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. 2022 Nov 18;126(47):9771–9780. doi: 10.1021/acs.jpcb.2c05729

Unexpected Heme Redox Potential Values Implicate an Uphill Step in Cytochrome b6f

Mateusz Szwalec 1, Łukasz Bujnowicz 1, Marcin Sarewicz 1, Artur Osyczka 1,*
PMCID: PMC9720722  PMID: 36399615

Abstract

graphic file with name jp2c05729_0007.jpg

Cytochromes bc, key enzymes of respiration and photosynthesis, contain a highly conserved two-heme motif supporting cross-membrane electron transport (ET) that connects the two catalytic quinone-binding sites (Qn and Qp). Typically, this ET occurs from the low- to high-potential heme b, but in photosynthetic cytochrome b6f, the redox midpoint potentials (Ems) of these hemes remain uncertain. Our systematic redox titration analysis based on three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse electron paramagnetic resonance (EPR) and optical spectroscopies) allowed for unambiguous assignment of spectral components of hemes in cytochrome b6f and revealed that Em of heme bn is unexpectedly low. Consequently, the cross-membrane ET occurs from the high- to low-potential heme introducing an uphill step in the energy landscape for the catalytic reaction. This slows down the ET through a low-potential chain, which can influence the mechanisms of reactions taking place at both Qp and Qn sites and modulate the efficiency of cyclic and linear ET in photosynthesis.

Introduction

Cytochrome b6f (cytb6f) is a homodimeric, membrane-embedded enzyme playing a crucial role in oxygenic photosynthesis, in which it provides a functional connection between photosystems II and I.1−3 Cytb6f is believed to operate according to the Q cycle mechanism originally formulated to describe the catalytic action of cytochrome bc1 (cytbc1), a counterpart of cytb6f involved in the molecular respiration in mitochondria and some bacteria. Both cytb6f and cytbc1 catalyze the electron transfer from membrane-soluble electron carriers plastoquinones (PQs) or ubiquinones (UQs), respectively, to water-soluble electron carriers plastocyanins or cytochromes (cyts) c, depending on the organism. The energy released during the electron transfer process is utilized to transport protons across the lipid membrane, thus contributing to the proton motive force required for ATP synthesis.4−6

The Q cycle is based on a joint operation of two catalytic sites: the quinone oxidation site (Qp or Qo in cytb6f and cytbc1, respectively) and the quinone reduction site (Qn or Qi).7−10 The Qp site catalyzes a bifurcation reaction, which directs electrons originating from the oxidation of quinol into two separate cofactor chains: the high-potential and the low-potential chain (LPC). One electron reduces the Rieske cluster and subsequently cytochrome c in the high-potential chain. The other electron reduces heme bp (hbp) and is then transferred across the membrane to heme bn (hbn) in the LPC. An electron from this heme is eventually used to reduce quinone to quinol at Qn (because of the bifurcation, two reactions at Qp yield electrons required for the reduction of one quinone at Qn). Thus, electron transfer from hbp to hbn links functionally the two catalytic sites.1,4

The two hemes b form a highly conserved structural motif. Accordingly, the spatial arrangement of these hemes is nearly identical in cytochromes bc1 and b6f.11 Given the direction of electron transfer, it is generally expected that hbp possesses a lower redox midpoint potential (Em) than hbn.12−14 This indeed is the case for cytochrome bc1, for which the values of Em of hemes b are well-established, being at pH 8.0 around −120 mV and +50/+150 mV (two components) for hbp and hbn, respectively. For this reason, hbp and hbn are commonly denoted as hbl and hbh, respectively (where “l” and “h” stand for low- and high midpoint potential, respectively). However, in the case of cytb6f, the Em values of respective hemes are much less clear, and a discussion on the redox properties of these hemes is ongoing.15 For long, the organization of the LPC of cytb6f was considered to be highly similar to that of cytbc1, but since the discovery of a c-type high-spin (HS) heme cn (hcn) in cytb6f located very close to hbn,16,17 the discussion now includes this additional component possibly forming an interacting redox pair with hbn.18

The major uncertainty concerns the actual difference in Em between the hbp and hbn.15 The analogy to cytbc1 would imply a lower Em for hbp and a higher one for hbn, but there have been experimental indications that these hemes might in fact be isopotential. The experiments of Rich and Bendall19 suggested that midpoint potentials (Ems) of hemes b of cytb6f isolated from higher plants are distinct, with reported Ems of +85 and −90 mV (pH 7.0). Accordingly, Hurt and Hauska observed well-separated Ems of hemes b with values of −60 and −188 mV at pH 7.4.20 Later, they re-evaluated these values by low-temperature optical measurements obtaining two partially overlapping spectral components, which yielded values of ∼3 mV for high-potential heme and −146 mV for low-potential heme at pH 5.6.21 Contrarily, the redox titrations of cytb6f natively embedded in the photosynthetic membrane of spinach chloroplasts showed that hemes b are isopotential within 50 mV with Em = −45 mV at pH 8.0.22 In yet another experiment, Clark and Hind23 titrated isolated cytb6f at pH 7.5 and obtained Ems of −30 and −150 mV. Based on these values, Clark and Hind speculated that in order for cytb6f to obey the Q cycle, the heme with the lower potential must correspond to hbl of cytbc1, which is placed near the Qp site. At this point, however, hcn had not been discovered yet; thus, its contribution to the LPC of cytb6f could not have been estimated.

Studies based on cytb6f obtained from unicellular algae showed similar results to those based on higher plant enzymes. Flash-induced absorption experiments conducted by Joliot and Joliot corroborated the results of Hurt and Hauska for Ems of hemes b and introduced a new component to the LPC of cytb6f with Em of ∼0 mV.24 This new redox component “G”, discovered by Lavergne, is now known to originate from hcn.25

Studies with cytb6f isolated from Chlamydomonas reinhardtii showed different Em values for hemes b with Ems of −84 and −158 mV at pH 8.0 for hbn and hbp, respectively.26 Further studies performed after the discovery of hcn by Alric et al. led to the Em values of −150, −50, and +40 mV for hbp, hbn, and hcn, respectively (pH 8.0).27 While the values appear in line with those previously reported for higher plant enzymes, Alric et al. stated that there is no evidence allowing for an unambiguous assignment in which heme b (high- or low-potential) is located near hcn on the negative side of the membrane.

As hcn occupies a position of quinone bound at the Qi site of cytbc1, the mechanism of the quinone reduction in Qn of cytb6f might differ from that of the Qi of cytbc1. The latter occurs as a sequence of two one-electron transfer steps from hbh to the occupant of the Qi site (quinone or semiquinone) and involves a stable semiquinone (SQ) intermediate. An analogical mechanism has long been considered for the Qn site of cytb6f. However, the close proximity of hbp and hcn gave rise to an alternative concept that the two-electron reduction of PQ at Qn (PQn) might be a concerted reaction with two electrons coming simultaneously from both these hemes.2,28 A difficulty in detecting the SQ in Qn (SQn) of cytb6f would be in line with this mechanism; however, neither concerted nor sequential reaction has been proven yet. At the same time, recent studies on the mechanism of PQ oxidation at the Qp and Qo sites of cytb6f and cytbc1, respectively, show an unexpectedly stable SQ spin-coupled to a reduced 2Fe2S cluster,29,30 the formation mechanism of which remains to be elucidated.

In view of all of these concerns, it is clear that the unambiguous determination of Em values and their proper assignments to specific hemes in the LPC of cytb6f is critical for describing the energy landscape and the mechanism of the catalytic reaction. This requires experimental specificity difficult to achieve for several reasons. First, the resolution of the optical spectra of hemes b at room temperature is relatively low. Additionally, the HS hcn does not exhibit easily detectable optical components. While in bacterial cytbc1, the spectral components originating from individual hemes b can be identified by specific mutations designed to change the spectral properties of a particular cofactor; such protein engineering is not feasible in the case of plant cytb6f. Electron paramagnetic resonance (EPR) spectroscopy is generally more selective in the detection of the heme species, but equilibrium redox titrations and further detection by low-temperature EPR spectroscopy require several milliliters of isolated protein at a concentration of a few tens of micromoles per liter, which have not been achieved so far. Also, a knowledge of the origin of EPR transitions is needed to determine the particular redox-active centers.

In this work, to achieve the experimental specificity required for unambiguous determination of the Em values of the hemes of the LPC of cytb6f, we combined three independent spectroscopic methods of detection of the amount of the reduced species in isolated cytb6f as a function of the ambient redox potential Eh. Low-temperature optical spectrophotometry and continuous wave (CW) EPR spectroscopy were used to record the spectra of the hemes, while pulse EPR spectroscopy allowed us to monitor the distance-dependent phase relaxation enhancement of the Rieske cluster (2Fe2S) by the oxidized heme components.31 The latter method provided information not only on the redox states of the hemes but also on the spatial arrangement of the species of particular redox potential.

Our results indicate that, contrary to the current notion on the Ems of hemes b in cytb6f, the Em value of hbp is larger than that for hbn. This introduces an uphill step for ET in the LPC of cytb6f, so far never considered in the energy landscape for the catalytic reaction. The mechanistic consequences of this finding are discussed.

Experimental Section

Cytb6f was isolated from spinach leaves using a large-scale protocol that was based on protocols of Baniulis32 and Romanowska.33 Briefly, 10 kg of spinach leaves were homogenized using a whole slow juicer. After filtration and centrifugation (17 000 g, 20 min, 4 °C), the pellet was resuspended in low-ionic-strength buffer and pressed through a French Press, followed by centrifugation (5000 g, 10 min, 4 °C). The supernatant was ultracentrifuged (148 000 g, 30 min, 4 °C), and the resulting pellet containing thylakoid membranes was resuspended in buffer to obtain a 1.5 mg/mL final chlorophyll concentration. Then, the thylakoids were solubilized by adding an equal volume of octyl glucoside solution to a final concentration of 25 mM. After ultracentrifugation (148 000 g, 20 min, 4 °C), the supernatant was collected and applied to a propyl-Sepharose column. The hydrophobic chromatography step was repeated. Cytb6f eluted from the column was further purified by ultracentrifugation on a continuous sucrose gradient. This procedure yielded 6 mL of pure cytb6f at a concentration of ∼50 μM. A more detailed description of the isolation procedure is provided in the Supporting Information. Cytbc1 was isolated from Rhodobacter capsulatus strain as described in ref (34).

Equilibrium redox titration of both cytb6f and cytbc1 was performed in the dark as generally described in ref (35). About 6 mL of 50 μM purified cytb6f (bicine buffer, pH 8.0) was redox-poised at different Ehs, under anaerobic conditions, by injections of small aliquots of sodium dithionite. At selected Eh, ∼ 300 μL of the sample was withdrawn through a flat optical cuvette, and the solution was subsequently put into argon-flushed EPR quartz Q band and X band tubes, which were then immediately frozen in liquid nitrogen. To minimize water vapor condensations, the tubes were wiped with ethanol before inserting them into a cryostat.

Optical spectroscopy was performed using a home-built spectrophotometer consisting of a Hamamatsu L10290 light source (Hamamatsu L10290), a 0.05-nm-resolution monochromator (Optel M250), a photomultiplier powered by Biologic PMS250, and a homebuilt Arduino-based analog/digital converter. Samples were placed in flat tubes with I.D. of 0.4 mm × 4 mm (CM Scientific Ltd.) and inserted into an ESR900 cryostat, with temperature controlled by an ITC503S instrument (Oxford Instruments). Spectra were recorded with a home-written LabVIEW program at a resolution of 0.1 nm between 500 and 600 nm. Each wavelength point was averaged 8000 times.

CW EPR spectra were measured at 10 K on a Bruker Elexsys E580 spectrometer operating at X band, equipped with a SuperHQ resonator and an ESR900 cryostat. Measurement parameters were as follows: microwave frequency/power, 9.39 GHz/6.45 mW; modulation amplitude, 15 G; and sweep time/width, 671 s/4495 G.

Pulse EPR measurements were performed on a Bruker Elexsys E580 spectrometer operating at Q band (33.58 GHz) at 14 K as described in.31 The resonator ER5107/D2/0501 inserted in a CF935 helium cryostat (Oxford Instruments) was used, and the temperature was controlled using an ITC503 temperature controller unit. Electron spin echo decay curves (ESE DCs) were measured at g ∼ 1.90, i.e., at the field position of the maximum amplitude of the echo-detected EPR spectrum of the 2Fe2S cluster. This position was selected due to the fact that it encompassed the largest population of 2Fe2S orientations with respect to the external magnetic field and provided the highest signal-to-noise ratio. Each ESE DC was measured using a π/2–t–π sequence, with a length of π/2 pulse of 16 ns. The microwave power was adjusted to obtain the maximum amplitude of the echo. The resonator dead time was 440 ns, and prior to analysis, the time axes for each ESE DC were shifted forward by the addition of the dead time. Further, ESE DCs were fitted with a single exponent function, and the fits were extrapolated to zero time to obtain amplitudes of the echo at the onset of the relaxation process. Then, amplitudes at zero time were used to normalize each ESE DC.

Results and Discussion

For clarity of the presentation of the results, we use terms hbn and hbp specifically for hemes b of cytb6f according to their position in the structure (i.e., hbn is close to Qn and the n-side of the membrane, while hbp is close to Qp and the p-side of the membrane). The terms hbh and hbl are reserved for respective hemes b of cytbc1. We emphasize upfront that, to explain the ensemble of the spectral and redox properties of hemes presented in this work, we had to abandon the long-standing notion about hbn having higher redox potential than hbp.

Resolving Optical Components of Low-Spin Hemes in cytb6f and cytbc1

The optical spectra of isolated cytb6f and cytbc1 were measured at different external redox potentials (Ehs) in the 500–600 nm wavelength range. To obtain a better spectral resolution of the components, all spectra were measured at 9 K. At the highest investigated Eh, the heme f (hf) in cytb6f and heme c1 (hc1) in cytbc1 were already fully reduced and thus were treated as constant components present in all of the analyzed spectra. The gradual lowering of Ehs led to the appearance of spectral components originating from the reduced low-spin (LS) hbn and hbp in cytb6f and LS hbh and hbl in cytbc1. A significant overlap of these components, especially in cytb6f, precluded direct determination of the changes in amplitudes of these hemes as a function of Eh; therefore, the global analysis fit (GAF) procedure was applied to find a minimum number of independent components that could reproduce the experimental spectra of both cyts (see details in the Supporting Information (SI)). The optimized parameters describing the shapes of spectral components of LS hemes b in cytb6f and cytbc1 are given in Table 1.

Table 1. Optical Components for Hemes b in cytb6f and cytbc1 at 9 Ka.

heme analytical function of shape global parameters of the Gauss functions
cytochrome b6f
bp Inline graphic λ1bp = 557.342 ± 0.020 nm
σ1bp = 1.49 ± 0.011 nm
λ2bp = 562.175 ± 0.018 nm
σ2bp = 1.2809 ± 0.0086 nm
r = 1.4446 ± 0.0078
bn Inline graphic λ1bn = 559.952 ± 0.019 nm
σ1bn = 1.1216 ± 0.021 nm
cytochrome bc1
bl Inline graphic λ1bl = 553.910 ± 0.038 nm
σ1bl = 1.363 ± 0.035 nm
λ2bl = 562.477 ± 0.020 nm
σ2bl = 1.666 ± 0.023 nm
r = 1.547 ± 0.043
bh Inline graphic λ1bh = 556.753 ± 0.011 nm
σ1bh = 1.768 ± 0.011 nm
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a

Ax is the local parameter describing the amplitude of the component x; λx, σx, and r are global parameters describing the position of maximum absorbance, the half-width of the absorption maxima, and the amplitude coefficient of the longer-wavelength peak of two-Gaussian spectra, respectively.

The experimental spectra and the emerging components in cytb6f and cytbc1 at the high (+265 and +175 mV, respectively), intermediate (−49 mV), and low Ehs (−255 mV) are shown in Figure 1. It is clear that within the investigated wavelength and Eh ranges, the optical spectra could be reproduced assuming the presence of LS hemes only. Although cytb6f contains an additional high-spin (HS) hcn, we did not identify a contribution that could be ascribed to this heme in this wavelength range. Comparison of the spectral shapes determined by GAF for hemes b revealed that hbp in cytb6f is similar to hbl in cytbc1; these two hemes required use of a two-Gaussian function with similar relative amplitudes of absorption peaks, albeit the positions and the widths of the peaks were different. The hbn in cytb6f showed qualitative similarity to hbh in cytbc1, and they were both reproduced by a single Gauss function, although the peak positions and widths of the bands were different. The presence of two types of components (one asymmetrical and split and one symmetrical) is in good agreement with previous observations of Hurt and Hauska.21

Figure 1.

Figure 1

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Optical spectra of the LS hemes of cytb6f (left column) and cytbc1 (right column) measured at 9 K for the samples redox-poised at the high (top), intermediate (middle), and low (bottom) external redox potential Eh. The experimental spectra (blue) were fitted with a sum of the respective spectral components (black). Gray lines show the components originating from hf and hc1. Hemes bp and bl are shown in red, while hemes bn and bh are shown as green lines. The values indicate the Eh of the particular sample measured.

Having defined the optical components, we examined the change of the amplitudes of hemes b as a function of Eh to obtain their respective Ems. The results of the fitting of the Nernst curves to the separated optical components are shown in Figure 2. The components ascribed to hbp and hbn (see Table 1) could be fitted with a single Nernst function yielding −80 mV, n = 0.63 and −111 mV, n = 0.74, respectively. A similar analysis performed for cytbc1 yielded Em of hbl of −124 mV, n = 0.8, while hbh exhibited the Nernst curve split into two fractions both with n = 1. The first fraction, contributing to 60% of total hbh, has Em = −32 mV, while the remaining 40% fraction has Em = +114 mV. Such a split in hbh is well defined and described as an inherent feature of this heme.36−38

Figure 2.

Figure 2

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Equilibrium redox titrations of LS hemes of cytb6f (a, b) and cytbc1 (c, d) obtained by decomposition of the optical spectra. (a) Spectral component of hems bp in cytb6f at different Ehs (left), and the Nernst curve fitted to the reduced fraction of the heme (right). (b) Same data as in (a) but for heme bn. (c) and (d) show the same type of analysis as in (a) and (b) but for hemes bl and bh of cytbc1. Statistical analysis based on confidence intervals is described in the SI (Figure S1).

The results obtained from the optical redox titrations of cytbc1 comply with the literature data. At the same time, the results obtained for cytb6f suggested that hbp, which in the structure occupies a position corresponding to that of hbl of cytbc1, has higher Em than hbn. This means that the redox potentials of hbp and hbn in cytb6f are in a reverse relationship compared to their counterparts hbl and hbh in cytbc1. Therefore, the optical spectrum of LS heme b in cytb6f, which appears as a first component upon lowering Eh, originates from hbp and not hbn. This implies an incorrect assignment of the spectra of these hemes in the literature.

Of note, the n parameters obtained for hemes b in cytb6f and hbl in cytbc1 are significantly lower than 1. This does not mean that the number of electrons involved in the redox reactions is not an integer. It suggests the presence of Coulombic interactions between closely separated hemes where the reduction state of one of the heme influences the redox potential of the other and vice versa.39 If Ems of the hemes are separated by less than approximately 120–140 mV, such interactions may be responsible for effective n less than 1 and the apparent stretching of the Nernst curves. Given the distances between hemes in the structures and differences in Ems, one would expect significant interactions between the following heme b pairs: hbn–hbp and hbp–hbp in cytb6f and hbl–hbl in cytbc1.40 This indeed was observed in our experiments, and in the case of the hbl–hbl pair, also reported in the literature.41

Redox Titrations of cytb6f and cytbc1 Analyzed by CW EPR Spectroscopy

All of the samples measured by optical spectroscopy were subsequently measured by CW EPR spectroscopy. In the titration analysis, we considered the following EPR transitions. In the case of cytbc1, we used well-defined g = 3.78 and 3.43, corresponding to LS highly axial low-spin (HALS) oxidized hbl and hbh, respectively. In the case of cytb6f, we used g = 3.65 ascribed to the gz transition of the oxidized LS, HALS hbp.42 As hbn does not show a separate, clearly identifiable transition, we chose g = 12.4 and 4.73 from the signals at g > 4.3 ascribed to hcn spin-coupled to hbn42,43 (see the SI for a more detailed description of the EPR spectrum of cytb6f) to monitor the redox-dependent changes in the amplitude of the hcn–hbn pair.

The amplitude of the signal at g = 3.65 gradually decreased upon lowering Eh (Figure 3a). A fit of the Nernst function to the data yielded Em = −73 mV and n = 0.41. We note that this transition is relatively weak, and a contribution of background is likely to introduce uncertainties, especially in the low Eh range, causing an additional decrease in the n parameter. Nevertheless, the Em value is in good agreement with the Em of −80 mV obtained for hbp from the optical titration (Figure 2a).

Figure 3.

Figure 3

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Equilibrium redox titrations of paramagnetic species by CW EPR spectroscopy at 10 K and different Ehs. (a) Spectra of the oxidized hbp of cytb6f (left) and its respective Nernst curve (right). (b) Low-field transition (g = 12.4) of oxidized hcn spin-coupled to hbn (left) and its respective Nernst curve (right). (c, d) show similar analysis for spectra of oxidized hbl and hbh in cytbc1, respectively. Statistical analysis based on confidence intervals is described in the SI (Figure S2).

The g = 12.4 (and 4.73) signals were taken to construct the Nernst curve of either hcn or hbn, since the reduction of at least one of these hemes in a coupled pair is expected to abolish this signal (Figure 3b). The analysis revealed that the g = 12.4 signal decreased upon lowering Eh, and the determined Em value for this species was +46 mV, n = 1. As there is no optical spectra component of a LS heme species having such a large Em, we concluded that the disappearance of g = 12.4 and 4.73 signals resulted solely from the one-electron reduction of hcn and not hbn. This Em value for hcn is in good agreement with the literature data.27 We note that the species with Em of around −111 mV (as determined for hbn by optical spectroscopy) was not identified in our CW EPR experiments, confirming the notion that this heme does not show a separate transition. Overall, CW EPR spectroscopy identified two Em values of −73 and +46 mV, corresponding to hbp and hcn, respectively.

Fitting the Nernst curve to the data of HALS g = 3.78 and 3.43 signals in cytbc1, originating from hbl (Figure 3c) and hbh (Figure 3d), respectively, yielded results consistent with data from optical spectroscopy. The amplitude of the EPR signal of hbl decreased upon lowering Eh, and the Nernst curve yielded Em = −120 mV, n = 0.49. In the case of hbh, the EPR signal also decreased upon lowering Eh and, similar to the optical data, there were two redox fractions detected with Em +152 and −30 mV, both with n = 1.

Redox properties of hemes b determined by optical and CW EPR spectroscopies for both cytb6f and cytbc1 are summarized in Table 2.

Table 2. Redox Properties of Hemes b in cytb6f and cytbc1 Determined by Optical and EPR Spectroscopies.

heme Em [mV] (optical spectroscopy) Em [mV] (CW EPR)
cytochrome b6f
cn n.d. +46 ± 5, n = 1.02 ± 0.17
bp –80 ± 2, n = 0.63 ± 0.02 –73 ± 6, n = 0.41 ± 0.04
bn –111 ± 2, n = 0.74 ± 0.04 n.d.
cytochrome bc1
bl –124 ± 6, n = 0.80 ± 0.13 –120 ± 6, n = 0.49 ± 0.06
bh –32 ± 19, n = 1.0 ± 0.5 –30 ± 20, n = 1.0 ± 0.7
+114 ± 30, n = 1.0 ± 0.7 +152 ± 13, n = 1.0 ± 0.5
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Eh-Dependent Enhancement of the Phase Relaxation of 2Fe2S in cytb6f and cytbc1

In addition to the analysis of redox titration data for cytb6f and cytbc1 obtained by optical and CW EPR spectroscopies, we performed measurements of the ESE DCs of the 2Fe2S cluster at 14 K for samples poised at different Ehs.

Within the investigated Eh range, 2Fe2S and hf of cytb6f remained fully reduced and had S = 1/2 and S = 0 spin states, respectively. The other hemes changed their spin states upon reduction from S = 1/2 to S = 0 (hemes b in cytb6f and cytbc1) or from S = 5/2 to S = 2 (hcn in cytb6f). Since ESE decay rates depend on the strength of dipolar interactions (defined by average dipolar relaxation time constants Tdip) with fast-relaxing paramagnetic centers in a distance-dependent manner,44−46 we expected to observe a gradual slowing down of the phase relaxation of 2Fe2S upon reduction of hemes b and hcn with decreasing Eh.31Figure 4 shows distances between 2Fe2S and hemes b and hcn in a monomer of cytb6f and cytbc1. Clearly, hbp or hbl is positioned closer to 2Fe2S than hbn or hbh. Therefore, in the most simplified case, it can be anticipated that hbp or hbl should exert a stronger effect on the relaxation of 2Fe2S as defined by shorter Tdip compared to hbn or hbh.

Figure 4.

Figure 4

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Comparison of spatial arrangement of 2Fe2S and hemes in monomers of cytb6f (a) and cytbc1. The LS hemes are shown as red sticks, while the HS hcn in a is shown as blue sticks. Dotted black lines show the distances between the 2Fe2S cluster (orange-yellow sticks) and heme iron ions. The distances were measured on the basis of the PDB structures of cytb6f (ID:6RQF) and cytbc1 (ID:1ZRT).

Performing GAF for ESE DCs (see details of the model, basic assumptions, and analytical approach in the SI) to estimate Tdip values provided a crude approximation of the distance-dependent enhancement of the phase relaxation of 2Fe2S induced by dipolar interactions with hemes b in cytb6f and cytbc1 and hcn in cytb6f. In the case of cytb6f, we used nine global adjustable parameters: three representing Tdip values and six representing the parameters of the Nernst curves (Em and n). Fixing n parameters to those obtained from optical and CW EPR spectroscopies led to instability of the fit; thus, estimated Tdip and particularly Em values were not sensible due to the stretching of the Nernst curves used for fitting. This effect caused the mixing of the contributions from different species to the relaxation enhancement of 2Fe2S. Therefore, we simplified GAF by fixing n values to 1 for all of the species. The results of the GAF of the ESE curves are shown in Table 3.

Table 3. Dipolar Decay Time Constants and Em Determined from Pulse EPR Spectroscopy of 2Fe2S in cytb6f.

heme Tdip [μs] (14 K) Em [mV], n = 1
cn 1.6 19
bp 2.2 –95
bn 3.4 –145
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Application of GAF to ESE DCs included three different species that may contribute to the enhancement of phase relaxation of 2Fe2S with Em values optimized at +19, −95, and −144 mV, with the strongest, intermediate, and the weakest impact on the relaxation, respectively. Although the Em values obtained from ESE DC seem approximately 20–30 mV lower than those from optical and CW EPR spectroscopies, they generally stay in agreement with the spectral analysis. The relatively small differences in Ems may result from several experimental limitations of ESE measurement and the simplified model used for GAF. Therefore, the results, especially in regard to Tdip, should be treated only as quasi-quantitative approximations of dipolar enhancement of the 2Fe2S relaxation. Comparison of Ems obtained from pulse EPR spectroscopy to data from optical and CW EPR spectroscopies suggests that the strongest effect on relaxation results from interactions of the cluster with hcn, an intermediate for hbp, and the weakest for hbn. Despite the fact that HS hcn is the most remote from 2Fe2S, its influence can be the strongest, as its magnetic moment and spin-lattice relaxation rate are the highest among paramagnetic hemes in cytb6f.47,48 Additionally, after the reduction, this heme is not converted into a diamagnetic species. Conversely, both hbn and hbp paramagnetic centers are more similar to each other than to hcn; they are LS hemes and undergo conversion to diamagnetic species upon reduction, completely abolishing their effects on the 2Fe2S relaxation. Hence, we assume that the effect of hbp and hbn on 2Fe2S is more directly associated with the distance to the cluster. The results shown in Table 3 suggest that in fact hbp, for which Em determined from pulse EPR is ∼ −90 mV, is closer to 2Fe2S than hbn for which Em is lower.

To verify this conclusion we performed analogous experiments on cytbc1 for which Ems and spectral properties are well defined. The results of GAF for ESE DCs measured at 14 K in cytbc1 provided an estimation of Tdip, when Em values obtained from optical spectroscopy were fixed at −120 mV for hbl and for hbh at −30/+120 mV (with a contribution of 60/40%, respectively). For hbl, Tdip is ∼0.82 μs, while for the more distant hbh, it is ∼1.38 μs. Such semiquantitative results for cytbc1 are expected, considering that these hemes have similar paramagnetic properties and hbl is closer to 2Fe2S than hbh. It generally shows that the species closer to 2Fe2S exerts a stronger effect on its relaxation. Thus, a comparison of the results obtained for cytb6f to those for cytbc1 seems to justify the conclusion that in fact the LS heme species in cytb6f with the lowest Em is more distant from 2Fe2S. Based on the spatial arrangement of hemes in cytb6f shown in Figure 4, this must be hbn.

Mechanistic Consequences of an Uphill Step in the LPC of cytb6f

The proximity of hcn to hbn and the difficulty in detecting SQ at Qn suggested the intriguing new possibility that the two-electron PQ reduction at the Qn site might follow the concerted mechanism rather than a sequential mechanism long considered in analogy to the well-defined reaction of ubiquinone reduction at the Qi site of cytbc1 (Figure 5a). In the concerted reaction, the two electrons are assumed to reduce PQ simultaneously without the formation of a SQ intermediate. As these electrons would have to come from hcn and hbn, the concerted reaction is primed by the state in which both these hemes are reduced (Figure 5b). However, our titration results indicate that the occupancy of this state is much lower than that originally assumed: if two electrons are available in the LPC, they will preferably reside on hcn and hbp rather than on hcn and hbn, due to the lowest Em of the latter (Figure 5c). This in itself does not dismiss the concerted electron transfer to PQn, as the reaction sequence would largely depend on the stability of SQn. If SQn is stable (i.e., the Em values of PQn/SQn and SQn/PQH2 couples are similar,49 assuming that the Em of PQn/SQn is lower), the energy landscape proposed in this work favors the sequential mechanism of PQn reduction due to spatial separation of two electrons on the LPC. However, if SQn is unstable (i.e., large separation between Ems of PQn/SQn and SQn /PQH2 pairs, still, assuming that Em of the first one is lower), the low Em of hbn might in fact favor initiation of the PQn reduction, which would proceed rapidly in a concerted reaction. At present, the stability of SQn is unknown and therefore discriminating between these two scenarios is not possible.

Figure 5.

Figure 5

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Energy landscapes of LPCs in cytbc1 (a) and cytb6f (b, c). (a) and (c) show energy landscapes resulting from this study, while (b) shows energy landscapes previously assumed for cytb6f. Energies are expressed in midpoint redox potentials of hemes. Green arrows indicate downhill steps, while the red arrow indicates the newly established uphill step in the energy profile of cytb6f. Gray inlets show the general profiles of the energy landscapes.

Regardless of the quinone reduction mechanism at the Qn site, the uphill step at the level of hbn is likely to affect electron distribution in the LPC41 and slow down electron transfer from the Qp site through the LPC to the Qn site. Based on the Moser–Dutton ruler,50 the estimated rate of the ET from hbn to PQn in the classical scheme (all downhill reactions) would be of the order of 106 s–1. The presence of the uphill step reduces this rate to 105 s–1. This shift in the estimated ET rate might be of physiological significance. Photosynthesis in plants involves two electron transfer paths that need to be appropriately balanced: a linear electron flow (LEF), which links two photosystems through cytb6f, and a cyclic electron flow (CEF), which reduces PQ using electrons introduced from the N-side of the membrane. The electron transfer from Qp to Qn sites, as part of the catalytic cycle of cytb6f, is an inherent step of LEF. If hcn provides an entry for the electrons in CEF to reduce PQ at Qn (as some models assume16,17), it is reasonable to assume that the uphill step at the level of hbn might provide an important element of regulation of the rate of CEF vs LEF in photosynthesis. Assuming that CEF involves ET from a potential n-side electron donor (possibly ferredoxin) to PQn, the rate of this reaction (considering the closest possible distance from the ferredoxin iron–sulfur cluster to PQn and their redox potentials) would be of the order of 105–106 s–1. As this estimated rate is comparable to the rate of the ET through the LPC, an uphill step could be seen as an adaptation of the LPC to facilitate both LEF and CEF.

Another consequence of an uphill step in LPC is that electrons might tend to reside at the hbp/Qp site for a prolonged time, which in turn might increase the probability of the formation of SQ radicals at this site.51,52 Indeed, a SQ spin-coupled to 2Fe2S was observed in native, noninhibited cytb6f during catalysis.30 To observe such a state in cytbc1, which has no uphill step in LPC, it is necessary to add a Qi site inhibitor that blocks electron transfer through LPC.

Conclusions

Application of three independent and comprehensive low-temperature spectroscopies (continuous wave and pulse EPR and optical spectroscopies) in equilibrium redox titrations of cytb6f resolved a long-standing uncertainty concerning the Em values of hemes b that support the cross-membrane electron transfer in photosynthesis. Optical spectroscopy identified unambiguously components originating from reduced hemes b, revealing that heme bp is described by a sum of two while bn is by one Gauss function. The component ascribed to heme bn is of particular importance given that there is no other reliable indicator of its oxidation state. The components corresponding to heme bp and bn were titrated at pH 8 with the Em values of −80 and −111 mV, respectively.

The g = 12.4 transition resulting from spin–spin interactions between oxidized hcn and hbn disappears upon decreasing Eh and reports the reduction of hcn with Em = +44 mV and not the reduction of hbn. The signal at g = 3.65, originating from HALS heme bp, was found to titrate with Em ∼ −73 mV (pH 8), which stayed in line with the Em of −80 mV, determined from optical spectroscopy. In accordance with the previous analysis, no EPR transition could be ascribed to hbn.

Pulse EPR measurements of distance-dependent relaxation enhancement of the phase relaxation of the 2Fe2S cluster revealed that the component originating from heme b having higher Em is positioned closer to the cluster than that having lower Em. This result, in view of the known distances of cofactors in the structures of cytb6f, confirmed that the redox midpoint potential of heme bp is higher than the Em of heme bn.

Our conclusion dismisses the long-standing assumption that hbp has Em lower than that of hbn, in analogy to the well-established Em values of the respective hemes b (bl and bh) of homologous cytbc1. It is thus clear that the terms hbl and hbh (where “l” and “h” stand for low and high potential, respectively) hold true only for hemes b of cytbc1 but not cytb6f.

The defined order of Ems implies the existence of an uphill step for electron transfer from heme bn to heme bp. It also indicates that in the spin-coupled pair of hemes bn and cn, the former heme has an approximately 150 mV lower Em than the latter. This is expected to impact the probabilities of the occurrence of specific reactions in both the Qn and Qp sites. In the Qn site, it may favor a sequential or concerted mechanism of PQ reduction, depending on the actual stability of SQn. It also slows down the electron transfer from the Qp to Qn site and increases the probability of the formation of SQp. All of these effects help in balancing the rates of the linear and cyclic electron transfer through cytb6f, thus contributing to the regulation of photosynthetic electron flow.

Acknowledgments

This work was supported by Foundation for Polish Science (TEAM, POIR.04.04.00-00-5B54/17-00 to A.O.).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jpcb.2c05729.

  • Detailed description of the isolation protocol; statistical analysis of data obtained by equilibrium redox titration; EPR spectra of cytb6f; and the details of the global analysis fit procedure for pulse EPR data (PDF)

Author Contributions

M.Sz., Ł.B., and M.S. designed, performed, and analyzed the data. M.Sz., Ł.B., M.S. and A.O. wrote the manuscript. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

Supplementary Material

jp2c05729_si_001.pdf (638.5KB, pdf)

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