Metastable Radical State, Nonreactive with Oxygen, Is Inherent to Catalysis by Respiratory and Photosynthetic Cytochromes Bc1/B6

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Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc1/b6f Marcin 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; and bDepartment 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 decrease the stationary level of SQ to avoid reactive oxygen reactions face a danger of wasteful energy dissipation by diversion species (ROS) (9–12). This destabilization of SQ, in turn, in- of the productive electron transfer pathway through the genera- evitably leads to an increase in the rate constant for the reaction tion of reactive oxygen species (ROS). Nevertheless, the widespread of SQ with oxygen, which might have deleterious consequences, quinone oxido-reductases from the cytochrome bc family limit the especially for enzymes exposed to the relatively high local oxygen amounts of released ROS to a low, perhaps just signaling, level concentrations associated with oxygenic photosynthesis (8, 13). through an as-yet-unknown mechanism. Here, we propose that a Here, it is shown that both cyt b6f and cyt bc1 generate a metastable radical state, nonreactive with oxygen, safely holds metastable radical state, nonreactive with oxygen, under steady- electrons at a local energetic minimum during the oxidation of state turnover. This result sheds light on thermodynamic prop- b f plastohydroquinone catalyzed by the chloroplast cytochrome 6 . erties of intermediates of electronic bifurcation at the Qp,imply- This intermediate state is formed by interaction of a radical with a ing a mechanism that explains how cyt b6f/bc1 maintain a balance metal cofactor of a catalytic site. Modulation of its energy level on between energy-conserving reactions and ROS production to se-

the energy landscape in photosynthetic vs. respiratory enzymes cure the enzymatic reactions at physiologically competent rates. BIOCHEMISTRY provides a possible mechanism to adjust electron transfer rates This mechanism provides a thermodynamic basis for the signifi- for efficient catalysis under different oxygen tensions. − cant difference in the O2 generation in the two enzymes, and advances our understanding of the molecular mechanism of con- cytochrome b6f | reactive oxygen species | semiquinone | electron trol of electron flow through the photosynthetic and respiratory paramagnetic resonance | electron transport chains and its contribution to ROS generation, which are postulated to function as signaling mediators released from bioenergetic hotosynthetic and respiratory cytochromes bc1/b6f (Fig. 1A) organelles. Pgenerate a proton-motive force (pmf) that powers cellular metabolism by using the Gibbs free energy difference (ΔG) be- Materials and Methods tween hydroquinone (QH2) derivatives (Fig. 1B) and oxidized Reagents. Equine cytochrome c, decylubiquinone (DB), decylplastoquinone soluble electron transfer proteins (e.g., cytochrome c or plasto- (PQ), antimycin A, dibromothymoquinone (DBMIB), sodium borohydride, cyanin) (1, 2). To increase the efficiency of this process, which is sodium dithionite, potassium ferricyanide (PFC), and other reagents were critical for the yield of the generated pmf, one part of the enzyme recirculates electrons to the quinone pool in the membrane Significance (Q pool), whereas the second part steers the electrons to the cytochrome c pool, powering the electron recirculation (Fig. 1C). Photosynthesis and respiration are crucial energy-conserving This mechanism, which is best established for the cytochrome bc1 processes of living organisms. These processes rely on redox (cyt bc1) (3, 4), with supporting data for the cytochrome b6f (cyt reactions that often involve unstable radical intermediates. In b6f) (5), discussed in ref. 2, is based on bifurcation of the route an oxygenic atmosphere, such intermediates present a danger for two electrons released upon oxidation of QH2 at one of the of becoming a source of electrons for generation of reactive — – catalytic sites the Qp site, (Qp), (Fig. 1D)(35). A model for oxygen species. Here, we discover that cytochrome b6f, a key the energetics of this reaction assumes that one electron derived component of oxygenic photosynthesis, generates a meta- from the two-electron QH2 donor is transferred, through the stable state nonreactive with oxygen during enzymatic turn- high-potential cofactor chain (“steering part” in Fig. 1C)to over. In this state, a radical intermediate of a catalytic cycle plastocyanin or cytochrome c, whereas the second electron is interacts with a metal cofactor of a catalytic site via spin-spin transferred across the membrane through low-potential cofactors exchange. We propose that this state is a candidate for regu- (“recirculation” part in Fig. 1C). lation of cyclic vs. noncyclic photosynthesis and also allows The electronic bifurcation process requires formation of a photosynthetic and respiratory cytochrome bc complexes to short-lived and reducing redox intermediate—ubisemiquinone function safely in the presence of oxygen. (USQ) or plastosemiquinone (PSQ) (4, 6, 7). However, such an intermediate in an oxygenic environment would readily reduce 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 oxygen to form superoxide radical, (O2 ), compromising the A.O. wrote the paper. efficiency of energy conservation (8). Even in cyt b6f where the level of superoxide production through this pathway is at least an The authors declare no conflict of interest. This article is a PNAS Direct Submission. order of magnitude greater than that from yeast cyt bc1, the − branching ratio for electron transfer to O2 forming O2 is only 1– Freely available online through the PNAS open access option. − 1 2% of the total flux (6). The low absolute level of O2 production M.S. and Ł.B. contributed equally to this work. in native proteins implies the existence of a mechanism that is 2To whom correspondence should be addressed. Email: [email protected]. not understood. In fact, contemporary models are based on at- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tempts to decrease stability of semiquinone (SQ) as a means to 1073/pnas.1618840114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1618840114 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Fig. 1. Structural and functional elements of cyt b6f/bc1 catalysis. (A) Cofactors and catalytic sites (red circles) overlaid on the protein surfaces (based on crystal structures of cyt b6f and cyt bc1 (PDB ID codes: 1VF5 and 1ZRT, respectively). (B) Chemical structures of hydroxyquinones with estimated average Em values of Q/QH2 couples at pH 7. Red, redox-active groups. (C) General scheme showing the “steering” and “recirculation” parts. (D) Simplified sequential scheme 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.

purchased from Sigma-Aldrich. Dodecyl-maltoside detergent was purchased and 190 μM, respectively. Samples with cyt bc1 for Q-band measurements

from Anatrace. PQ and DB were suspended in ethanol and DMSO, re- (Fig. 2B) were obtained similarly as for cyt b6f, as shown in Fig. 2A. Final

spectively, and reduced to hydroquinone form with H2 gas released from concentrations of cyt bc1, antimycin, PFC, and DBH2 were 300 μM, 500 μM, acidic water solution of sodium borohydride in the presence of platinum. 2.5 mM, and 4 mM, respectively. Spectra in Fig. 3 A and B were obtained for Ethanolic stock of reduced PQ was mixed with DMSO in 1:2 (vol/vol) ratio to samples containing 90 μM cyt b6f or 130 μMcytbc1 supplemented with decrease the rate of spontaneous oxidation of plastohydroquinone (PQH2). 2 mM DBMIB. Both substrates were kept at −80 °C until used.

Preparation of Samples Under Anoxic Conditions. Samples containing cyt b6f Enzymes. Cyt bc1 was isolated according to the procedure described in ref. 14 and cyt bc1 in aerobic and anaerobic conditions were prepared similarly as from wild-type Rhodobacter capsulatus grown under semiaerobic condi- those for X-band experiments presented in Fig. 2 A and B, respectively, with tions. Thylakoid membranes were isolated from spinach as described in ref.

15. Cyt b6f was isolated according to the procedure described in SI Materials and Methods.

Electron Paramagnetic Resonance Spectroscopy. Electron paramagnetic resonance (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. 2A were prepared by manual injection of PQH2 to the EPR tube containing a mixture of cyt b6f, plastocyanin (PC), and PFC. After the addition 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 Fig. 2. EPR spectra of FeS of cyt b6f (A) and cyt bc1 (B) at two resonance concentrations of cyt b6f, PC, PFC, and PQH2 were 140 μM, 25 μM, 680 μM, frequencies (X and Q). Blue, spectra measured for samples obtained by rapid μ and 910 M, respectively. Samples for measurements shown in Fig. 2B (X freezing of the reaction solution 2 s after mixing with respective QH2. Black, band) were prepared by the freeze-quench method as described in ref. 17 spectra of the same samples measured after reaching the equilibrium (5 min with the exception that glycerol was not present in the buffer. Final con- after mixing). Vertical dashed lines, rounded g values of major EPR transi-

centrations of cyt bc1, cytochrome c, antimycin, and DBH2 were 20, 190, 130, tions (see Fig. S1 for details).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1618840114 Sarewicz et al. Downloaded by guest on October 2, 2021 observation implies that the probability of formation of SQ-FeS in noninhibited enzymes is greater in cyt b6f than in cyt bc1.

Detection of the Spin-Coupled State Between High-Potential Quinone

Analog (DBMIB) and Reduced FeS in cyt b6f/bc1 Under Equilibrium. A similar paramagnetic state can be generated in cyt b6f and cyt bc1 under equilibrium in the presence of the halogenated quinone derivative, DBMIB (Fig. 3 A and B, respectively), possessing a relatively high average redox mid-point potential (Em) (see comparison of Ems of quinone/hydroquinone couples for ubi- Fig. 3. EPR spectra of FeS measured for samples of cyt b6f (A) and cyt bc1 quinone (UQ), PQ, and DBMIB in Fig. 1B). In fact, this signal (B) after 5 min incubation of the enzymes with DBMIB. Vertical dashed lines, was observed in spectra of DBMIB-inhibited cyt b6f or cyt bc1 rounded g values of major EPR transitions. (22–25). However, the nature of the g transitions in the presence of DBMIB, as we now propose, was misinterpreted as an alter-

the exception that the final concentration of cyt b6f was 50 μM. To obtain ation of protein structure around FeS. If DBMIB changed the g anaerobic conditions, glucose oxidase (final activity 1 U/mL) and glucose tensor of FeS, the g values of the spectra for DBMIB-altered FeS (final concentration 15 mM) were added to the reaction mixture. Reagents should have the same value regardless of the spectrometer fre- after glucose addition were incubated for 30 min and mixed under atmo- quency used for detection. The clear frequency dependence of sphere of sulfur hexafluoride gas. the g values measured for samples in the presence of DBMIB (Fig. 3 A and B) indicates that it binds to reduced FeS as a Results semiquinone, and these two centers are subject to similar spin– b f Detection of PSQ Spin-Coupled to Reduced FeS in Noninhibited cyt 6 spin exchange interaction as USQ-FeS (17) or PSQ-FeS coupled Under Nonequilibrium Conditions. Fig. 2A shows that noninhibited centers in cyt bc1 and cyt b6f, respectively. cyt b6f, exposed to its substrates, PQH2 and oxidized plastocyanin, generates an intermediate of Qp detected by EPR at a Redox State of Hemes b Under Conditions Favoring Generation of SQ- characteristic spectral line position defined by an approximate g FeS. Fig. 4 examines the redox state of the hemes b in cyt bc1 value ∼1.95 (see Fig. S1 for exact g values). The g value of this complex under three different conditions relevant to experi-

transition strongly depends on the resonance frequency. The ments described above. Before mixing the enzyme with ubihy- BIOCHEMISTRY shift in the g value indicates that the transition must be a result of droquinone (UQH2) b-type hemes located at n and p side of magnetic interactions between at least two paramagnetic centers membrane (bn and bp, respectively) are oxidized (Fig. 4A, green). (18, 19) and not an effect of structural changes that lead to However, after 2 s required to generate a relatively large USQ- modifications of g values of [2Fe-2S] Rieske cluster (FeS). Fur- FeS signal (Fig. 2B) heme bn undergoes complete reduction, thermore, it closely resembles a transition (also frequency- whereas heme b is still fully oxidized (Fig. 4A, blue). This result ∼ p dependent) found earlier in cyt bc1 (g 1.94 in Fig. 2B) and appears unexpected because it means that semiquinone that is in assigned as USQ magnetically coupled to reduced FeS via spin– spin–spin exchange interaction with reduced FeS is unable to spin exchange interaction (designated as USQ-FeS) (17). By reduce heme bp. Even less reducing power is observed when analogy to cyt bc1, we propose that the signal in cyt b6f originates semiquinone form of DBMIB creates the spin-spin coupled state from PSQ that undergoes electron spin–spin exchange interac- with FeS. In this case, both hemes remain oxidized (Fig. 4B) tion with reduced FeS (PSQ-FeS). The occupancy of SQ-FeS because they are unable to take electrons from the high-potential center in cyt b f and cyt bc (Fig. 2 A and B, blue) was estimated 6 1 synthetic semiquinone at the Qp. as 13% and 42% of the total Qp sites, respectively (see details in SI Materials and Methods). Testing the Reactivity of PSQ-FeS and USQ-FeS with Molecular To record SQ-FeS (this term corresponds to either USQ-FeS Oxygen. Because the experiments revealing SQ-FeS (shown in or PSQ-FeS) in cyt b6f, as in cyt bc1, the steady-state enzymatic Fig. 2) were performed in the presence of oxygen, we infer that reaction must be interrupted, and the reaction mixture frozen SQ-FeS cannot be highly reactive with oxygen. It follows that the before equilibrium between substrates (PQH2 and oxidized yield of generated SQ-FeS should not be significantly sensitive to plastocyanin) and products (PQ and reduced plastocyanin) are the presence or absence of O2. Indeed, in cyt b6f, the amount of reached. When the enzymes used all substrates (at equilibrium), PSQ-FeS generated under oxygenic and anoxygenic conditions is the signals were no longer present (Fig. 2 A and B, black). similar (Fig. 5A). However, in the case of cyt bc1, the amount of Nevertheless, the SQ-FeS in both enzymes, despite being far USQ-FeS is almost always higher when the reaction is carried from the global energy minimum, is relatively long-lived in out in the presence of oxygen (see example in Fig. 5B). The comparison with a putative unstable SQ that is commonly de- reasons why USQ-FeS is more efficiently formed in the presence scribed (9, 10, 20, 21). A series of control experiments (Fig. S2) of oxygen is not understood. However, one may speculate that a − verified that the detected signal did not result from nonspecific portion of O2 reduces UQ that stays hydrogen-bonded to and nonenzymatic reactions between substrates and/or buffer constituents. These measurements confirmed that generation of the g ∼ 1.95 transition is possible only when cyt b6f catalyzes net electron transfer from PQH2 to plastocyanin. Until now, all reports of detection of intermediates of Qp, including USQ-FeS in cyt bc1, 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 Qp, because electrons entering the low-potential path must find an alternate route that restores oxidizing equivalents in Qp Fig. 4. EPR gz transitions of oxidized hemes bp and bn of cyt bc1.(A) Anti- necessary to support turnover. However, PSQ-FeS in cyt b6f was mycin-inhibited enzyme before (green) and 2 s after initiation of reaction by

detected in the noninhibited enzyme (Fig. 2A), which shows that addition of UQH2 (blue). (B) Enzyme incubated with DBMIB in the absence of this state can be formed in the absence of inhibitors. This antimycin.

Sarewicz et al. PNAS Early Edition | 3of6 Downloaded by guest on October 2, 2021 semiquinone, because of its relatively oxidizing potential, can never 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 thermodynamic diagram of electronic bifurcation (Fig. 6). This diagram 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 bp to Q at Qp (transition from state 3). This state protects the enzyme against ROS production by: (i) the fact that electron transfer from SQ-FeS to molecular oxygen (to state 8) is energetically unfavorable and (ii) blocking Qp for the next QH2 oxidation until electrons are Fig. 5. Comparison of the amplitudes of g ∼ 1.95/1.94 transition, in oxy- removed from the low-potential chain. The metastable state exists genic and anoxygenic environment for cyt b6f (A) and cyt bc1 (B), obtained until electrons from the low-potential chain are removed through under conditions similar to those of Fig. 2. the 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 from reduced FeS. As a result, O is converted to oxygen and UQ to 2 the low-potential chain to the Q pool, in relation to the rate of the USQ that strongly interacts with FeS. In other words, the portion − reduction of this chain by Qp, creates conditions that favor ap- of USQ-FeS is a product of O2 scavenging by UQ that is bound pearance of the SQ-FeS metastable state. Such conditions may to FeS. Such the effect is clearly visible in cyt bc1, but not in cyt b6f, exist in some physiological states, e.g., in the presence of a high as only in cyt bc1 do the conditions, antimycin-inhibited cyt bc1 vs. transmembrane potential or a high concentration of QH2 in the Q 2% in noninhibited cyt b6f (6, 26, 27), used for detection of USQ- pool. It is intriguing that a similar g = 1.94 transition of unknown FeS, represent the conditions in which the enzyme produces sig- − – origin, which might reflect the USQ-FeS state, was reported to nificant amount of O2 per single oxidized QH2 (10 18%). appear under ischemia and disappear under reperfusion of rat Discussion hearts (29). The free energy (ΔG) diagram, shown in Fig. 6, which depends One of the important issues associated with the understanding of not only on E but also on other processes, e.g., reconfiguration fundamentals of oxidative metabolism concerns elucidation of m of H bonds within Qp, provides a possible explanation for sig- the mechanism by which enzymes catalyzing reactions that pro- nificant occupation of the energetic state representing PSQ-FeS ceed through highly unstable radical intermediates have adapted in noninhibited cyt b f. Although the difference in E between to safely function in the presence of molecular oxygen (8). In 6 m hemes bn and bp in cyt b6f is not well defined (shown as the width addressing this issue for the cyt bc1/b6f family, a concept emerges in the level of state 5 in Fig. 6A), the average is somewhat more from the unexpected discovery of the PSQ-FeS state in Qp of negative than in hemes b in cyt bc1 (30, 31). This difference, noninhibited cyt b6f (Fig. 2A) and subsequent comparative together with the fact that PQ possesses a more positive Em than analysis of the conditions favoring appearance of this state in cyt UQ (13) makes the energetic gap between state 4 (with SQ-FeS) b6f and cyt bc1 under oxygenic and anoxygenic environments and state 5 (with reduced heme bn) smaller in cyt b6f compared (Figs. 2–5). Based on the results and analysis presented in this with cyt bc1. In other words, in cyt bc1, the electron transfer from work, it is concluded that the generation of SQ-FeS in cyt bc or 1 USQ-FeS (state 4) to heme bn (state 5) is energetically much cyt b6f is an inherent part of enzymatic catalysis that can be more favorable than the electron transfer from PSQ-FeS to described as a metastable state nonreactive with oxygen. The heme bn in cyt b6f. In addition, the existence of high-spin heme cn concept of stabilization of SQ in the Qp site by interaction with is possibly a bottleneck for electron flow from the low-potential reduced FeS of cyt bc was proposed by Link (28), who consid- 1 chain to the Q pool, or reduction of PQ in Qn requires a co- ered antiferromagnetic coupling of high energy between these operative two-electron transfer from hemes bn/cn (32, 33). two centers as a possible explanation for the failure to detect an With the proposed mechanism, it can be appreciated that SQ intermediate in this site. However, such a strong interaction energetic states associated with oxidation of QH2 by cyt b6f/cyt bc1 should result in disappearance of the EPR signal of FeS, which are positioned at levels that allow smooth catalysis while limiting was not observed. Furthermore, this concept was in opposition to released ROS to perhaps just signaling levels that carefully report a more popular view explaining the lack of SQ detection by a the dynamically changing redox state of cofactors (6, 34). It is high instability of SQ at Qp (9, 10, 12, 20). Our results indicate proposed that the SQ-FeS metastable state serves as a “buffer” that coupling between reduced FeS and SQ exists in both cyt bc1 for electrons that are unable to be relegated from Qp through the and cyt b6f, but its energy is small enough that, regardless of the low-potential chain. Availability of the buffer allows the enzyme antiferromagnetic or ferromagnetic character of coupling, it pro- to be held in the state that protects against energy-wasting reac- duces detectable EPR transitions of SQ-FeS (with a characteristic tions including the short circuits (two electrons from QH2 going g ∼ 1.94) at temperatures higher than a few degrees Kelvin. to the same cofactor chain) and the leaks of electrons to mo- Quite importantly, SQ-FeS in cyt bc1 is observed along with lecular oxygen (1, 7, 8). At the molecular level, the stabilization of reduced heme bn and oxidized heme bp (Fig. 4A). On thermo- SQ by its spin-coupling to reduced FeS is likely to occur through dynamic grounds, this observation implies that electron transfer the creation of an H bond between SQ and histidine ligating from SQ-FeS to heme bp is an uphill step. As a consequence, if reduced FeS (transition from 2 to 4 in Fig. 6) (35). heme bp is unable to transfer electrons further to heme bn, the The stabilization of SQ-FeS can occasionally be broken (depic- electron moves back to quinone (Q) at Qp and SQ-FeS is re- ted in the Fig. 6 as the transition from state 4 to 2), resulting in the formed. After reaching equilibrium, no net reactions that lead to formation of a highly unstable semiquinone (SQ not spin-coupled significant occupation of the metastable state occur and, conse- to FeS), which is able to reduce molecular oxygen. This semi- quently, this state is no longer detected spectroscopically. To quinone remains the most likely state responsible for limited su- observe it at equilibrium, one must use a high potential quinone peroxide release. In the reversible transition between SQ-FeS and analog (DBMIB in our case). However, a large increase in Em SQ that is not spin-coupled to FeS (states 4 and 2, respectively), dramatically stabilizes this state to the point that it becomes in- stationary levels of these two states will be proportional. It follows hibitory, at least for cyt b6f. The electron from DBMIB that the amount of ROS will correlate with the amount of the

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1618840114 Sarewicz et al. Downloaded by guest on October 2, 2021 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 on

the respective cofactor or quinone molecule. Yellow squares, Qp empty or occupied by substrate; purple squares, Qp with bound DBMIB semiquinone. Green BIOCHEMISTRY and red arrows, downhill and uphill transitions between the states, respectively. Red frame indicates the states that are inaccessible in antimycin-inhibited cyt

bc1. State 1 represents the most populated initial state for QH2 oxidation under steady-state turnover (in antimycin-inhibited cyt bc1 heme bn is already reduced 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 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 bn) 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 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.

detected SQ-FeS, despite its nonreactivity with oxygen. Indeed, stabilization of the PSQ-FeS blocks the oxidation of another noninhibited cyt b6f produces larger amounts of superoxide than PQH2 in Qp, and, thus, creates a condition in which the proba- noninhibited cyt bc1 (6), which stays in line with our observation bility of delivering the second electron needed to complete the that in the case of noninhibited enzymes SQ-FeS can only be reduction of PQ in Qn by ferredoxin is significantly increased. detected in cyt b6f (Fig. 2A). Nevertheless, when semiquinone is The phenomenon of spin–spin exchange interactions between present in Qp, the equilibrium is always shifted toward its sta- semiquinones and metal centers has been observed many times bilization by formation of the SQ-FeS spin-coupled center in different biological systems, including a coupling between tightly + whereby ROS release is limited to the level of ∼2 molecules of bound semiquinone Q and Fe2 in photosynthetic reaction centers − A O2 per 100 electrons transferred to FeS. Such residual ROS (38, 39), between flavin semiquinones and metal cofactors (18, 40), generation detected in cyanobacterial cyt b6f has been proposed and between iron-sulfur cluster N2 and ubisemiquinone in mito- to activate the p side of the chloroplast transmembrane Stt7 (36) chondrial complex I. However, the role of metal cofactors in the and to carry out longer range signaling in the plant cell (37). vicinity of radicals is not always clear and remains a subject of de- The probability of creation of the SQ-FeS metastable state bate, as exemplified by recent discussion on possible origin on the may vary in different species depending on the relative Em values unusual properties of the SQ signals in complex I (41). In light of the of quinones and low-potential cofactors. Perhaps it is adjusted to present study, we envisage that FeS in cyt bc1/b6f has a dual role in the oxygen tension in the cellular environment (8). Indeed, cyt electron transfer. Its obvious role is to accept electrons from sub- b6f, which experiences more than an order of magnitude higher strate but besides this function, it offers a mean of stabilization of level of oxygen in chloroplasts than cyt bc1 in mitochondria, has a potentially dangerous intermediates that are inherently associated greater tendency to reside in this buffered state. Also, a possible with the stepwise quinone redox reactions. This metal center behaves consequence of the existence of PSQ-FeS in noninhibited cyt b6f as a Lewis acid with electrophilic properties. When it creates a bond is that it may be responsible for regulation of the electron transfer with SQ, its electron-withdrawing properties decrease the probability pathways of oxygenic photosynthesis. As proposed, cyt b6f in of reaction of SQ with oxygen. We propose that this feature is not native chloroplasts can catalyze PQH2 oxidation according to two restricted to the iron-sulfur cluster of cyt bc1/b6f, but may be common alternative mechanisms: (i) noncyclic, in which one molecule of for other metal cofactors that, besides having a role in electron PQ in Qn undergoes a sequential reduction by two electrons transfer, stabilize potentially reactive radicals. derived from Qp and (ii) cyclic, in which one electron is delivered to Qn from Qp, whereas the second electron comes from reduced ACKNOWLEDGMENTS. This work was supported by The Wellcome Trust ferredoxin (2). We speculate that creation of the metastable state Grant 095078/Z/10/Z (to A.O.). Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National PSQ-FeS may serve as a factor that changes the efficiency of Research Center supported by Ministry of Science and Higher Education, part cyclic vs. noncyclic electron transfer between photosystem I and of which included Grant 35p/1/2015 (to M.S.) and a scholarship (to Ł.B.). II. This inference is explained by the fact that transient Studies of W.A.C., S.B., and S.K.S. were supported by NIH Grant GMS-038323.

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