria et al., 2013;Pietras et al., 2016;Crofts et al., 2017;Sarewicz et al., 2017;Sarewicz et al., 2018;Bujnowicz et al., 2019). Additionally, the inability to generate USQ o in the equilibrium redox titrations (Takamiya and Dutton, 1979;Sarewicz et al., 2018) implicated a concept of a high instability of the semiquinone and its reactivity was proposed to be a reason for superoxide production by Cytbc 1 . ...
... Our previous work reported an unusual EPR signal with an average g value less than 2, which originated from the Q o site of the antimycin-inhibited Cytbc 1 , isolated from Rhodobacter capsulatus (Sarewicz et al., 2013;Sarewicz et al., 2017;Bujnowicz et al., 2019). This signal was detected only when the Q o site was able to catalyze the UQH 2 oxidation and the cytochrome c reduction before the system reached equilibrium. ...
... At X-band, the most prominent "derivative-shaped" transition of SQ o -2Fe2S in Cytbc 1 was detected at g ∼1.94 and was found to shift to ∼1.96 at Q-band. Remarkably, the SQ o -2Fe2S signal was also detected in isolated but non-inhibited spinach cytochrome b 6 f during the oxidation of synthetic decylplastoquinol and the reduction of plastocyanin (Sarewicz et al., 2017). ...
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Cytochrome bc 1 (mitochondrial complex III) catalyzes electron transfer from quinols to cytochrome c and couples this reaction with proton translocation across lipid membrane; thus, it contributes to the generation of protonmotive force used for the synthesis of ATP. The energetic efficiency of the enzyme relies on a bifurcation reaction taking place at the Q o site which upon oxidation of ubiquinol directs one electron to the Rieske 2Fe2S cluster and the other to heme b L . The molecular mechanism of this reaction remains unclear. A semiquinone spin-coupled to the reduced 2Fe2S cluster (SQ o -2Fe2S) was identified as a state associated with the operation of the Q o site. To get insights into the mechanism of the formation of this state, we first constructed a mutant in which one of the histidine ligands of the iron ion of heme b L Rhodobacter capsulatus cytochrome bc 1 was replaced by asparagine (H198N). This converted the low-spin, low-potential heme into the high-spin, high-potential species which is unable to support enzymatic turnover. We performed a comparative analysis of redox titrations of antimycin-supplemented bacterial photosynthetic membranes containing native enzyme and the mutant. The titrations revealed that H198N failed to generate detectable amounts of SQ o -2Fe2S under neither equilibrium (in dark) nor nonequilibrium (in light), whereas the native enzyme generated clearly detectable SQ o -2Fe2S in light. This provided further support for the mechanism in which the back electron transfer from heme b L to a ubiquinone bound at the Q o site is mainly responsible for the formation of semiquinone trapped in the SQ o -2Fe2S state in R. capusulatus cytochrome bc 1 .
... A relatively recent study by Sarewicz/Bujnowicz et al. revealed that the EPR spectrum of 2Fe2S in DBMIB-supplemented spinach Cytb 6 f is frequency-dependent, which is a strong indication that DBMIB semiquinone at the Q p site is spin−spin coupled to the reduced 2Fe2S. 482 Besides being extensively used to characterize electron flow in the photosynthetic chain, DBMIB has also been largely employed to study cell biological responses in photosynthetic organisms. Combining DBMIB with the PSII specific inhibitor DCMU allows the PQ(H 2 ) pool to be either reduced (DBMIB) or oxidized (DCMU) in the light. ...
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... 258,575,597 Interestingly the SQ-2Fe2S state is not restricted to Cytbc 1 but has as it was also observed in isolated spinach Cytb 6 f exposed to its substrates, PQH 2 and PC. 482 In this case, however, unlike in Cytbc 1 , inhibition of the Q n site was not required. This revealed that the probability of SQ-2Fe2S state formation when the enzyme operates without inhibition is higher in the Q p site of Cytb 6 f compared to the Q o site Cytbc 1 . ...
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... Its operation is prone to bypass reactions which generate reactive oxygen species (ROS) via a semiquinone radical, resulting in energy waste and deleterious radicals 13 . Unproductive reactions can be triggered through an unbalanced redox-state of the quinone pool, hypoxia, or inherited diseases [14][15][16][17] . In cyt c oxidases, the key to energy conversion is a coupling of dioxygen reduction at the canonical binuclear centre (BNC) with proton pumping 2 . ...
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... In our model, the entrance for quinones occurs at a site never considered before. Nonetheless, 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), a potent inhibitor of Q p that forms a stable semiquinone-2Fe2S state (39), was found in the crystal structure bound in the region of PQ1 ( fig. S7) (40). ...
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... 51,52 Indeed, a SQ spincoupled to 2Fe2S was observed in native, noninhibited cytb 6 f during catalysis. 30 To observe such a state in cytbc 1 , which has no uphill step in LPC, it is necessary to add a Q i site inhibitor that blocks electron transfer through LPC. ...
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... In this process, the Q o site quinol releases two protons to the P-side of the membrane and the complete reduction and protonation of a quinone molecule in the Q i site needs oxidation of a second quinol at the Q o site and proton uptake from the N-side of the membrane. Consequently, bifurcated electron transfer must be achieved upon quinol oxidation to enable the Q cycle, i.e. the highly reactive SQ • at the Q o site must be controlled to avoid short circuits [2,[29][30][31][32][33][34] which lead to futile bypass reactions which would lower the efficiency of cellular respiration and can generate reactive oxygen species [29] that can cause oxidative damage to the cell [35]. ...
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... More recent studies, however, suggest that the oxygen reductant is the semiquinone formed in the so-called semi-reverse reaction in which cytochrome b L reduces the fully oxidized quinone [245,246]. Osyczka [247,248] proposes that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of ubiquinol. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site, presumably the FeS cluster, under physiological conditions. ...
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On looking back at a lifetime of research, it is interesting to see, in the light of current progress, how things came to be, and to speculate on how things might be. I am delighted in the context of the Mitchell prize to have that excuse to present this necessarily personal view of developments in areas of my interests. I have focused on the Q-cycle and a few examples showing wider ramifications, since that had been the main interest of the lab in the 20 years since structures became available, − a watershed event in determining our molecular perspective. I have reviewed the evidence for our model for the mechanism of the first electron transfer of the bifurcated reaction at the Qo-site, which I think is compelling. In reviewing progress in understanding the second electron transfer, I have revisited some controversies to justify important conclusions which appear, from the literature, not to have been taken seriously. I hope this does not come over as nitpicking. The conclusions are important to the final section in which I develop an internally consistent mechanism for turnovers of the complex leading to a state similar to that observed in recent rapid-mix/freeze-quench experiments, reported three years ago. The final model is necessarily speculative but is open to test.
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The mechanisms of action of reactive oxygen species (ROS) in plant and animal cells, in chloroplasts, mitochondria, and other sub-cellular compartments such as the endoplasmic reticulum and nucleus, which result in deleterious effects as well as intra-organelle and metabolically significant signaling reactions, are discussed, as well as physiological effects induced by the signaling functions of ROS. The role of ROS in chloroplast retrograde signaling and modulation of abiotic stress response and pathogen defense is discussed. Attention is focused on mechanisms of ROS production, and particularly on the intra-organelle reactions mediated through reduction of oxygen to superoxide in the mitochondrial cytochrome bc 1 and the chloroplast b 6f complexes, for which understanding of the mechanistic details is facilitated by the existence of high resolution crystal structures. Attention is directed to the structure-based details that underlie the more than tenfold larger specific rate of superoxide production in the b 6f complex compared to that of the mitochondrial bc 1 complex. Because plastoquinol oxidation by the cytochrome b 6f complex is known to initiate chloroplast state transitions, the effect of superoxide on the intra-membrane chlorophyll protein kinase that is integral to the cytochrome b 6f complex, and which underlies the photosynthetic state transitions that regulate the distribution of light energy between the two photosystems, is considered.
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The two-electron ubiquinol oxidation or ubiquinone reduction typically involves semiquinone (SQ) intermediates. Natural engineering of ubiquinone binding sites of bioenergetic enzymes secures that SQ is sufficiently stabilized, so that it does not leave the site to membranous environment before full oxidation/reduction is completed. The ubiquinol oxidation Qo site of cytochrome bc1 (mitochondrial complex III, cytochrome b6f in plants) has been considered an exception with catalytic reactions assumed to involve highly unstable SQ or not to involve any SQ intermediate. This view seemed consistent with long-standing difficulty in detecting any reaction intermediates at the Qo site. New perspective on this issue is now offered by recent, independent reports on detection of SQ in this site. Each of the described SQs seems to have different spectroscopic properties leaving space for various interpretations and mechanistic considerations. Here, we comparatively reflect on those properties and their consequences on the SQ stabilization, the involvement of SQ in catalytic reactions, including proton transfers, and the reactivity of SQ with oxygen associated with superoxide generation activity of the Qo site.
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Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex - cytochrome bc1 - are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1(-1)) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1(-1) (T) over the range 5-120 K yielded the values of the Orbach energy (EOrb), Debye temperature θD and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While θD was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin-orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin-orbit coupling but was also influenced by other factors - possibly the modification of protein rigidity and therefore the vibrational modes around the Fe-S cluster that change upon the movement of the iron-sulphur head domain.
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Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. Copyright © 2015 the American Physiological Society.
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The electrochemical parameters of all cofactors in the supercomplex formed by the Rieske/cytb complex and the SoxM/A-type O2-reductase from the menaquinone-containing Firmicute Geobacillus stearothermophilus were determined by spectroelectrochemistry and EPR redox titrations. All redox midpoint potentials (Em) were found to be lower than those of ubi- or plastoquinone-containing systems by a value comparable to the redox potential difference between the respective quinones. In particular, Em values of +200 mV, -360 mV, -220 mV and -50 mV (at pH 7) were obtained for the Rieske cluster, heme bL, heme bH and heme ci, respectively. Comparable values of -330 mV, -200 mV and +120 mV for hemes bL, bH and the Rieske cluster were determined for an anaerobic Firmicute, Heliobacterium modesticaldum. Thermodynamic constraints, optimization of proton motive force build-up and the necessity of ROS-avoidance imposed by the rise in atmospheric O2 2.5 billion years ago are discussed as putative evolutionary driving forces resulting in the observed redox upshift. The close conservation of the entire redox landscape between low and high potential systems suggests that operation of the Q-cycle requires the precise electrochemical tuning of enzyme cofactors to the quinone substrate as stipulated in P. Mitchell's hypothesis.
Chapter
The cytochrome b 6f complex (f, derived from “leaf” in Latin, “folium,” or French, “feuille”, see D. S. Bendall, this volume) is responsible for the proton-coupled electron transfer reactions that link the two light-trapping photosystem reaction centers in the electron transport chain of oxygenic photosynthesis, and contains the rate-limiting step of the entire electron transport chain. The understanding of the functions of the intra-membrane cytochrome b 6f complex in its role as the central electron transport complex in the photosynthetic electron transport chain has greatly increased in recent years because of the solving of the atomic structure of the complex, as well as its extrinsic electron transfer domains solved separately, by X-ray crystallographic analysis. The electron transfer and proton translocation functions of the cytochrome b 6f complex are discussed in a context that focuses on function in the context of structure. The structure considerations are based on the recently determined high resolution (2.50 Å) crystal structure (PDB, protein data base, accession 4OGQ). The complex consists of a 280 kDa hetero-oligomeric intra-membrane lipo-protein complex containing, per monomer, 13 trans-membrane α-helices and seven prosthetic groups, the latter consisting of five hemes, one chlorophyll a molecule and one β-carotene. In addition, each monomer contains 23 lipid binding sites, the function of which crosses into a completely new area of structure-function, both for the b 6f complex and for the entire broader field of membrane proteins. Considering energy-transducing membrane protein complexes, the b 6f complex, along with other hetero-oligomeric electron transfer membrane proteins provides a unique perspective in the broad field of membrane protein structure-function that encompasses transport and channel proteins, and extends beyond photosynthesis and bioenergetics to biomedicine. The additional perspective arises from the fact that the function of the cytochrome complexes, along with those of other redox proteins, can be assayed quantitatively through various high resolution spectroscopies. A major theme in the present article is the comparison of structure and function of the b 6f and the cytochrome bc 1 complex, which is located in the mitochondrial respiratory chain and in purple photosynthetic bacteria. One purpose of the comparison is to illustrate that although the two complexes are phylogenetically related, and there are many similarities in structure-function, there are many differences. An analysis and comparison of structures and functions implies that all charge mechanisms determined for bc 1 should not apply without exception or modification to the b 6f complex.
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The cytochrome (cyt) bc(1), b(6)f and related complexes are central components of the respiratory and photosynthetic electron transport chains. These complexes carry out an extraordinary sequence of electron and proton transfer reactions that conserve redox energy in the form of a trans-membrane proton motive force for use in synthesizing ATP and other processes. Thirty years ago, Peter Mitchell proposed a general turnover mechanism for these complexes, which he called the Q-cycle. Since that time, many opposing schemes have challenged the Q-cycle but, with the accumulation of large amounts of biochemical, kinetic, thermodynamic and high-resolution structural data, the Q-cycle has triumphed as the accepted model, although some of the intermediate steps are poorly understood and still controversial. One of the major research questions concerning the cyt bc(1) and b(6)f complexes is how these enzymes suppress deleterious and dissipative side reactions. In particular, most Q-cycle models involve reactive semiquinone radical intermediates that can reduce O(2) to superoxide and lead to cellular oxidative stress. Current models to explain the avoidance of side reactions involve unprecedented or unusual enzyme mechanisms, the testing of which will involve new theoretical and experimental approaches.
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Three important classes of redox centers involved in electron transfer (ET) processes are reviewed. Together, however, they can cover the whole range, with cytochromes in the middle, Fe-S centers toward the lower end, and the cupredoxins toward the higher end. All three redox centers have structural features that make them unique, and yet they also show many similarities that make them excellent choices for ET processes. Because the redox-active iron is fixed into a rigid porphyrin that accounts for four of the iron's six coordination sites, most of the electronic structure and redox properties remain similar between different cytochromes. In completing the primary coordination sphere of the iron, cytochromes typically use a combination of nitrogen and sulfur ligations from histidine or methionine side chains, respectively; terminal amine ligation has also been observed. For iron-sulfur proteins, the reduction potential ranges are influenced to some extent by the number of irons because it affects the redox states and transitions.