The cytochrome b₆f complex: plastoquinol oxidation and regulation of electron transport in chloroplasts

Abstract

In oxygenic photosynthetic systems, the cytochrome b₆f (Cytb₆f) complex (plastoquinol:plastocyanin oxidoreductase) is a heart of the hub that provides connectivity between photosystems (PS) II and I. In this review, the structure and function of the Cytb₆f complex are briefly outlined, being focused on the mechanisms of a bifurcated (two-electron) oxidation of plastoquinol (PQH₂). In plant chloroplasts, under a wide range of experimental conditions (pH and temperature), a diffusion of PQH₂ from PSII to the Cytb₆f does not limit the intersystem electron transport. The overall rate of PQH₂ turnover is determined mainly by the first step of the bifurcated oxidation of PQH₂ at the catalytic site Q_o, i.e., the reaction of electron transfer from PQH₂ to the Fe₂S₂ cluster of the high-potential Rieske iron–sulfur protein (ISP). This point has been supported by the quantum chemical analysis of PQH₂ oxidation within the framework of a model system including the Fe₂S₂ cluster of the ISP and surrounding amino acids, the low-potential heme b₆^L, Glu78 and 2,3,5-trimethylbenzoquinol (the tail-less analog of PQH₂). Other structure–function relationships and mechanisms of electron transport regulation of oxygenic photosynthesis associated with the Cytb₆f complex are briefly outlined: pH-dependent control of the intersystem electron transport and the regulatory balance between the operation of linear and cyclic electron transfer chains.

Introduction

Oxygenic photosynthetic organisms (plants, algae, and cyanobacteria) assimilate carbon dioxide and produce molecular oxygen due to the energy of light quanta absorbed by the light-harvesting pigments of photosynthetic apparatus. The light-induced excitation of Photosystems I and II (PSI and PSII) initiate electron transfer along the chloroplast electron transport chain (ETC) from the water-oxidizing complex (WOC) of PSII to NADP⁺, a terminal physiological acceptor of PSI (Nelson and Yocum 2006; Mamedov et al. 2015). Two photosystems, PSII and PSI, are interconnected via the membrane-bound cytochrome b₆f complex (Cytb₆f), and mobile electron carriers, plastoquinone (PQ) and plastocyanin (Pc). The Cytb₆f complex belongs to the Cytbc family of Cyt complexes (Berry et al. 2000; Crofts 2004a; Cramer et al. 2006; Tikhonov 2014, 2018; Malone et al. 2019, 2021; Sarewicz et al. 2021, 2023). This complex stands between PSII and PSI and operates as plastoquinol:plastocyanin oxidoreductase, oxidizing PQH₂ and reducing Pc (Fig. 1). Two electrons, extracted from H₂O by WOC (Bhowmick et al. 2023), are used to reduce PQ to plastoquinol (PQH₂, the double-reduced form of PQ): H₂O + PQ + 2 ${\text{H}}_{{{\text{out}}}}^{ + }$ → ½O₂ + PQH₂ + ${\text{2H}}_{{\text{in}}}^{ + }$. Here, ${\text{H}}_{{{\text{out}}}}^{ + }$ and ${\text{H}}_{{\text{in}}}^{ + }$ symbolize the protons taken up from the chloroplast stroma and released into the thylakoid lumen. The PQH₂/PQ pool is a hub that mediates the intersystem electron transfer and serves as one of the crucial factors regulating oxygenic photosynthesis. PQH₂ donates two electrons to the Cytb₆f, which reduces cytochrome f (Cytf). The operation of the Cytb₆f complex as the PQH₂ oxidase is associated with the following events: (1) the formation of PQH₂ in PSII, (2) the PQH₂ diffusion in the thylakoid membrane toward the Cytb₆f, (3) the penetration of PQH₂ into the catalytic site of the Cytb₆f, and (4) PQH₂ oxidation followed by electron transfer to Cyt f. Reduced Cyt f is oxidized by Pc; reduced Pc donates an electron to P₇₀₀⁺ (the oxidized primary electron donor in PSI) (Haehnel et al. 1980; Gross 1993). Thus, two electrons extracted from H₂O by WOC of PSII are transferred sequentially via PSI to NADP⁺ (through ferredoxin, Fd, and ferredoxin-NADP-oxidoreductase, FNR), reducing NADP⁺ to NADPH. The Cytb₆f complex plays a crucial role in regulation of oxygenic electron transport.

Fig. 1

A schematic presentation of the linear and cyclic electron transport pathways in chloroplasts. a Depicts the arrangement of the membrane-bound protein complexes (Photosystem I, Photosystem II, Cytb₆f, NDH-1, PTOX); FNR, ferredoxin-NADP-reductase; FQR, illusive ferredoxin-plastoquinone-reductase; PTOX, photosynthetic terminal oxidase; and the diffusion routes of mobile electron carriers: plastoquinone (PQ), plastoquinol (PQH₂), plastocyanin (Pc), and ferredoxin (Fd). Reduced molecules NADPH are consumed in the Calvin-Benson cycle (CBC). Electron transport processes are shown by red arrows, they are accompanied by translocation of hydrogen ions into the thylakoid lumen. b Presents the enlarged side view of the monomer of the dimeric Cytb₆f complex from Chlamydomonas reinhardtii (PDB entry 1Q90, Stroebel et al. 2003). This view is perpendicular to the membrane plane. Symbols Q_o and Q_i depict the catalytic centers involved into the reactions of PQH₂ oxidation and PQ reduction, respectively. Plastoquinone binding site Q_o is positioned between heme b₆^L and the Fe₂S₂ cluster of the iron–sulfur protein (ISP). Plastoquinone binding site Q_i is placed between hemes b₆^H and c_n. Color code of main polypeptides: cyan, Cyt f; grey, Cyt b₆; purple, the iron–sulfur (Fe₂S₂) protein; blue, subunit IV. Cofactors: red, hemes b₆^L, b₆^H, and c_n, as indicated; orange, heme f; Fe atoms are shown as dark red spheres; green, Chl a. For true colors of subunits and cofactors see the online version of this article. Figures were produced using Accelerys DV visualizer software package (http://www.accelrys.com)

The oxidation of PQH₂ by the Cytb₆f is the step that virtually determines the rate of the intersystem electron transport (Haehnel 1984; Berry et al. 2000; Crofts 2004a; De Vitry et al. 2004; Tikhonov 2013, 2014; Höhner et al. 2020; Malone et al. 2021; Johnson and Berry 2021; Sarewicz et al. 2021). The Cytb₆f complex works as a proton pump: two protons, taken up from the stroma upon the reduction of PQ (PQ + 2e⁻ + 2H⁺_out → PQH₂), are released into the lumen (the internal volume of thylakoids) as a result of PQH₂ oxidation at the Q_o site (PQH₂ → PQ + 2e⁻ + 2H⁺_in). Thus, the Cytb₆f contributes to generation of $\Delta {\tilde \mu _{{{\rm{H}}^ + }}}$, the trans-thylakoid difference in electrochemical potentials of hydrogen ions, which serves as the driving force for ATP formation from ADP and inorganic phosphate P_i (Mitchell 1969, 2011; Williams 1988; Boyer 1997; Walker 2013; Romanovsky and Tikhonov 2010; Junge and Nelson 2015). According to the Q-cycle model first suggested by Peter Mitchell (Mitchell 1975, 1976), the overall number of protons translocated through the Cytb₆f complex is two times higher than a number of electrons delivered from PQH₂ to PSI (H⁺/e⁻ = 2; for review, see Sacksteder et al. 2000). ATP and NADPH (the macroergic products of photosynthesis) are used mainly in biosynthetic reactions of the Calvin-Benson cycle (CBC) (Edwards and Walker 1983).

Despite the progress in understanding the molecular structure of the Cytb₆f complex, its interaction with PQH₂ is far from being satisfactorily understood (for comprehensive reviews, see (Malone et al. 2021; Sarewicz et al. 2021). In this paper, I briefly overview the molecular architecture of the Cytb₆f complex in chloroplasts and mechanisms of PQH₂ oxidation. The analysis of a bifurcated (two-electron) oxidation of PQH₂ suggests that the first step of PQH₂ oxidation (electron transfer from PQH₂ to the iron–sulfur protein, ISP) is the basic factor that determines the rate of electron transfer between PSII and PSI. The second step of the bifurcated oxidation of PQH₂ would be prompted by the plastosemiquinone radical (PSQ^·) movement toward the low-potential heme of Cyt b₆^L (an electron acceptor) and the carboxy group of the Glu78 residue (a proton acceptor). Finally, the participation of the Cytb₆f complex in regulation of oxygenic photosynthesis is briefly outlined.

Photosynthetic electron transport in chloroplasts

Lateral heterogeneity of thylakoids and the routes of photosynthetic electron transfer

Lateral heterogeneity of thylakoid membranes is characteristic of chloroplasts (Anderson and Anderson 1980; Anderson et al. 1988, 2012; Albertsson 2001; Staehelin 2003; Dekker and Boekema 2005; Pribil et al. 2014; Ruban and Johnson 2015). Figure 2 shows schematic representation of electron transport complexes locations in granal and stromal domains of thylakoids. The piles of granal thylakoids are comprised of appressed circular disk-like vesicles (typically, an average size of ~ 0.4–0.6 μm). Grana has been found to be plastic structure, with variable diameter and a number of layers, depending on the intensity and spectral quality of light (Rozak et al. 2002; Wood et al. 2018, 2019; Gu et al. 2022). Most of PSII complexes are positioned in appressed thylakoids of grana, while PSI and ATP synthase complexes are localized mainly in stroma-exposed domains of thylakoids and grana margins. Cytb₆f complexes are distributed between stacked (granal) and unstacked (stromal) regions of chloroplasts. The populations of Cytb₆f complexes localized in granal and stromal domains of thylakoid membranes can be involved into different routes of electron flow, linear and cyclic pathways. Due to a high mobility of PQH₂ in thylakoid membranes and a rapid lateral diffusion of Pc within the lumen (Höhner et al. 2020), granal Cytb₆f complexes ensure the long-distance linear electron flow (LEF) from PSII to PSI. The stroma-exposed Cytb₆f complexes can participate in cyclic electron flow (CEF) around PSI, supporting the trans-thylakoid proton transfer and ATP synthesis, but without the reduction of NADP⁺. In the course of CEF, electrons return from PSI to the intersystem ETC via the illusive ferredoxin-plastoquinone reductase (FQR) (Bendall and Manasse 1995; Joliot and Joliot 2002; Munekage et al. 2002, 2004, 2008; Puthiyaveetil et al. 2016); this route of electron transfer may be termed as a ‘short’ cycle (CEF1). There are good reasons to believe, based on biochemical experiments, that the illusive FQR can be identified with the electron transfer mediators PGR5 and PGRL-1 bound to PSI (DalCorso et al. 2008). The operation of the CEF1 pathway is likely to proceed through a supercomplex formed by the PSI and Cytb₆f complexes (Buchert et al. 2020; Yadav et al. 2017). Within this supercomplex, PSI and Cytb₆f are able to exchange electrons, mediating the electron flow around PSI (Iwai et al. 2010). Since CEF1 returns electrons from Fd to the PQ/PQH₂ pool through the Cytb₆f complex, one can say that the Cytb₆f virtually plays the role of the FQR.

Fig. 2

A schematic representation of the location of electron transport complexes in the granal and stromal domains of thylakoid membranes. The alternative pathways of electron transport, LEF (linear electron flow), CEF1 and CEF2 denote the “short” and “long’ routes of cyclic electron transfer around PSI. Electron transport processes are shown by red arrows. Abbreviations: CBC, the Calvin-Benson cycle; PSI, PSII, photosystems I and II; b₆f designates the Cytb₆f complex; CF₀-CF₁ denote the ATP synthase complex; FNR, ferredoxin-NADP-reductase; FQR, illusive protein ferredoxin-plastoquinone reductase; NDH-1, NADH-dehydrogenase-like complex type-1; PQ and PQH₂ denote plastoquinone and plastoquinol; PQ_A and PQ_B are the PQ molecules bound to PSII. LEF, CEF1 and CEF2 symbolize alternative pathways of electron transport (for other details, see explanations in the main body of the text)

Another route of cyclic electron transfer around PSI (nominated as CEF2, Fig. 1) is realized through the photosynthetic NADH dehydrogenase-like complex type-1 (NDH-1), which accepts an electron from reduced Fd (not NADPH) and then donates it to the PQ/PQH₂ pool (Shikanai 2007, 2016; Strand et al. 2016, 2017; Laughlin et al. 2019, 2020; Schuller et al. 2019). Similar to mitochondrial Complex I, the NDH-1 complex operates as a Fd-dependent proton-pumping oxidoreductase, which pumps two protons into the lumen per electron transferred to PQ (for review, see Strand et al. 2017; Laughlin et al. 2020). A proper partitioning of electron fluxes between the LET, CEF-1 and CEF-2 pathways would support an optimal balance between ATP and NADPH, which is necessary for functioning the Calvin-Benson cycle (ATP/NADPH = 3/2; Edwards and Walker 1983).

The rate-limiting steps in the intersystem electron transfer

It is generally recognized that the rate of the intersystem electron transfer is determined mainly by the rate of PQH₂ oxidation (Rumberg and Siggel 1969; Haehnel 1984; Tikhonov et al. 1984; Ryzhikov and Tikhonov 1988; Hope et al. 1994; Kramer et al. 1999; Takizawa et al. 2007; Foyer et al. 2012; Hasan and Cramer 2012; Malone et al. 2021). The rate of PQH₂ turnover is determined by the following events: (i) the PQH₂ diffusion from PSII to the Cytb₆f, (ii) the penetration of PQH₂ into the quinol-oxidizing catalytic site Q_o of the enzyme and the formation of the PQH₂-ISP complex, and (iii) the oxidation of PQH₂ by the ISP. In this section, we start with the diffusion-controlled steps of the intersystem electron transfer. Then, after a brief consideration of the Cytb₆f architecture, we will focus on the mechanism of PQH₂ oxidation in the catalytic center of the Cytb₆f.

Plastoquinone diffusion

Exchange of PQH₂ and PQ molecules at the catalytic sites of the enzyme needs the percolation of quinol/quinone molecules through the lipid membrane and their diffusion inside the protein moiety of the Cytb₆f. The PQH₂ diffusion in the thylakoid membrane over-crowded with protein obstacles may retard electron transfer from PSII to the Cytb₆f (Kirchhoff et al. 2000, 2002, 2011). In chloroplasts, however, under a wide range of physiological conditions (pH, temperature), the overall rate of the intersystem electron transport is not limited by PQH₂ diffusion, being determined predominantly by the processes proceeding after the PQH₂ binding to the catalytic site Q_o of the Cytb₆f complex (Haehnel 1976; Tikhonov et al. 1984). Note that the PSII and Cytb₆f complexes may be localized in distant domains of thylakoid lamellas; however, significant amounts of these complexes are close to each other (e.g., the complexes localized in and nearby the grana). This provides shortening a way for obstructed diffusion of PQH₂ from PSII to the nearest Cytb₆f. In plants, grana diameter varies within a small range (about 360–600 nm; for references, see Rozak et al. 2002; Staehelin 2003; Ruban and Johnson 2015). According to (Höhner et al. 2020), the restriction of the grana diameter might exert a strong evolutionary pressure, providing a fast communication between PSII and the Cytb₆f complexes.

In chloroplasts, the formation of PQH₂ in PSII and its diffusion toward the Cytb₆f complex occur within ∆τ ≤ 2–4 ms (at room temperatures; for references, see Haehnel 1976; Tikhonov et al. 1984, 2014; Höhner et al. 2020). This time is shorter than the half-time of electron transfer from PQH₂ to ${\text{P}}_{700}^{ + }$ (via the Cytb₆f and Pc, t_1/2 ≥ 5–20 ms), demonstrating that the overall rate of PQH₂ turnover is determined mainly by direct interaction of PQH₂ with the Cytb₆f, but not the PQH₂ diffusion from PSII to the Cytb₆f complexes. This statement has been first proved by measuring the flash-induced redox transients of P₇₀₀ in chloroplasts with the PSI electron acceptor methylviologen used to support efficient electron efflux from PSII to PSI and further to O₂ (Haehnel 1976; Tikhonov et al. 1984). In order to illustrate this point, I reproduce below the results of our earlier study of bean chloroplasts (Fig. 3). In this set of experiments, before kinetic measurements, chloroplasts were pre-conditioned either by the far-red (FR) pre-illumination by light exciting predominantly PSI (λ_max ≈ 707 nm) or by continuous white light (WL) exciting both PSI and PSII. In the first case, the plastoquinone pool and P₇₀₀ centers were kept oxidized; in response to a short pulse of WL, oxidized centers P₇₀₀⁺ reduced with the half-time τ_1/2 ~ 15–20 ms. The reduction of P₇₀₀⁺ started, however, only after a lag-phase Δτ (Fig. 3a). The length of the lag-phase involves the times of PQH₂ formation in PSII and its traffic toward the Cytb₆f. The release of PQH₂ from PSII after a short light flash takes about 0.6 ms (at room temperatures; Haehnel 1984); thus, the lag-phase mainly reflect the migration of PSII from PSII to the Cytb₆f complex. Otherwise, parameter τ_1/2 is determined by the events that occur after the PQH₂ molecules reached the Cytb₆f complex: the oxidation of PQH₂ by the ISP and further electron transfer to P₇₀₀⁺ (PQH₂ → b₆f → Pc → PSI).

Fig. 3

Effects of chloroplast pre-conditioning on the time-course of the amplitude the EPR signal of P₇₀₀⁺ in bean chloroplasts with 20 μM methylviologen, pH 7.5. Redox changes of P₇₀₀ were induced either by the pulse (t_1/2 = 750 μs) of white light given simultaneously with a background far-red (λ_max = 707 nm) light (a), or recorded after switching off a continuous white light, illumination time 12 s, in the presence of 10 mM NH₄Cl (b). Kinetic curves represent the modified time-courses of P₇₀₀⁺ borrowed from the original publication (Tikhonov et al. 1984)

After the pre-illumination of chloroplasts by WL exciting both photosystems, the PQ/PQH₂ pool becomes reduced. In this case, the post-illumination reduction of P₇₀₀⁺ with the half-time τ_1/2 ≥ 15–20 ms (depending on temperature and pH) starts immediately after switching the WL off, without the lag-phase Δτ (Fig. 3b). The loss of the lag-phase after the WL pre-illumination can be explained: reduced PQH₂ molecules already reached the Cytb₆f complexes, being able to deliver electrons to the Cytb₆f without the PQH₂ diffusion delay. The relationship Δτ < τ_1/2 has been observed under a wide range of experimental conditions (variations of temperature in the range from 5 to 35 °C, and pH variations between 5.0 and 8.5 (Tikhonov et al. 1984)). This gives clear evidence that the light-induced formation of PQH₂ in PSII and its diffusion toward the Cytb₆f complex proceed more rapidly than the intrinsic events of PQH₂ oxidation after the PQH₂ binding to the catalytic center Q_o.

Recently, in order to elucidate the influence of the thylakoid architecture on the diffusion-dependent electron transport mediated by PQH₂ and Pc, Höhner et al. (2020) assayed the Arabidopsis mutants with different grana diameters (varied in the range from ≈370 to 1,600 nm).

They examined electron transport in chloroplasts of three genotypes of Arabidopsis, in which thylakoid architecture was modified by inducing membrane curvature. The curt1abcd mutant was characterized by significantly extended grana diameter (up to ~ 1,600 nm), in the overexpressor mutant CURT1A-oe grana diameter shrinked to ~ 350 nm (Armbruster et al. 2013; Pribil et al. 2018). It has been found that the time of the PQH₂ diffusion from PSII to the Cytb₆f, estimated from the length of the lag phase, is not affected (Δτ  ~ 3.2–3.6 ms) with significant variations of grana diameter (from ~ 370 to 1,600 nm). This is in agreement with the conclusion that the rate of PQH₂ oxidation is determined predominantly by the intrinsic events of PQH₂ oxidation inside the Cytb₆f complex, rather than the PQH₂ diffusion in the lipid domains of the thylakoid membrane.

The notion of a relatively fast diffusion of PQH₂ in thylakoid membranes is consistent with theoretical evaluations of the plastoquinone diffusion coefficient based on the random work modelling of plastoquinone motions in two-dimensional lipid systems. According to (Tremmel et al. 2003), the apparent coefficient of the PQ diffusion in the thylakoid membrane approaches to D_PQ ~ 2 × 10⁻⁸cm² s⁻¹. This implies that PQH₂ could travel farther than 400 nm in 20 ms, suggesting that PQH₂ migration from PSII to the Cytb₆f complexes located near the grana margins should not limit linear electron transport between PSII and PSI, i.e., PQ-diffusion limitations would be mitigated by close localization of the Cytb₆f and PSII complexes.

Note that the literature data on the rates of partial reactions of electron transfer in the Cytb₆f complex are often scattered, depending on the plant species and plant pre-illumination history. Relatively short times of Cyt f and Cyt b reduction (t_½ ≈ 3–6 ms) are typical of intact C. reinhardtii cells (Soriano et al. 1996; Ponamarev and Cramer 1998) and the cyanobacterium Synechococcus sp. PCC 7002 (Yan and Cramer 2003). One of the reasons for the dispersion of kinetic data might be related to differences between the species and variability of stoichiometry between the PSII, Cytb₆f and PSI complexes (Schöttler et al. 2015). Different capacities of donor and acceptor species interacting with the Cytb₆f may also influence the apparent rates of kinetic processes, exaggerating or underestimating rapid and slow phases post-illumination reduction of P₇₀₀⁺. At any rate, however, we can safely state that the oxidation of PQH₂ inside the Cytb₆f is one of basic factors that determine the rate-limiting step in the chain of electron transport between PSII and PSI.

Plastocyanin diffusion

Obstructed diffusion of Pc within a narrow gap of the lumen may restrict electron flow from the Cytb₆f to PSI. Changes in the chloroplast architecture can influence the long-range diffusion of Pc inside the thylakoid lumen (Kirchhoff et al. 2011; Höhner et al. 2020). The shortening or elongation of the distance between PSII to the Cytb₆f could proceed due to variations in the grana diameter (Höhner et al. 2020). In particular, plants grown at low light conditions reveal somewhat reduced grana diameter (Wood et al. 2019; Flannery et al. 2021) and the concomitant shortening of an average distance between PSII to the Cytb₆f complexes localized in grana.

There have been made two important observations. First, the light-induced swelling of thylakoids releases the restrictions for the Pc movements inside the lumen, providing the acceleration of the long-range lateral diffusion of Pc (Kirchhoff et al. 2011). Second, the widening of the grana disk diameter, and concomitant elongation of the Pc diffusion pathway, can lead to a significant increase in the half-time of Pc movement inside the lumen from the grana-hosted Cytb₆f complexes to PSI (Höhner et al. 2020). The latter implies that significant elongation of the Pc diffusion path slowed down the operation of Pc. It should be noted, however, that under a wide range of experimental conditions, the Pc-dependent electron transport from PSII to P₇₀₀⁺ occurs more rapidly (t_1/2 ≤ 300 μs) than the oxidation of PQH₂ by the Cytb₆f (τ_1/2 ≥ 5–20 ms, Stiehl and Witt 1969; Haehnel 1984; Tikhonov et al. 1984; Harbinson and Hedley 1989; Laisk et al. 2016; Ptushenko et al. 2019; Höhner et al. 2020).

Architecture and overview of the Cytb ₆ f structure

Electron carriers and catalytic sites

First three-dimensional structures of the Cytb₆f have been first obtained by the X-ray analysis (at a resolution of 3.0–3.1 Å) of crystal samples from the thermophilic cyanobacterium Mastigocladus laminosus (PDB code 1FV5; Kurisu et al. 2003) and the green alga Chlamidomonas reinhardtii (PDB code 1Q90; Stroebel et al. 2003). The Cytb₆f complexes from different photosynthetic organisms reveal similar architecture. These complexes are organized as the dimers of multisubunit monomers (Fig. 4a) peculiar to the Cyt bc family of electron transport complexes (for review, see Berry et al. 2000; Crofts 2004a; Cramer and Hasan 2016; Malone et al. 2019, 2021; Sarewicz et al. 2021). Each monomer consists of eight polypeptide subunits with 13 trans-membrane helixes. The monomers include four “large” subunits (16–31 kDa): the Rieske iron–sulfur protein (ISP), the Cyt b₆ and Cyt f proteins, and subunit IV (subIV). Each multisubunit monomer reveals two prosthetic groups, chlorophyll a (Chl a) and β-carotene, associated with the subIV. Several “small” hydrophobic subunits (3.3–4.1 kDa) are arranged at the outside periphery of the monomer ensembles. Dimeric structure of the Cyt b₆f complex provides the formation of a large protein-free intermonomer cavity (~ 30 Å × 25 Å × 15 Å) through which PQH₂ and PQ can penetrate into the quinone-binding sites. The potential interactions between the electron transport chains localized in two monomers of the dimeric b₆f complex have been discussed in the literature (for references, see Crofts et al. 2008; Nawrocki et al. 2019; Crofts 2021; and references therein). Below, I focus on the primary PQH₂ oxidation processes at the Q_o catalytic site located within a single multisubunit monomer.

Fig. 4

The molecular architecture of the spinach dimeric Cytb₆f complex reconstructed from the cryo-EM data (PDB code 6RQF; Malone et al. 2019). a Present the side view of the Cytb₆f complex, demonstrating the protrusion of the external domains of Cyt b₆ and Cyt f into the bulk phase of the thylakoid lumen. b and c show the trans-membrane and top (from the stromal side) views of the Cytb₆f complex, and the locations of prosthetic groups and plastoquinone molecules, PQ1, PQ2 and PQ3. Figures were produced using Accelerys DV visualizer software package (http://www.accelrys.com)

The Cytb₆f complex contains several electron carriers, which perform the catalytic functions associated with redox changes of PQH₂ and PQ. Figure 4b and c depict the trans-membrane and top views of the native spinach Cytb₆f complex (PDB code 6RQF; Malone et al. 2019), indicating the positions of electron carriers participating in PQH₂ oxidation and PQ reduction at the catalytic sites Q_o and Q_i, respectively.¹ Each monomer ensemble contains the following carriers: the Fe₂S₂ cluster of the ISP, two hemes of the Cyt b₆ (the low-potential heme b₆^L and the high-potential heme b₆^H), and an atypical heme c_n positioned in close proximity to the high-potential heme b₆^H on the stromal side of the complex. A c-type heme f protrudes into the thylakoid lumen. Along with the redox cofactors, there are two Chl a molecules positioned inside the dimer complex.

Crystallization of the Cytb₆f complexes with the quinone analogue inhibitors, tridecyl-stigmatellin (TDS) and NQNO (2n-nonyl-4-hydroxy-quinoline-N-oxide), revealed two sites for quinone binding: the Q_o site (quinol oxidase) and the Q_i site (quinone reductase) (Yamashita et al. 2007). The quinol-binding portal of the Q_o site represents a hydrophobic cavity (~ 11 Å × 12 Å) covered inside by lipid molecules (Hasan and Cramer 2014; Cramer and Hasan 2016; Bhaduri et al. 2019). The quinone exchange portal of the catalytic site Q_o is positioned near the Fe₂S₂ cluster of the ISP. One lobe of the Q_o volume is oriented toward the Fe₂S₂ cluster while the other side extends toward heme b₆^L. TDS was found in the close vicinity of the Rieske protein, forming the hydrogen bond with the His residue ligating one of the Fe atoms of the Fe₂S₂ cluster. Figure 5 shows a fragment of the Q_o site, which contains the inhibitor TDS located near the Fe₂S₂ cluster (PDB code 1Q90). Note that the position of the ring of the 2,3,5-trimethylbenzoquinol molecule (TMBQH₂, the tail-less analog of PQH₂), is nicely fitted to the TDS molecule resolved in the crystal structure of the Cytb₆f. Both species, TMBQH₂ and TDS, form the hydrogen bond (–O–H∙∙∙N_ε<) with the His155 residue of the ISP.

Fig. 5

The arrangement of an inhibitor tridecyl-stigmatellin (TDS) hydrogen-bonded to atom N_ε of His155 in the crystal structure of the Cytb₆f complex (PDB entry 1Q90, Stroebel et al. 2003). Green balls show the position of the head group atoms of plastoquinone

The second quinone-binding center (site Q_i) is located on the stromal side of the Cytb₆f, at the interface between an “atypical” heme c_n and the large inter-protein quinone exchange cavity between hemes b₆^H and c_n (Kurisu et al. 2003; Stroebel et al. 2003; Yamashita et al. 2007; Malone et al. 2019, 2021; Sarewicz et al. 2021, 2023). It is believed that heme c_n participates in cyclic electron transport around PSI, mediating electron transfer from the acceptor side of PSI (via Fd) to PQ located in the Q_i site (Strand et al. 2016, 2017; Schuller et. al., 2019).

According to the Mitchell’s Q cycle (Mitchell 1975, 1976; Berry et al. 2000; Crofts 2004a, 2021; Osyczka et al. 2005; Cramer et al. 2006, 2011; Mulkidjanian 2010), in the catalytic site Q_o two electrons are extracted from PQH₂ and directed to the high-potential and low-potential redox chains (Fig. 6a). The very idea that in the course of quinol oxidation two electrons are directed into separate electron transfer chains was suggested by Wikström and Berden, who had found that external oxidant (O₂) induced the reduction of Cyt b in mitochondria in the presence of antimycin (Wikström and Berden 1972). In the Cytb₆f complex, one electron is transferred to the Fe₂S₂ cluster of the ISP (reaction 1), another electron is directed to the low-potential heme b₆^L (reaction 2). Reduced ISP (ISP_red) donates an electron to Pc and further to P₇₀₀⁺ via the high-potential chain: ISP_red → Cyt f → Pc → P₇₀₀⁺. From the plastosemiquinone (PSQ^·) formed after the first step of PQH₂ oxidation, an electron reduces the low-potential heme b₆^L (Joliot and Joliot 1988). Reduced heme b₆^L donates an electron to the high-potential heme b₆^H positioned near the PQ-binding center Q_i, which operates as the PQ reductase (Kramer and Crofts 1993; Crofts 2004c, 2021). At the Q_i center, according to modified Q cycle, the PQ molecule accepts one electron from Cyt b₆ and serves as the recipient of the second electron coming from the acceptor side of PSI through the cyclic electron transfer chain: PSI → Fd → FQR → c_n → (PQ)_i. Here, FQR and c_n denote the illusive Fd-quinone reductase (Munekage et al. 2004) and c-type cytochrome (Kurisu et al. 2003; Stroebel et al. 2003), respectively. The fully reduced PQH₂ molecule dissociates from Q_i and then can bind to the vacant center Q_o. Both reactions of the bifurcate oxidation of PQH₂ proceed as the proton-coupled electron transfer (PCET) processes:

$${\text{PQH}}_{{2}} \to {\text{PSQ}}^{ \cdot } + {\text{e}}^{ - } + {\text{H}}_{{{\text{in}}}}^{ + } ,$$

(1)

$${\text{PSQ}}^{ \cdot } \to {\text{PQ}} + {\text{e}}^{ - } + {\text{H}}_{{{\text{in}}}}^{ + } .$$

(2)

Fig. 6

a A schematic diagram of a bifurcated (two-electron) oxidation of plastoquinol (PQH₂) at the catalytic site Q_o of the Cytb₆f complex. Numbers 1 and 2 indicate the reactions of electron transfer from PQH₂ to the Fe₂S₂ cluster (1) and from semiquinone PQH^· to the low-potential heme b₆^L (2). Modified Fig. 2 from Ustynyuk and Tikhonov (2022). b A diagram of the midpoint redox potentials of electron carriers illustrating electron transfer along the high-potential (ISP, Cyt f, Pc, and P₇₀₀) and low-potential redox (hemes b₆^L, b₆^H, and c_n) branches of electron transfer in the Cytb₆f complex. The redox potential levels depicted by red triangle and horizontal lines represent the average values taken from the literature for Chlamydomonas reinhardtii (Pierre et al. 1995; Zito et al. 1998; Alric et al. 2005; Nelson and Yocum 2006; Hasan et al. 2013a); blue triangle and horizontal lines correspond to cyanobacteria (Nakamura et al. 2011). Green circles and horizontal lines depict the redox potentials measured by low-temperature EPR spectroscopy in spinach chloroplasts (Szwalec et al. 2022)

Here, PSQ^· denotes the semiquinone species (protonated or deprotonated, PQH^· or PQ^·−) formed after the first step of the bifurcated reaction. The term proton-coupled implies that the reactions (1) and (2) are tightly coupled with the proton transfer to appropriate proton-accepting groups (Mayer and Rhile 2004).

Thermodynamic reasons for the assignment of electron carriers to the high- and low-potential branches are based on measurements of the midpoint redox-potentials (E_m) of these carriers. Figure 6b presents a diagram illustrating this point (for references, see (Tikhonov 2014, 2018; Malone et al. 2021)). The driving force for the first reaction of PQH₂ oxidation by the ISP_ox is characterized by a high redox-potential of the redox pair PQH₂/PQ^· (E_m ~ 450 mV). The lower value of E_m of the redox pair ISP_red/ISP_ox (E_m ~ 300–320 mV; Nitscke et al. 1992) implies that electron transfer from PQH₂ to ISP_ox is the up-hill (energy-accepting) process that would limit the overall rate of PQH₂ oxidation (for more details see below Section "Oxidation of PQH₂ by the Cytb₆f complex, the Q cycle"). Further reactions of electron transfer along the high-potential branch to P₇₀₀⁺ occur as the down-hill reactions (Fig. 6b).

The very essence of the second reaction (the oxidation of PSQ^·) is that the plastosemiquinone radical PSQ^· is a rather strong reductant capable of reducing electron carriers standing in the low-potential chain. The potential of the redox pair PSQ^·/PQ is lower (E_m ~ − 250 mV) (Rich and Bendall 1980) than the E_m values of electron carriers of the low-potential branch (Joliot and Joliot 1988). In general, the E_m values for the hemes the low-potential chain reported in the literature reveal a tendency of increasing in the line b₆^L → b₆^H → c_n (Fig. 6b). This is in agreement with the traditional point of view on the Q cycle operation. It should be noted, however, that specific E_m values of the hemes b₆^L, b₆^H and c_n reported in the literature are sometimes scattered, depending on the system investigated and the methods used for determination of E_m (Rich and Bendall 1980; Hurt and Hauska 1982, 1983; Clark and Hind 1983; Joliot and Joliot 1988; Furbacher et al. 1989; Pierre et al. 1995; Zito et al. 1998; Alric et al. 2005; Nakamura et al. 2011). In particular, recent redox titration of spinach chloroplasts (Szwalec et al. 2022) based on independent methods, optical spectroscopy and low-temperature EPR, revealed unexpected low value (E_m ≈ − 111 mV) attributed to heme b₆^H, which was lower than the E_m value of heme b₆^L (E_m ≈− 73 mV). The authors conclude that this result may dismiss the long-standing assumption that heme b₆^L has lower E_m value than heme b₆^H. This also implies that in the Cytb₆f complex electron flow between hemes b₆^L and b₆^H may slow down electron flow along the low-potential chain. It is conceivable that this effect might be one of the factors contributing to the regulation of electron flow through the Cytb₆f complex.

Plastoquinone molecules inside the Cytb ₆ f complex and the enter/exit pathways

Crystal structures

The dimer structure of the Cytb₆f provide the formation of a rather large cavity (~ 30 × 25 × 15 Å) through which PQH₂ and PQ molecules enter into the quinone-binding centers (Cramer et al. 2006; Hasan et al. 2013c). The PQH₂ binding portal Q_o, where the PQ/PQH₂ exchange occurs, lies inside the cavity covered by lipid molecules (about 23 potential sites of lipid binding per monomer; Hasan and Cramer 2014; Cramer and Hasan 2016). Figure 7 shows a fragment of the Q_o site in the crystal structure of the Cytb₆f complex (PDB code 1Q90; Stroebel et al. 2003), which involves heme b₆^L, Glu78, and the Fe₂S₂ cluster of the ISP surrounded by two His residues and several amino acids residues. The structure was supplemented by TMBQH₂, the tailless analog of plastoquinol. The position of TMBQH₂ near the ISP was determined as described in (Ustynyuk and Tikhonov 2022). The formation of the hydrogen bond between the –OH group of the quinol molecule and the atom N_ε of the His residue, liganding one of the Fe atoms of the Fe₂S₂ cluster (His155 in Fig. 7), is considered as the preconditioning for the formation of substrate-enzyme complex (PQH₂-His) at the catalytic site Q_o (for references, see Crofts et al. 1983; Crofts 2004a, 2004b, 2004c, 2021; Mulkidjanian 2010; Cramer and Hasan 2016; Sarewicz et al. 2021, 2023). The atom N_ε is assumed to be the primary recipient of the proton donated by quinol. Alternative model, suggesting that the primary acceptor of the proton may be a water molecule, has been suggested in (Postila et al. 2013; Barragan et al. 2016). The PQ binding site Q_i is located in the stroma-exposed domain of Cytb₆f, at the interface between heme c_n and the inter-protein quinone exchange cavity (Fig. 4). The unique heme c_n exists in the Cytb₆f, but it is absent in the Cytbc₁ complexes. According to the modified Q-cycle, heme c_n may participate in cyclic route of electron flow around PSI, mediating the reduction of PQ bound to Q_i (Kurisu et al. 2003; Stroebel et al. 2003; Munekage et al. 2004; Shikanai 2007).

Fig. 7

A fragment of the crystal structure of the Cytb₆f complex from Chlamidomonas reinhardtii (PDB entry 1Q90) used to illustrate the primary reactions of a bifurcated oxidation of quinol at the Q_o site of the Cytb₆f complex. Red and blue arrows show the directions of the electron and proton transfer reactions related to plastoquinol oxidation. Figures were produced using Accelerys DV visualizer software package (http://www.accelrys.com)

Cryo-EM structures

PQH₂ molecules formed in PSII travel to the intermonomer cavity of the Cytb₆f by lateral diffusion in the membrane. Penetration of PQH₂ into the quinol-binding portal Q_o is associated with its diffusion within the Cytb₆f lipoprotein complex: it is likely that PQH₂ molecules with flexible isoprenoid chains reach the quinone-binding catalytic sites by percolation through the intermonomer cavity and curved intraprotein pathway inside the Cytb₆f complex. In first crystal structures of the Cytb₆f complex, neither plastoquinol nor plastoquinone have been resolved within the Q_o portal. Significant progress in understanding the structure and function of the native Cytb₆f complex has been recently achieved with the use of the cryo-EM technique (Malone et al. 2019, 2021; Proctor et al. 2022; Sarewicz et al. 2023). The first native structure of the spinach Cytb₆f complex, obtained without the crystallization procedure and in the lack of inhibitors (PDB code 6RQF; Malone et al. 2019), revealed three PQ molecules (designated as PQ1, PQ2 and PQ3) fixed at different positions inside the intermonomer cavity (Fig. 8). The benzene ring of PQ1 is adjacent to the heme b₆^L and Chl a; PQ2 is located near the hemes b₆^H and c_n; PQ3 is situated between the hemes b₆^H and c_n bound to different monomers of the dimeric Cytb₆f complex.

Fig. 8

The location of plastoquinone molecules in the stroma-oriented cavity of the dimeric Cytb₆f complex from spinach (PDB code 6RQF) recovered by the cryo-EM (Malone et al. 2019). Arrows indicate the positions of two Fe₂S₂ clusters, hemes of Cyt f, the low- and high-potential hemes b₆^L and b₆^H, hemes c_n, and two Chl a molecules. Symbols PQ1, PQ2, and PQ3 denote plastoquinone molecules positioned in the intermonomer cavity at a distance from the Fe₂S₂ clusters of the ISPs

In the first cryo-EM structure of the native spinach Cytb₆f complex all plastoquinone molecules were resolved away from the catalytic center Q_o (Malone et al. 2019). No doubt it might seem very surprising that neither PQ nor PQH₂ molecules were found in the Q_o portal. The mystery of the lost plastoquinones has been recently solved. The breakthrough in this field was made by Osyczka and collaborators (Sarewicz et al. 2023). They resolved the high-resolution cryo-EM structures of the spinach Cytb₆f homodimer with the worm-like intra-protein pathway for traffic of endogenous plastoquinones, providing the passage of PQH₂ to the catalytic center Q_o and the exit of PQ to lipid domains. In each multisubunit monomer, the authors visualized three plastoquinone molecules arranged one after another (a tail-head–tail-head–tail-head arrangement). Such a traffic of quinones inside the Cytb₆f complex never been considered before. The head group of one of three PQH₂ molecules (PQ1) was positioned very close to the Fe₂S₂ cluster. A model developed by Sarewicz et al. (2023) suggests the one-way diffusion of quinones through the intra-protein channel during the catalytic cycle. According to the model, two plastoquinone molecules, PQ1 and PQ3, occupy the entry and exit moieties of the long intra-protein channel. PQ2 is located in the middle part of the channel. It has been proposed that the entering of PQ1 into the channel entrance involves a slip of its tail inside the protein cavity. It is likely that the re-arrangement of PQ1 inside the channel can be accompanied by a flip of its head group toward the ISP, providing the formation of the hydrogen bond between PQ1 and the ISP. The penetration of PQ1 into the quinone-conducting channel further proceeds toward the position of PQ2, initiating the sequence of events associated with the bifurcated oxidation of PQH₂ at the Q_o site. It has been found that the head group of PQ1 is proximal to the Fe₂S₂ cluster. It seems, however, that the position of the head group of PQ1 was not optimal for its bifurcated oxidation (~ 6.5 Å from His128). The authors propose that the catalytically active position of PQ1 is highly transient and the bifurcated reaction occurs when PQH₂ travels between PQ1 and PQ2 positions. Oxidized PQ molecule moves to the next position in the chain (PQ3), from which it can travel to the bulk of the lipid domain of the thylakoid membrane (Fig. 9b).

Fig. 9

Schematic routes of plastoquinone diffusion inside the Cytb₆f complex. Traces of plastoquinone traffic are shown by double-line blue arrows. a PQH₂ molecules reduced in PSII enter the intermonomer cavity. b The one-way diffusion of plastoquinone molecules (PQ1, PQ2, and PQ3) from the thylakoid membrane through the intraprotein channels toward the catalytic Q_o centers of the spinach Cytb₆f complex. The structures of the Cytb₆f complex and the trace of the channel for plastoquinone diffusion were discovered by Sarewicz et al. (2023) using high-resolution cryo-EM (PDB code 6RQF)

An interesting observation reported by Malone et al. (2019) is that the phytyl tail of Chl a lie on the way of plastoquinone passage to Q_o (Figs. 4c and 8). According to (Sarewicz et al. 2023), the phytyl tail of Chl a could occupy the intraprotein PQ-conducting channel. It has been found that the phytyl tail may have two conformations (Malone et al. 2019). This suggests that the phytyl tail might operate as a mechanical “gate”, providing the admission of PQH₂ to the catalytic site Q_o. The traffic of PQH₂ through the intra-propein quinone-conducting channel would be controlled by conformational changes of the phytyl tail of Chl a. In one case, the phytyl tail restricts the PQH₂ diffusion, preventing the access of PQH₂ to the catalytic site Q_o (Malone et al. 2019; Sarewicz et al. 2023). After the phytyl chain turn, the penetration of PQH₂ to the Q_o site becomes possible (Malone et al. 2019; Sarewicz et al. 2023).

Oxidation of PQH₂ by the Cytb ₆ f complex, the Q cycle

The oxidation of PQH₂ at the Q_o center starts with the transfer of the H atom from the −OH group of PQH₂ to oxidized ISP_ox (Crofts 2004a, 2004b, 2004c; Crofts et al. 2013). One of the Fe ions of the Fe₂S₂ cluster is ligated by two His residues (Fig. 5). Structural data suggest that the N_ε atom of deprotonated His (His155 in C. reinhardtii (Stroebel et al. 2003), His129 in M. laminosus (Kurisu et al. 2003), or His128 in spinach (Malone et al. 2019)) is the prime candidate for the role of the proton recipient from PQH₂. The formation of the H-bond between the –OH group of PQH₂ and the N_ε atom of deprotonated His is considered as a pre-requisite for quinol oxidation. The existence of this bond has been demonstrated by spectroscopic methods (EPR, NMR, and ATR-FTIR) in Cyt bc₁ complexes (Samoilova et al. 2002; Zu et al. 2003; Iwaki et al. 2005; Lin et al. 2006; Hsueh et al. 2010), which are akin to the Cyt b₆f. The fully oxidized Fe₂S₂ cluster is the EPR silent diamagnetic species (the total spin S = 0) due to the antiferromagnetic coupling between two paramagnetic Fe³⁺ ions (S = 5/2). After the one-electron reduction of ISP_ox, the Fe₂S₂ cluster becomes paramagnetic and reveals the EPR signal characterized by S = 1/2 (Sarewicz et al. 2021; Ruuge and Tikhonov 2022).

Electron transfer from the reduced ISP (ISP_red) to Cyt f (or Cyt c₁ in the Cytbc₁ complex) is associated with the large-scale conformational changes in the ISP. After the reduction of the ISP, its mobile domain, which contains the cluster Fe₂S₂, displaces from the plastoquinone-binding site and moves toward heme f. The long-range “tethered” diffusion of the mobile fragment enables electron transfer to heme f, which further reduces Pc. It is likely that the tethered diffusion of the ISP extrinsic domain does not limit the overall rates of the Cytb₆f and Cytbc₁ turnover. For example, in Rb. sphaeroides the binding/dissociation reactions and movements of ISP occur more rapidly (~ 30–60 μs) than the rate-limiting reactions in the high-potential redox chain (Crofts 2004b). A rapid movement of the Fe₂S₂ cluster away from the Q_o site precludes the donation of the second electron from PSQ^· to the high-potential branch, directing the PSQ^· radical to reduce heme b₆^L.

The radical pairs PSQ^·–Fe₂S₂^· formed after the first step of quinol oxidation have been detected in Cytbc₁ and Cytb₆f complexes by the electron paramagnetic resonance (EPR) method (Sarewicz et al. 2013, 2017, 2018, 2021; Pietras et al. 2016; Bujnowicz et al. 2019). The low-temperature EPR signals detected at cryogenic temperatures were attributed to the reduced cluster Fe₂S₂^· of the Rieske protein (a triplet with the central line at g = 1.95), the semiquinone radical PSQ^· (a singlet line at g = 2.045), and a new EPR signal related presumably to the radical pair PSQ^·–Fe₂S₂^·. A semiquinone PSQ^· is coupled to the reduced cluster Fe₂S₂^· via spin–spin exchange interaction. The coupling energy, evaluated on the basis of EPR data, appears to be of a rather small value (~ 3.5 GHz or ~ 1 K). This suggests that the radical pair PSQ^·-Fe₂S₂^· to be an unstable transient structure. After the breakdown of the radical pair, the highly reactive semiquinone species PSQ^· can donate an electron to heme b₆^L of the low-potential branch of electron transfer in the Cytb₆f (Kramer and Crofts 1993). Further electron transfer along the low-potential chain (PSQ^· → b₆^L → b₆^H → c_n → PQ_i) would provide the reduction of PQ molecule (PQ_i) bound to the redox site Q_i. According to a model of modified Q cycle in the Cytb₆f, the PQ_i molecule is believed to receive a second electron from the acceptor side of PSI through the chain of CET around PSI (Munekage et al. 2004; Strand et al. 2016). Fully reduced PQH₂ molecule releases from the site Q_i and then can rebind to the vacant center Q_o. Thus, due to the cyclic operation of the Cytb₆f complex, associated with the PQH₂ return from the Q_i site to the catalytic center Q_o, one turnover of this complex will provide enhanced stoichiometry of the trans-thylakoid proton transfer (H⁺/e⁻ = 2), when two protons are translocated per one electron delivered from PQH₂ to PSI through the high-potential chain (Mitchell 1975, 1976).

Electron transfer along the high-potential chain is associated with significant conformational changes in the Cytb₆f complex. The Fe₂S₂ cluster of the ISP is separated from heme f by ~ 26 Å; this excludes direct electron transfer from the ISP_red to Cyt f by the mechanism of quantum mechanical tunneling (Moser et al. 1997; Page et al. 1999). There is a number of experimental evidence that the mobile domain of the ISP_red, which contains the Fe₂S₂ cluster, moves from the Q_o site toward heme f by the mechanism of the tethered-diffusion. Electron transfer by domain movement was strongly documented by the X-ray analysis of the crystal structures of the Cytbc₁ complex (Zhang et al. 1998). In the crystals of the Cytbc₁ complex with some inhibitors, the positions of the mobile ISP domain containing the Fe₂S₂ cluster were resolved in different places of the complex, reflecting the flexibility of this domain. After the re-oxidation of the ISP_red by Cyt f, the mobile extrinsic domain of the ISP with the oxidized cluster Fe₂S₂ returns to its initial (proximal) position at the Q_o site. In the Cytb₆f complex, the conformational mobility of the ISP subunit has long been a subject of debate. The hinge fragment and the extrinsic mobile domain of the ISP were disordered in the crystallographic structures of the Cytb₆f, indicating a significant flexibility of the ISP mobile fragment (Baniulis et al. 2009; Hasan et al. 2013b). The disorder of the ISP extrinsic domain is usually considered as the evidence of its high conformational mobility. It has been suggested that the conformational mobility of the ISP is determined by its lipid environment (Hasan et al. 2013b; Hasan and Cramer 2014). There are indications that the shuttle movements of the flexible extrinsic domain of the ISP between the Q_o site and heme f occur more rapidly than the electron transfer from PQH₂ to Fe₂S₂ rapidly (Breyton 2000; Yan and Cramer 2003; de Vitry et al. 2004; Hasan et al. 2013b), and, therefore, these movements do not limit the overall rate of the Cytb₆f turnover.

The breakdown of the unstable radical pair PSQ^·–Fe₂S₂^· would facilitate the oxidation of the radical PSQ^· due to its movement inside the intra-protein cavity (Δl ~ 1 nm) toward heme b₆^L and the proton-accepting −COO⁻ group of Glu78 (Fig. 10a). Molecular dynamics calculations performed by Cramer and collaborators (Hasan et al. 2014; Ness et al. 2019) confirm the possibility of a fast displacement of PQH^· from the Q_o-binding site (Δτ ~ 10 ns). If this is the case, reaction (2) would proceed almost simultaneously with reaction (1). Once the radical shifts toward b₆^L and Glu78, PSQ^· becomes oxidized to PQ. A rapid oxidation of PSQ^· (reaction 2) should preclude the donation of the second electron to the high-potential branch. Short life-time of PQH^· would also reduce a probability of superoxide (O₂^·−) formation due to O₂ interaction with PQH^· (Mubarakshina et al. 2006; Ivanov et al. 2018).

Fig. 10

a A fragment of the crystal structure of the spinach Cytb₆f complex (PDB code 6RQF) involved into a bifurcated oxidation of PQH₂ (HO-Q-OH) at the Q_o site. Red and blue arrows designate the electron and proton transfer from the H(1) atom of plastoquinol to His128, and the electron and proton transfer from the H(2) atom of plastosemiquinone (HO-Q-O^·) to heme b₆^L and the −COO⁻ group of Glu78. b A tentative pathway of the H⁺ transfer from the protonated group −COOH of Glu78 to the bulk phase of the thylakoid lumen

Both steps of a bifurcated oxidation of PQH₂ are tightly coupled to proton transfer to appropriate proton-accepting groups. One of the His residues of the ISP is considered as the primary recipient of a proton donated by PQH₂. The −COO⁻ group of highly conserved Glu78 (spinach numbering) may serve as the acceptor of the second proton donated by PQH^· (−COO⁻ + H⁺ →−COOH; Zito et al. 1998; Osyczka et al. 2006; Hasan et al. 2013c; Victoria et al. 2013). Glu78 stands in the position proximal to heme b₆^L. In the Cytb₆f complex, the mobility of the −COO⁻ group is limited due to a salt bridge between Glu78 и Arg87. This group is oriented toward a throat of the hydrophilic tunnel that forms a proton-conducting pathway for the proton liberated from plastosemiquinone and transferred to the thylakoid lumen. The proton transferred passes through the channel, which includes the proton-binding groups of Glu3 and Glu58 (Fig. 10b).

Concerning the second step of PQH₂ oxidation, it is worth noting that the heme b₆^L and the −COO⁻ group of Glu78 stand at a rather long distance (by ~ 6.5 Å) from the place where the radical PQH^· appears after the first step of the PQH₂ oxidation. The remoteness of the electron and proton acceptors from PQH^· would preclude its oxidation, and only after a rapid displacement of PQH^· toward heme b₆^L and Glu78 the oxidation of PQH^· could be efficiently realized. The assumption about rapid movements of a semiquinone within the Q_o portal, based on the analysis of kinetic data, has been proposed by Antony Crofts and collaborators, who studied the ubiquinol oxidation inside the Cytbc₁ complex (Crofts 2004b, 2021; Crofts et al. 2017). This proposal was also supported by estimations performed within the framework of a simple model based on conventional kinetic approaches (Tikhonov 2014) and quantum chemical modeling of PQH₂ oxidation at the Q_o site (Ustynyuk and Tikhonov 2022).

Quantum chemical modeling of a bifurcated oxidation of PQH₂

The density function theory (DFT) approach has been used to analyze quinol oxidation by Cytbc complexes (Shimizu et al. 2008; Frolov and Tikhonov 2009; Postila et al. 2013; Barragan et al. 2015, 2016; Husen and Solov’yov 2016; Ustynyuk et al. 2018; Ustynyuk and Tikhonov 2018, 2022). One of somewhat truncated quantum chemical models, used in (Ustynyuk and Tikhonov 2022) for modelling a bifurcated oxidation of PQH₂ at the catalytic Q_o site, contained four functional groups: 1) the Fe₂S₂ cluster surrounded by two His residues; 2) heme b₆^L; 3) Glu78, and 4) 2,3,5-trimethylbenzoquinol (TMBQH₂), a tail-less analog of PQH₂. Results of quantum chemical calculations are consistent with experimental data. Oxidized cluster Fe₂S₂ is diamagnetic (total spin S = 0) due to antiferromagnetic interaction of two paramagnetic ions, Fe³⁺(1) and Fe³⁺(2), with individual spins S = 5/2, demonstrating that two Fe ions in the oxidized and reduced states of the ISP have opposite projections of their spins (Noodleman et al. 1995, 2002; Siegbahn and Blomberg 1999; Sarewicz et al. 2021). Reduced cluster Fe₂S₂ is a paramagnetic species with the total spin S = 1/2. The oxidized heme b₆^L is also paramagnetic, this is consistent with experiment (Palmer 1985; Sarewicz et al. 2021). Calculated spin of the TMBQH^· radical was determined as S = 1/2 (Ustynyuk and Tikhonov 2018, 2022).

The first step of PQH₂ oxidation

Figure 11 reproduces the plot of the system energy versus the distance between the atom H(1) of TMBQH₂ and the N_ε atom of His155 (C. reinhardtii numbering, PDB code 1Q90). The two local minima correspond to the initial and the final positions of H(1), characterizing the formation of the hydrogen bond H(1)−N_ε ($R_{{{\text{H}} - {\text{N}}_{{\upvarepsilon }} }}$ = 1.11 Å). The oxidation of TMBQH₂ can be considered as the proton coupled electron transport (PCET) process, when the electron is directed to the Fe³⁺(1) ion of the Fe₂S₂ cluster, while the proton is accepted by the N_ε atom of His155 (Fig. 11a). This process needs the overcoming of the energy barrier ΔE^≠ (Fig. 11b). After a rapid (nonadiabatic) transfer of a proton to N_ε, the energy of the system increases by ΔE = 25 kJ mol⁻¹. The rise of energy is followed by its decrease by ΔE_rel = 8 kJ mol⁻¹, which occurs in the result of geometry “relaxation” followed the H atom transfer (for detail, see Ustynyuk and Tikhonov 2022). The overall change in the system energy can be evaluated as ΔE₁ = ΔE—ΔE_rel ≈ 17 kJ mole⁻¹. Similar values of the energy rise have been reported for the Cytbc₁ complex (Crofts et al. 2000; Zu et al. 2003; Barragan et al. 2016). An increase in the system energy is consistent with experimental evidence that the first step of the bifurcated oxidation of quinol at the Q_o site of the Cyt bc complexes is the endergonic (energy-accepting) process (Crofts 2004a, 2000c, 2021; Crofts et al. 2000, 2013). Thermodynamic analysis of the first electron transfer in Rb. sphaeroides, performed in terms of the Marcus-Brønsted equation, suggested that the overall reaction of an electron and a proton transfer from quinol to the ISP favored a proton first then electron sequence (for details, see Crofts et al. 2000; Zu et al. 2003; Crofts 2021). The overall free energy change in the reaction His_ox + QH₂ → His(H⁺)_red + QH^· was estimated as ΔG ~ 9–10 kJ mol⁻¹.

Fig. 11

DFT modelling of the first stage of 2,3,5-trimethylbenzoquinol (TMBQH₂) oxidation by the ISP. a Depicts a fragment of the Cytb₆f structure (PDB code 1Q90) used as a model system for analyzing the H(1) transfer from TMBQH₂ (the tail-less plastoquinol analog) to the ISP. b Shows the dependence of the system energy versus the distance between the H(1) atom donated by TMBQH₂ and the N_ε atom of His155 (PDB entry 1Q90). The energy profile was reconstructed on the basis of data presented in (Ustynyuk and Tikhonov 2022)

The second step of quinol oxidation

The endergonic nature of the first step of the quinol oxidation suggests that the semiquinone product SPQ^· requires its rapid removal to ensure sufficiently high rate of the forward reaction of electron transfer. The semiquinone species TMBQH is further oxidized to TMBQ; heme b₆^L acts as the electron acceptor, the −COO⁻group of Glu78 fulfills the role of the proton recipient (Fig. 12a). The question arises: is it possible that the radical TMBQH formed in the vicinity of the Fe₂S₂ cluster might efficiently donate an electron to heme b₆^L without its movement toward heme b₆^L and Glu78? The distances between TMBQH positioned near the Fe₂S₂ cluster and the participants of the second reaction (heme b₆^L and the −COO⁻ group of Glu78) are too long to provide efficient oxidation of TMBQH without its shift toward heme b₆^L. DFT computations revealed that the direct oxidation of TMBQH by remote heme b₆^L and the proton transfer to the −COO⁻ group (~ 6 Å) would be strongly restricted due to a high energy barrier (ΔE^≠  ~ 270 kJ mole⁻¹; Ustynyuk and Tikhonov 2022). However, as the radical TMBQH get closer to Glu78 and heme b₆^L, its oxidation becomes possible, because the reaction becomes exergonic. Figure 12b demonstrates that an energy of the model system decreases with shortening the distance between TMBQH and Glu78. The energy of the system further decreases with the transfer of the proton H(2) of TMBQH from its initial position, corresponding to minimum of energy in Fig. 12b, to the −COO⁻ group of Glu78 (Fig. 12c, ΔE₂ ≈ − 100 kJ mole⁻¹).

Fig. 12

DFT modelling of the second stage of a bifurcated oxidation of plastoquinol. a Depicts a fragment of the crystal structure of the Cytb₆f complex (PDB code 1Q90) used as a model system for the analysis of the radical TMBQH oxidation by heme b₆^L. b demonstrates how an energy of the model system changes with shortening the distance between TMBQH and Glu78. The distance between the H(2) atom of TMBQH and the nearest atom O of the –COO⁻ group of Glu78 is used as the parameter characterizing the shift of TMBQH toward Glu78. c shows changes in the energy of the model system upon the transfer of the proton H(2) of TMBQH from its initial position, corresponding to minimum of energy in b, to the −COO⁻ group of Glu78. The energy profile was reconstructed on the basis of data presented in (Ustynyuk and Tikhonov 2022)

The outcomes of DFT calculations support the notion that the first step of the bifurcated reaction is the energy-accepting process which determines the overall rate of PQH₂ oxidation. A rapid movement of plastosemiquinone toward heme b₆^L and the –COO⁻ group of Glu78 would accelerate its oxidation, thereby facilitating the overall rate of PQH₂ oxidation. In favor of a high mobility of plastoquinone inside the intraprotein cavity of the portal Q_o may serve the results of molecular dynamics simulations, which predict that PQH₂ can move throw the cavity (about 5-7 Å) within a few ns (Hasan et al. 2014).

Energetics and kinetics of the PQH₂/PQ turnover inside the Q_o portal

The cycle of PQH₂/PQ turnover includes: the PQH₂ enter into the quinol-binding portal Q_o, the PQH₂ oxidation per se, and the release of PQ from the portal Q_o. The steric constraints in the Cyt b₆f structure could limit diffusion-controlled processes associated with the PQH₂ turnover, imposing the limitations on the rate of electron transfer from PQH₂ to Cyt f. The influence of diffusional restrictions in the Q_o portal on the quinol turnover has been clearly demonstrated in Cramer’s laboratory (Ness et al. 2019). Using genetic modifications of subIV (Pro105Ala and Pro112Ala) in cyanobacteria Synechococcus sp. PCC 7002, the authors were able to narrow the entrance into the portal Q_o, thereby creating an obstacle for the penetration of PQH₂ to the quinol-binding site. In the result of this manipulation, they observed a marked deceleration of the Cyt f reduction (average half-times 6.7 ± 1.3 ms → 20.7 ± 5.2 ms) and a two-fold slowing down in the rate of cell growth.

The redox steps of PQH₂ turnover inside the Cytb₆f complex are the basic events that determine the overall rate of the intersystem electron transport. The uphill electron transfer from PQH₂ to ISP_ox is assumed to determine the rate of the two-electron quinol oxidation at the Q_o site (Crofts 2004a, 2004b, 2021). The energy uptake (ΔE₁ > 0) during this reaction would be regained due to the energy-donating step of the plastosemiquinone (PSQ^·) oxidation (ΔE₂ < 0), because PSQ^· is a strong electron donor capable of reducing heme Cyt b₆^L. The overall energy balance should be favorable for PQH₂ oxidation (ΔE₁ + ΔE₂ < 0). From the physical point of view, the coupling between the energy-accepting and energy-donating reactions (1) and (2) could be realized, provided both reactions occur as concerted processes (Blumenfeld and Tikhonov 1994; Snyder et al. 2000; Osyczka et al. 2004, 2005; Zhu et al. 2007; Reece and Nocera 2009). A simple kinetic model suggests that the rate constant k₂ of electron transfer from PSQ^· to heme b₆^L may be about k₂ ~10⁸ s⁻¹ (Tikhonov 2014). According to molecular dynamics simulations for the Cytbc₁ (Crofts et al. 2017), it is feasible that semiquinone can rapidly move (~ 4 Å/ns) in the cavity of the hydrophobic portal Q_o. It is highly likely that the dissociation of the radical pair and semiquinone movements occur rapidly, in the range < 10 μs. Since the oxidation of PSQ^· would proceed much more rapidly than the first step of PQH₂ oxidation by the ISP (τ_1/2 ≥ 4–20 мc), we could suggest that both steps of PQH₂ oxidation might be virtually considered as synchronous processes.

Note that plastosemiquinones may also serve as the electron donors for superoxide radical generation in the Cytbc complexes that might proceed via the side channel of SPQ^· oxidation due to electron transfer to molecular oxygen and the formation of harmful superoxide radicals (Cape et al. 2006, 2007; Baniulis et al. 2013, 2016; Bujnowicz et al. 2019; Sarewicz et al. 2021). The removal of semiquinone species due to their rapid oxidation by Cyt b₆^L would minimize the production of reactive oxygen species (ROS). Rapid disappearance of chemically active semiquinone radicals would diminish a probability of O₂ reduction by PSQ^·, thereby precluding the formation of ROS (for review, see Asada 2006; Mubarakshina et al. 2006; Halliwell and Gutteridge 2007; Ivanov et al. 2018). Relatively short life-time of PSQ^· may explain why it is difficult to detect semiquinone radicals in the Cytbc₁ and Cytb₆f complexes by EPR (Sarewicz et al. 2017, 2021). Note that the specific rate of superoxide formation in the Cytb₆f complexes is higher by an order of magnitude than in the Cytbc₁ complex (Baniulis et al. 2013). It has been proposed that the retention of PSQ^· due to the steric effects of the Chl phytyl tail should stimulate the superoxide production in the Cytb₆f.

Summing up, we can state that: (1) the second reaction of PQH₂ oxidation occurs much more rapidly (by a factor of ~ 10³) than the first reaction (for references, see Crofts 2021); (2) the intermediate complex ISPH^·-PSQ^· is an unstable structure; after the radical pair break, the tethered diffusion of the extrinsic domain of the reduced ISP_red toward heme f occurs; (3) electron transfer from PSQ^· to heme b₆^L should be stimulated by migration of PSQ^· toward heme Cyt f to shorten the distance between the electron donor and acceptor. Using the Moser–Dutton ruler for the rate of electron tunnelling between redox centers (Moser et al. 1997; Page et al. 1999), expanded by Crofts (2004b) for proton-coupled electron transfer reactions, we could evaluate the rate constant k₁ of PQH₂ oxidation within the framework of a simple kinetic model as k₁ ~ 40–170 s⁻¹; these values reasonably agree with experimental data on electron transfer from PQH₂ to P₇₀₀⁺ (Tikhonov 2018).

Regulation of the Cytb ₆ f functions in chloroplasts

The pH-dependent control of the Cytb ₆ f turnover

The light-induced acidification of the lumen (pH_in↓) is one of the major regulators of the intersystem electron flow. The feedback control of electron transport provides optimal functioning of photosynthetic apparatus, preventing an excessive acidification of the lumen and the over-excitation of PSII (Rumberg and Siggel 1969; Tikhonov et al. 1981, 1984; Nishio and Whitmarsh 1993; Schönknecht et al. 1995; Kramer et al. 1999; Jahns et al. 2002). A decrease in pH_in decelerates the oxidation of PQH₂ at the Q_o site and attenuates the activity of PSII due to an enhancement of heat dissipation of light energy in the light-harvesting antenna of PSII (Rees et al. 1989; Müller et al. 2001; Li et al. 2009).

The ISP operates as a switch controlling the rate of the intersystem electron flow (for illustration, see Fig. 13). Electron transfer from PQH₂ to the ISP is governed by a “proton-gated” affinity mechanism, determined by the ability of proton binding to the His group of the ISP (Brandt 1996; Link 1997, 1999). The intrinsic mobile fragment of the ISP is positioned in the lumen-oriented domain of the enzyme (the Q_o site of the Cytb₆f), and connected with the bulk of the lumen via two intra-protein proton-conductive “trails” (Tikhonov 2014, 2018). The oxidized ISP (ISP_ox) is characterized by pK_ox ~ 6–6.5 (Finazzi 2002; Soriano et al. 2002). At pH_in > pK_ox, the proton-binding His residue of the ISP (e.g., His155 in C. reinhardtii or His128 in spinach) is deprotonated, being able of accepting a proton from PQH₂. Increased activity of hydrogen ions inside the lumen (e.g., at pH_in < 6.2) will induce the protonation of the functional His residue of the ISP. The reduction of the ISP induces an increase in the affinity of the ISP to a proton; the pK_red value of His increases with the reduction of ISP up to pK_red ≈ 8.3 − 8.9 (Zu et al. 2003; Iwaki et al. 2005; Lin et al. 2006; Hsueh et al. 2010; Lhee et al. 2010). In illuminated chloroplasts, the relationship pH_in ≤ pK_red holds true. The protonated His is unable to accept a proton; therefore, the acidification of the lumen would impede the oxidation of PQH₂. Thus, the back-pressure of hydrogen ions from the lumen would retard the proton dissociation from the ISP(H⁺), thereby slowing down the oxidation of PQH₂ and decelerating electron flow through the Cytb₆f. The oxidation of the ISP by Cyt f would lead to a decrease in pK_ISP, promoting the proton release from the ISP into the lumen.

Fig. 13

A diagram illustrating the redox-dependent protonation/deprotonation events in the ISP protein of the Cytb₆f complex. Blue and red lines show tentative pH-dependences for the reduced and oxidized forms of the ISP. Dashed line depicts the pH-dependence of electron flow through the Cytb₆f complex as calculated according to formula (3) with the model parameters pK_ox = 6.2 and pK_red = 8.7 (see text for explanations)

The above reasonings allow to figure out the pH-dependence of the intersystem of electron flow through the Cytb₆f (J_b6f). The overall electron flux should be determined by two factors: (i) a probability p(ISP_ox) of finding the oxidized ISP_ox in deprotonated state, and (ii) a probability of finding ISP in reduced and protonated state, p(ISP(H⁺)_red. The latter factor implies that the reduction of ISP by PQH₂ occurs simultaneously with the ISP protonation (the PCET reaction). Obviously, the steady-state electron flux through the Cytb₆f will be proportional to the product of the two probabilities, J_b6f ~ p(ISP_ox) × p(ISP(H⁺)_red. Simple calculations lead to the following expression:

$$J_{{b{6}f}} \left( {{\text{pH}}} \right) \sim {1}0^{{{\text{p}}K{\text{red}} - {\text{pH}} }} \times ({1 } + { 1}0^{{{\text{p}}K{\text{red}} - {\text{pH}}}} )^{{ - {1}}} \times ({1 } + { 1}0^{{{\text{p}}K{\text{ox}} - {\text{pH}}}} )^{{ - {1}}} .$$

(3)

The model predicts the bell-shape pH-dependence of J_b6f. In Fig. 13, green dashed line presents the function J_b6f (pH) calculated for pK_ox = 6.2 and pK_red = 8.7 (for detail, see Tikhonov 2014, 2018). This curve adequately describes the experimental pH-dependence of the rate of electron flow through the Cytb₆f complex in chloroplasts (Hope et al. 1994; Hope 2000).

The pH-dependent regulation of electron flow through the Cytb₆f lies in the basis of the so-called photosynthetic control phenomenon (Rumberg et. al., 1968; West and Wiskich 1968; Schönknecht et al. 1995; Kramer et al. 1999; Foyer et al. 2012; Tikhonov 2012, 2013; Colombo et al. 2016). The term photosynthetic control implies that the rate of photosynthetic electron transport depends on metabolic state of chloroplasts, which is determined by the adenylate status of chloroplasts (the ATP/ADP ratio) (for references, see Tikhonov 2014, 2018).

In the context of pH-dependent control of PQH₂ oxidation in chloroplasts, it necessary to note that the lateral heterogeneity of lamellar membranes and uneven distribution of the ATP synthase complexes may lead to a pH gradient in the lumen from the granal to the stromal thylakoids, with more significant acidification of the granal lumen. A critical analysis of experimental data and the results of computer modelling (Tikhonov and Vershubskii 2014; Vershubskii et al. 2017) support the notion that the long-range diffusion of protons within the lumen and obstructed diffusion of mobile electron carriers (PQH₂ and Pc) could influence the lateral profiles of pH along the thylakoid membranes. The model predicts significant alkalization of the inter-thylakoid gap and the establishment of nonuniform lateral profiles of ΔpH under the photophosphorylation conditions. As it was suggested by Kirchhoff et al. (2017), the partial decoupling of the proton concentrations between the lumen compartments of granal and stromal thylakoids might have important implications for independent regulation of LEF and CEF.

Another mechanism of pH-dependent down-regulation of the intersystem electron flow is accomplished by attenuating PSII turnover caused by the lumen acidification known as the non-photochemical quenching, NPQ (for references, see Rees et al. 1989; Lazar 1999; Müller et al. 2001; Li et al. 2009; Jahns and Holzwarth 2012; Demmig-Adams et al. 2012; Ruban et al. 2012; Rochaix 2014; Colombo et al. 2016). The NPQ mechanism is realized by the light-induced enhancement of thermal dissipation of energy in the light-harvesting antenna of PSII. Both down-regulation mechanisms (the pH-dependent deceleration of Cytb₆f turnover and generation of NPQ) are characterized by close pK values (~ 6.0–6.5), providing similar contributions to the pH-dependent attenuation of electron flow from PSII to PSI.

Plastoquinone pool capacity

PQH₂/PQ traffic in the thylakoid membrane over-crowded with protein complexes (about 70–80% protein in spinach) is one of the factors that could influence the rate of electron transport between PSII and PSI (Kirchhoff et al. 2000). The diffusion-controlled turnover of PQH₂ and the overall rate of the intersystem electron transport may depend on the relative size of the photo-reducible plastoquinone pool (Kurreck et al. 2000). A high content of plastoquinone molecules would enhance the connectivity between spatially separated PSII and Cytb₆f complexes (Haehnel 1984; Siggel et al. 1972; Tikhonov and Vershubskii 2017). A relative capacity of the PQH₂/PQ pool may be determined by the plant growth conditions and their physiological state (McCauley and Melis 1986; Suslichenko and Tikhonov 2019; Flannery et al. 2021; Suslichenko et al. 2022). The regulatory feedbacks, e.g., the activation/deactivation of PSI and PSII complexes, activation of the CBC reactions, and the pH-dependent control of the Cytb₆f turnover, will modulate dynamics of PQH₂/PQ redox transients (Buchanan 1980; Foyer et al. 2012; Rochaix 2014). In chloroplasts, variability of the photo-reducible plastoquinone pool capacity is provided by plastoglobules, the lipid storing particles, which serve as the reservoirs contributing to the plastoquinone pool (Pralon and Kessler 2016). Plastoglobules are structurally and functionally associated with the thylakoid membranes: they are attached to stromal thylakoids and contain about 70–75% of total plastoquinone. An increase in the relative size of this pool would stimulate the connectivity between spatially separated PSII and Cytb₆f complexes, thereby supporting high rates of the intersystem electron transfer (Rumberg et al. 1968; Stiehl and Witt 1969; Siggel et al. 1972; Haehnel 1984; Tikhonov and Vershubskii 2017). Enhanced size of the photo-reducible PQH₂/PQ pool is characteristic of plants grown in low-light (LL) conditions (Suslichenko and Tikhonov 2019). Therefore, the LL-acclimated plants with increased size of the plastoquinone pool in chloroplasts would support efficient electron transport even with the light attenuation. Otherwise, the reduction of the PQH₂/PQ pool in high light-grown plants should prevent their too high photochemical activity, thereby avoiding the oxidative stress at strong light.

Sub-localization of the Cytb ₆ f complexes in thylakoid membranes

Standing at the crossroad of electron transport pathways, the Cytb₆f complex performs a role of a hub which directs electrons to noncyclic or cyclic routes of electron flow (Munekage et al. 2004; Shikanai 2007; Joliot and Johnson 2011; Hertle et al. 2013; Tikhonov 2014; Strand et al. 2016; Malone et al. 2021). In chloroplasts, one of the structure-dependent mechanisms of short-term regulation of electron transport (in a timescale of seconds to minutes) may be associated with a redistribution of electron transport complexes between the granal and stromal thylakoids (for review and references, see Ruban and Johnson 2015; Wood et al. 2019). Changes in plant acclimation conditions, e.g., spectral composition and light intensity, may influence the composition and lateral distribution of photosynthetic complexes, including the Cytb₆f (Boardman 1977; Anderson et al. 1988, 2012; Flannery et al. 2021). Sub-localization of the Cytb₆f complexes in granal and stromal domains is dynamic, depending on the functional state of plants and architecture of chloroplasts (Vallon et al. 1991; Johnson et al. 2014; Dumas et al. 2016; Wood et al. 2018, 2019; Flannery et al. 2021; Hepworth et al. 2021). In particular, Wood et al. (2019) reported about rapid (t_1/2 ~ 10 min) reversible changes in grana size: WL of low-intensity (LL) reduced size of grana in Arabidopsis and spinach, while WL of high-intensity (HL) increased grana size. The dynamics of thylakoid stacking is primarily determined by phosphorylation of the light-harvesting complex II (LHCII) (Wood et al. 2018, 2019). The relative abundance of the Cytb₆f varied upon the long-term (weeks) acclimation of plants to LL and HL growth light intensity, positively correlating with changes in PSII activity. In HL-grown plants, an increase in CEF has been observed (Flannery et al. 2021; Benkov et al. 2023).

The re-modelling of the chloroplast lamellar system may regulate the partition of electrons between LEF and CEF pathways. However, the question about sub-localization of the Cytb₆f and the rate of the intersystem electron transport remains a matter of discussions. There is no consensus about the light-induced re-localization of the Cytb₆f between the granal and stromal domains of the thylakoid lamellas, although certain correlations between the grana size and the abundance of the Cytb₆f complexes have been observed (Höhner et. al., 2020; Flannery et al. 2021). As noted above, in the Arabidopsis mutants with extremely wide grana (up to 1,600 nm in diameter) an average time of plastoquinol diffusion between the PSII to Cytb₆f complexes markedly increased, but it remained independent of granum diameter close to native (Höhner et al. 2020). The re-localization of Cytb₆f complexes from the granal to stromal domains of the chloroplast membranes could enhance the CEF capacities. The re-distribution of the Cytb₆f might be induced by changes in the distance between adjacent membranes in stacked piles of granal thylakoids (Kirchhoff et al. 2017).

State transitions

The Cytb₆f complex is involved in regulation of the light energy partitioning between the light-harvesting antennas of PSI and PSII, using the state transitions mechanism of the redistribution of mobile light-harvesting complexes between PSII and PSI (Allen 1981, 1992; Lemeille and Rochaix 2010; Minagawa 2011; Rochaix et al. 2012). A macromolecular device, which receives a signal from the reduced PQH₂ pool and induces the state I → state II transition, is inherent to the Cytb₆f complex and specific protein subunits bound to it. The signal transducer, which actuates the LHCII kinase, is specific for the quinone bound and its redox state (Vener et al. 1997, 1998; Zito et al. 1999; Finazzi et al. 2001; Dumas et al. 2017).  Putative molecular mechanism of the kinase activation due to PQH₂-induced structural changes in the Cytb₆f complex has been suggested by Hasan et al. (2013a). There are the catalytic and regulatory domains of the kinase with two Cys residues, which are essential for the enzyme activity. The redox state of these residues may be regulated by Fd and thioredoxin through the membrane-bound thiol oxidoreductases (Lennartz et al. 2001; Motohashi and Hisabori 2006; Dietz and Pfannschmidt 2011). The over-reduction of the acceptor side of PSI will cause the reduction of Cys residues, thereby inducing the “State II → State I” transition, upon which a PSI activity will lessen.

Phosphorylation of the mobile light-harvesting subunits of PSII is a reversible process. After a decrease in the concentration of PQH₂, the dephosphorylation of phosphorylated complexes occurs due to the LHCII phosphatase TAP/PPH1 activity (state II → state I transition; Puthiyaveetil et al. 2016). Dephosphorylated light-harvesting subunits rebind to PSII antenna, and chloroplasts return to the initial state I. Finally, we must emphasize the regulatory role of the Cytb₆f complex associated with signaling and mediating gene expression in the plant cell (Depege et al. 2003; Bellafiore et al. 2005; Puthiyaveetil et al. 2016).

Concluding remarks

Standing between PSII and PSI, the Cytb₆f complex provides the intersystem electron transport and plays the pivoting role in regulation of electron transport in oxygenic photosynthesis. The bifurcated oxidation of PQH₂ at the Q_o site is one of the clue events that determine the rate of electron transfer from PSII to PSI. Experimental studies and theoretical analysis of the bifurcated oxidation of PQH₂ suggest that the overall rate of PQH₂ turnover is determined by the energy-accepting (endergonic) reaction of electron transfer from PQH₂ to the ISP. The further reaction of plastosemiquinone (PSQ^·) oxidation is the energy-donating (endergonic) process, the rate of which should increase upon its movement toward the low-potential heme b₆^L within the intraprotein cavity that includes the Q_o site. The movement of PSQ^· closer to the heme would allow about a 10³-fold increase in the rate of its oxidation (for references, see Crofts 2021). The bifurcated oxidation of PQH₂ is tightly coupled to the proton release into the lumen. This process is controlled by the intra-thylakoid pH_in, demonstrating the slowing down of PQH₂ oxidation with the lumen acidification (Tikhonov et al. 1981, 1984; Kramer et al. 1999).

The Cytb₆f complex is involved to directing electrons to alternative routes of electron transport (Munekage et al. 2004; DalCorso et al. 2008; Strand et al. 2016; Zhao et al. 2020). The Cytb₆f complexes localized in grana mediate linear electron transport (LET) from PSII to PSI, while the complexes localized in stroma-exposed domains of the thylakoid membranes participate in cyclic electron transport (CET) around PSI. Structural changes and/or metabolic shifts in chloroplasts would influence a balance between LET and CET, supporting optimal functioning of photosynthetic apparatus under variable growth conditions (Yamory and Shikanai 2016).

The Cytb₆f complex participate in partitioning the light energy absorbed between the light-harvesting antennas of PSI and PSII (state transitions), using the Stt7/STN7 kinase, which is inherent to the Cytb₆f. Modulating the photochemical activities of PSI and PSII, these transitions provide optimal functioning of ETC under variable metabolic conditions. The feedback regulation of the redox state of the PQH₂/PQ pool, caused by changes in activities of the PSI, PSII and Cytb₆f complexes, may be involved in the retrograde signaling in the plant cell (Allen and Pfannschmidt 2000; Fernández and Strand (2008); Foyer et al. 2012; Mielecki et al. 2020).