In vivo electron donation from plastocyanin and cytochrome c6 to PSI in Synechocystis sp. PCC6803.

Many cyanobacteria species can use both plastocyanin and cytochrome c6 as lumenal electron carriers to shuttle electrons from the cytochrome b6f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast re-reduction of P700in vivo after a single charge separation. Here, we show that both cytochrome c6 and plastocyanin can bind to PSI in the dark and participate to the fast phase of P700 reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c6 than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P700 oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c6, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.

Crystal structures of native cytochrome c6 from Thermosynechococcus elongatus in two different space groups and implications for its oligomerization.

Native cytochrome c6 was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25 Šresolution, respectively. To date, the structure of native cytochrome c6 from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomerization, and related structures have only previously been solved using X-ray crystallography after recombinant gene overexpression in Escherichia coli. The reported space groups of related cyanobacterial cytochrome c6 structures differ from those reported here. Interestingly, the protein-protein interfaces that were observed utilizing X-ray crystallography could also explain homo-oligomerization in solution; specifically, trimerization is indicated by infra-red dynamic light scattering and blue native gel electrophoresis in solution. Trimers were also detected by mass spectrometry. Furthermore, there is an indication of post-translational methylation in the crystal structure. Additionally, the possibility of modifying the crystal size and the redox activity in the context of photosynthesis is shaping the investigated cytochrome as a highly suitable model protein for advanced serial crystallography at highly brilliant X-ray free-electron laser sources.

Alanine to serine substitutions drive thermal adaptation in a psychrophilic diatom cytochrome c6.

In this study, we investigate the thermodynamic mechanisms by which electron transfer proteins adapt to environmental temperature by directly comparing the redox properties and folding stability of a psychrophilic cytochrome c and a mesophilic homolog. Our model system consists of two cytochrome c6 proteins from diatoms: one adapted specifically to polar environments, the other adapted generally to surface ocean environments. Direct electrochemistry shows that the midpoint potential for the mesophilic homolog is slightly higher at all temperatures measured. Cytochrome c6 from the psychrophilic diatom unfolds with a melting temperature 10.4 °C lower than the homologous mesophilic cytochrome c6. Changes in free energy upon unfolding are identical, within error, for the psychrophilic and mesophilic protein; however, the chemical unfolding transition of the psychrophilic cytochrome c6 is more cooperative than for the mesophilic cytochrome c6. Substituting alanine residues found in the mesophile with serine found in corresponding positions of the psychrophile demonstrates that burial of the polar serine both decreases the thermal stability and decreases the midpoint potential. The mutagenesis data, combined with differences in the m-value of chemical denaturation, suggest that differences in solvent accessibility of the hydrophobic core underlie the adaptation of cytochrome c6 to differing environmental temperature.

Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer.

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, - 0.033 ± 0.006, - 0.036 ± 0.008, and - 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.

Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus.

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.

Quantum yield measurements of light-induced H2 generation in a photosystem I-[FeFe]-H2ase nanoconstruct.

The quantum yield for light-induced H2 generation was measured for a previously optimized bio-hybrid cytochrome c 6-crosslinked PSI(C13G)-1,8-octanedithiol-[FeFe]-H2ase(C97G) (PSI-H2ase) nanoconstruct. The theoretical quantum yield for the PSI-H2ase nanoconstruct is 0.50 molecules of H2 per photon absorbed, which equates to a requirement of two photons per H2 generated. Illumination of the PSI-H2ase nanoconstruct with visible light between 400 and 700 nm resulted in an average quantum yield of 0.10-0.15 molecules of H2 per photon absorbed, which equates to a requirement of 6.7-10 photons per H2 generated. A possible reason for the difference between the theoretical and experimental quantum yield is the occurrence of non-productive PSI(C13G)-1,8-octanedithiol-PSIC13G (PSI-PSI) conjugates, which would absorb light without generating H2. Assuming the thiol-Fe coupling is equally efficient at producing PSI-PSI conjugates as well as in producing PSI-H2ase nanoconstructs, the theoretical quantum yield would decrease to 0.167 molecules of H2 per photon absorbed, which equates to 6 photons per H2 generated. This value is close to the range of measured values in the current study. A strategy that purifies the PSI-H2ase nanoconstructs from the unproductive PSI-PSI conjugates or that incorporates different chemistries on the PSI and [FeFe]-H2ase enzyme sites could potentially allow the PSI-H2ase nanoconstruct to approach the expected theoretical quantum yield for light-induced H2 generation.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.

[Brownian dynamics simulations of protein-protein interactions in photosynthetic electron transport chain].

The application of Brownian dynamics for simulation of transient protein-protein interactions is reviewed. The review focuses on theoretical basics of Brownian dynamics method, its particular implementations, advantages and drawbacks of the method. The outlook for future development of Brownian dynamics-based simulation techniques is discussed. Special attention is given to analysis of Brownian dynamics trajectories. The second part of the review is dedicated to the role of Brownian dynamics simulations in studying photosynthetic electron transport. Interactions of mobile electron carriers (plastocyanin, cytochrome c6, and ferredoxin) with their reaction partners (cytochrome b6f complex, photosystem I, ferredoxin:NADP-reductase, and hydrogenase) are considered.

Insights into the relationship between the haem-binding pocket and the redox potential of c6 cytochromes: four atomic resolution structures of c6 and c6-like proteins from Synechococcus sp. PCC 7002.

The structure of cytochrome c6C from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.03 Šresolution. This is the first structural report on the recently discovered cyanobacterial cytochrome c6-like proteins found in marine and nitrogen-fixing cyanobacteria. Despite high similarity in the overall three-dimensional fold between cytochromes c6 and c6C, the latter shows saliently different electrostatic properties in terms of surface charge distribution and dipole moments. Its midpoint redox potential is less than half of the value for typical c6 cytochromes and results mainly from the substitution of one residue in the haem pocket. Here, high-resolution crystal structures of mutants of both cytochromes c6 and c6C are presented, and the impact of the mutation of specific residues in the haem-binding pocket on the redox potential is discussed. These findings contribute to the elucidation of the structure-function relationship of c6-like cytochromes.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

Cytochrome c(6B) of Synechococcus sp. WH 8102--crystal structure and basic properties of novel c(6)-like family representative.

Cytochromes c are soluble electron carriers of relatively low molecular weight, containing single heme moiety. In cyanobacteria cytochrome c6 participates in electron transfer from cytochrome b6f complex to photosystem I. Recent phylogenetic analysis revealed the existence of a few families of proteins homologous to the previously mentioned. Cytochrome c6A from Arabidopsis thaliana was identified as a protein responsible for disulfide bond formation in response to intracellular redox state changes and c550 is well known element of photosystem II. However, function of cytochromes marked as c6B, c6C and cM as well as the physiological process in which they take a part still remain unidentified. Here we present the first structural and biophysical analysis of cytochrome from the c6B family from mesophilic cyanobacteria Synechococcus sp. WH 8102. Purified protein was crystallized and its structure was refined at 1.4 Å resolution. Overall architecture of this polypeptide resembles typical I-class cytochromes c. The main features, that distinguish described protein from cytochrome c6, are slightly red-shifted α band of UV-Vis spectrum as well as relatively low midpoint potential (113.2±2.2 mV). Although, physiological function of cytochrome c6B has yet to be determined its properties probably exclude the participation of this protein in electron trafficking between b6f complex and photosystem I.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms.

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000µmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Residues PsaB Asp612 and PsaB Glu613 of photosystem I confer pH-dependent binding of plastocyanin and cytochrome c(6).

The binding and electron transfer between plastocyanin (pc) or cytochrome c(6) (cyt c(6)) and photosystem I (PSI) can be described by hydrophobic as well as electrostatic interactions. The two α helices, l and l' in PsaB and PsaA, respectively, are involved in forming the hydrophobic interaction site at the oxidizing site of PSI. To obtain mechanistic insights into the function of the two negatively charged residues D612 and E613, present in α helix l of PsaB, we exchanged both residues by site-directed mutagenesis with His and transformed a PsaB deficient mutant of Chlamydomonas reinhardtii. Flash-induced absorption spectroscopy revealed that PSI harboring the changes D612H and E613H had a high affinity toward binding of the electron donors and possessed an altered pH dependence of electron transfer with pc and cyt c(6). Despite optimized binding and electron transfer between the altered PSI and its electron donors, the mutant strain PsaB-D612H/E613H exhibited a strong light sensitive growth phenotype, indicating that decelerated turnover between pc/cyt c(6) and PSI with respect to electron transfer is deleterious to the cells.

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A).

Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119.

Most organisms performing oxygenic photosynthesis contain either cytochrome c(6) or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown. In this article, we describe a second cytochrome c(6) (the so called cytochrome c(6)-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c(6A) than to cyanobacterial cytochrome c(6). In this article, we conclude that the cytochrome c(6)-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c(6) and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c(6) and plastocyanin. In any way, the Cyt c(6)-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.

Cloning, expression and purification of the luminal domain of spinach photosystem 1 subunit PsaF functional in binding to plastocyanin and with a disulfide bridge required for folding.

The photosystem 1 subunit PsaF is involved in the docking of the electron-donor proteins plastocyanin and cytochrome c6 in eukaryotic photosynthetic organisms. Here we report the expression, purification and basic characterization of the luminal domain of spinach PsaF, encompassing amino-acid residues 1-79. The recombinant protein was expressed in Escherichia coli BL21 (DE3) using a pET32 Xa/LIC thioredoxin fusion system. The thioredoxin fusion protein contained a His6 tag and was removed and separated from PsaF through proteolytic digestion by factor Xa followed by immobilized metal affinity chromatography. Further purification with size-exclusion chromatography resulted in a final yield of approximately 6 mg PsaF from one liter growth medium. The correct identity after the factor Xa treatment of PsaF was verified by FT-ICR mass spectrometry which also showed that the purified protein contains an intact disulfide bridge between Cys residues 6 and 38. Secondary structure and folding was further explored using far-UV CD spectroscopy indicating a α-helical content in agreement with the 3.3 Å-resolution crystal structure of photosystem I. and a helix-coil transition temperature of 29 °C. Thermofluorescence studies showed that the disulfide bridge is necessary to keep the overall fold of the protein and that hydrophobic regions become exposed at 50-65 °C depending on the ionic strength. The described expression and purification procedure can be used for isotopic labeling of the protein and ¹5N-HSQC NMR studies indicated a slow or intermediate exchange between different conformations of the prepared protein and that it belongs to the molten-globule structural family. Finally, by using a carboxyl- and amine-reactive zero-length crosslinker, we have shown that the recombinant protein binds to plastocyanin by a specific, native-like, electrostatic interaction, hence, confirming its functionality.

Structural and kinetic studies of imidazole binding to two members of the cytochrome c (6) family reveal an important role for a conserved heme pocket residue.

The amino acid at position 51 in the cytochrome c(6) family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c(6) Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe-S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k (obs) for imidazole binding to Arabidopsis thaliana cytochrome c(6A), which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c(6), was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s(-1)). For the cytochrome c(6) Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c(6A) was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c(6) family is additionally responsible for tuning the stability of the heme iron-Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.

Gated electron transfer of cytochrome c6 at biomimetic interfaces: a time-resolved SERR study.

The electron shuttle heme protein Cyt-c(6) from the photosynthetic cyanobacterium Nostoc sp. PCC 7119 was immobilized on nanostructured Ag electrodes coated with SAMs that mimic different possible interactions with its natural reaction partner PSI. The structure, redox potential, and electron-transfer dynamics of the SAM-Cyt-c(6) complexes were investigated by TR-SERR spectroelectrochemistry. It is shown that the heterogeneous electron-transfer process is gated both in electrostatic and hydrophobic-hydrophilic complexes. At long tunneling distances, the reaction rate is controlled by the tunneling probability, while at shorter distances or higher driving forces, protein dynamics becomes the rate-limiting event.

Purification and characterization of cytochrome c(6) from Acaryochloris marina.

Cytochrome c(6), (cyt c(6)) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E(m)) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.

Atomic-resolution structure of reduced cyanobacterial cytochrome c6 with an unusual sequence insertion.

The structure of the reduced form of cytochrome c(6) from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.2 A and refined to an R-factor of 0.107. This protein is unique among all known cytochromes c(6), owing to the presence of an unusual seven-residue insertion, KDGSKSL(44-50), which differs from the insertion found in the recently discovered plant cytochromes c(6A). Furthermore, the present protein is unusual because of its very high content (36%) of the smallest residues (glycine and alanine). The structure reveals that the overall fold of the protein is similar to that of other class I c-type cytochromes, despite the presence of the specific insertion. The insertion is located within the most variable region of the cytochrome c(6) sequence, i.e. between helices II and III. The first six residues [KDGSKS(44-49)] form a loop, whereas the last residue, Leu50, extends the N-terminal beginning of helix III. Several specific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein. The crystal structure contains three copies of the cytochrome c(6) molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupying barely 17.58% of the crystal volume.