First of all, the c-ring structure itself is important for two reasons. This is the first and the only high-resolution structure of c-rings from plants and this was the missing part in the picture of the high-resolution structures that were obtained from mitochondria and bacteria. The high-resolution structures of mitochondria and bacterial c-rings are already known. The fact that chloroplast c-rings are similar to that from mitochondria and bacteria is important. The second reason is that the structure of the c-ring revealed molecular mechanisms of intersubunit contacts which determine the size of the c-ring (Figs. S13–15). Although this result was discussed previously in literature1,2,28 and the fact that the c-ring from spinach chloroplasts has 14 subunits was also known22, in this work due to high-resolution crystallographic data we showed in details the network of hydrogen bonds, compared the intersubunit contacts of c-rings from plants, mitochondria and bacteria, and showed molecular mechanisms of how determination of c-ring stoichiometries occurs.

We described a striking feature of the structure of the c-ring ATP synthase from chloroplasts – the presence in the hydrophobic part of the internal pore of the c-ring of circular-like electron densities placed at the distance of about 5.4 Å from each other along the central axis and parallel to the membrane plane. As we stressed, the present observation becomes even more important due to the fact (as it appears) that it is a universal feature of all known high-resolution structures of other F 1 F O synthase c-rings.

We present the evidences that the densities might correspond to isoprenoid quinones. It is consistent with about 1.5 times larger hydrophobic thickness of hydrophobic surface of the inside of the c-ring than that of a lipid bilayer and a membrane protein. UV-Vis spectra (Fig. S6A) comprise isoprenoids presumably inside the c-ring and differential UV-Vis spectroscopy shows quinones presumably with trimethyl benzoquinone (TMBQ) (PQ-9 polar moiety) in the c-ring samples dissolved in EtOH (Fig. S6D–F). Although the noise is quite high in the differential spectrum of the c-ring samples (Fig. S6D) (about 0.01 a.u. of absorption) the peak at 260 nm can be clearly seen and the whole curve is close to the TMBQ differential spectrum. The high noise might be due to the low absolute concentration of the compound reacting with NaBH 4 because of the high dilution ratio of the c-ring samples in EtOH (~1:30). However, that was done to eliminate changes in the solvent (EtOH) caused by the buffer in which the c-ring protein is and for proper control experiments.

The schematic drawings showing possible fit of different isoprenoid molecules (Figs. 4 and 5) into c-rings from different isoprenoids also point towards the hypothesis. Finally, the universality of the additional densities inside different c-rings (Figs. S7–S9) suggest that the molecule inside the c-ring should have universality among different species, long hydrophobic tail, contain isoprenoids and be wide-spread among different species. Thus, taking into account the harsh conditions of preparation (+65 °C, NLS 1%) of the c-ring for the UV-Vis studies we conclude that the isoprenoids, in particular, plastoquinone or its derivatives might be one of the candidates. Moreover, since we do not see any fragmented electron densities of an isoprenoid molecule at the external surface of the c-ring we suggest that isoprenoids might be inside of the c-ring.

To our best knowledge this hypothesis since 200832 was not furthermore discussed and developed. There was no proof that carotenoids are inside of the c-ring pore. Indeed, the detergent treatment of membrane proteins does not remove (at least completely) native lipids surrounding the protein surface. An example of this is a squalene molecule at the hydrophobic surface of bacteriorhodopsin evidenced with high resolution crystallographic structures obtained with the crystals grown from the protein solubilized in a quite harsh detergent beta-octyl-glucoside34.

Our crystallographic data do not provide an evidence of the presence of isoprenoid molecules at the surface of the c-ring, in opposite, as we have shown, the characteristic for isoprenoid molecules electron densities are present inside the c-ring. Taking into account that usually only tightly bound amphiphilic molecules remain bound to a membrane protein and are simultaneously resolved on electron density maps we conclude that the molecules responsible for the color of the crystal are really placed inside the c-ring.

The color of the crystals is determined by absorption of light in the range of 400–500 nm. It is a characteristic of carotenoids, however, contribution to the absorption range is also characteristic of some other isoprenoids. Unfortunately, the analysis of the spectra is even more complicated due to the possible presence of chlorophyll molecules in the examined samples.

Thus, despite the fact that electron densities inside the c-ring, spectroscopic and HPLC data point toward the presence of an isoprenoid molecule inside the c-ring, all this itself is not sufficient to identify the exact type of the molecule.

In general, identification of lipid molecules on electron density maps of membrane proteins is a challenge. In the most cases the densities related to the lipids are weaker, fragmented and usually polar heads are not resolved at all35. However, in the present case there are hints, which make the identification more reliable. The unusually large thickness of the hydrophobic surface of the internal part of the c-ring means that the lipids should have unusually long hydrophobic chains. Carotenoids first emerged in archaebacteria as lipids reinforcing cell membranes. To serve this function their linear chain is comprising usually 9 to 11 conjugated C = C bonds with the length corresponding the thickness of the hydrophobic part of lipid membranes (30–35 Å). Thus, they alone cannot explain the hydrophobic thickness of the pore of the c-ring. Moreover, carotenoids are not found in mitochondria and therefore they can hardly explain similar electron densities in yeast mitochondria ATP-synthases c-rings (Fig. S6). In opposite, other polyisoprenes may fit the requirements. The best known of such polyisoprenes are ubiquinone in the mitochondrion and in prokaryotes36, and plastoquinone (PQ) and similar isoprenes are associated with the chloroplast and prokaryote photosynthetic membranes37.

We note that Meier et al. suggested that in their experiments29 phospholipids are incorporated into the central hole from one side of the cylinder during the reconstitution procedure, as the detergent-purified c-ring is completely devoid of phospholipids. The authors recognize that “the association with phospholipids during the reconstitution procedure merely indicates a strong affinity of the C 11 cylinder for hydrophobic molecules to its central cavity” Thus, Meier et al. support the idea of high affinity of c-ring for hydrophobic molecules to its central pore. Isoprenoids are specific, but also very hydrophobic lipids. Therefore, we should also note that a mixture of different lipids, for instance isoprenoids and phospholipids, may fill better the central pore of the c-ring. In addition, we have indirect evidence on UV-Vis spectra (Fig. S6) that there might be a mixture of different lipids and isoprenoids due to absorption peaks corresponding to beta-carotene and chlorophyll a molecules together with absorption peak at 335 nm, that does not correspond to beta-carotene or chlorophyll a, however might correspond to plastosemiquinone-9 or its derivatives33.

It is noted that additional electron densities in the internal part of the c-rings are not equally strong in all other known high-resolution structures of c-rings. However, there is a strong correlation between the level of additional electron densities inside the c-rings and their purification and crystallization conditions (Table S3). The structures obtained from intact ATP synthases, without using harsh conditions (NLS or/and high temperature) demonstrate pronounced additional characteristic electron densities in the inner pore (4F4S and 5BPS, Fig. S7). The c-rings for which harsh solubilization and purification conditions were used demonstrate less pronounced additional electron densities (4CBK and 2 × 2 V, Fig. S8). The only exception is the structure 2XQU, where well-ordered circles of additional electron densities take place, though harsh conditions were used to purify the c-ring (Fig. S9B).

A unique arrangement of electronic levels due to the polyene chain structure makes isoprenoids also efficient protectors of the cell against reactive oxygen species (ROS)38. The importance of quinone isoprenoids is not limited by the electron carrier functions, for instance, coenzyme Q is an essential component of the respiratory chain, a cofactor of pyrimidine biosynthesis, a proton permeability barrier39,40 and acts as an antioxidant in mitochondrial membranes. More recently CoQ has been identified as a modulator of apoptosis, inflammation and gene expression38.

Thus, we speculate that isoprenoid quinones, which present in membranes with F O F 1 ATP synthases may also present inside the c-rings. We suggest that they may play an important role as scavengers of ROS protecting not only external but also internal part of the c-rings, in particular against ROS. This is supported also by the fact that, in opposite to carotenoids, in vertebrates these molecules damaged by ROS are synthesized de novo41. In addition, by the studies of BCDO2-deficient mice and human cell cultures it indicates that carotenoids can impair respiration and induce oxidative stress. Mammalian cells thus express a mitochondrial carotenoid-oxygenase that degrades carotenoids to protect these vital organelles42.

We hypothesize that isoprenoid quinones, such as plastoquinone and coenzyme Q, may be universal cofactors of proton ATP synthases and their abnormalities may lead to cell and tissue dysfunctions not only through disturbance of the electron transport bioenergetics chain but also directly through dysregulation of ATP synthases. Although this hypothesis seems plausible, we recognize that additional experiments are necessary to verify it and other possible explanations should be considered. However, precise identification of what molecule corresponds to the electron densities inside the c-ring is possible only by high-resolution crystallography. It is a well-known problem in case of lipids bound to a membrane protein. Nevertheless, what is indeed clear is that inner part of c-rings of different F 1 F O ATP-synthases have universal unusual features, which may have a great functional importance, and isoprenoid quinones correspond well to obtained experimental data and their universality among different organisms.

It is not excluded that not only isoprenoid molecules could be placed inside c-ring but also in addition there might be some of F O subunits of ATP synthase partially entering the inner pore of the c-ring. Indeed, works8,10,43 show that in case of mitochondrial ATP synthases C terminus of F O subunit e might be extended to the center of the c-ring. Moreover, in c-rings of V-type ATP synthases44,45 the densities inside were assigned to two alpha helices. The work44 also shows that one of the alpha helices placed in the center of the pore belongs to V O part of the V-type ATP synthase. We speculate that the presence of an anchor in the center of the c-ring may have functional importance. Namely, one cannot exclude that this internal anchor is connected with a subunit of the stator F O part of ATP synthase. This would additionally stabilize precise positioning of the rotor to the stator in the region of proton transfer pathway (active site of the c-ring), (Fig. S10), similarly as in electrical motors, where the stator and rotor precise positioning is absolutely necessary. Reviews1,2 mention surprisingly small contact between a subunit and c-ring and it can be not sufficient to stabilize positioning of Glu61 active site relative to a subunit. Thus, we hypothesize that isoprenoid layer covering inner surface of the c-ring (Fig. 5) may also reduce frictions between the anchor of the stator and the surrounding upon rotation of the c-ring (Fig. S10).