A universal coupling mechanism of respiratory complex I.

Complex I is the first enzyme in the respiratory chain, which is responsible for energy production in mitochondria and bacteria1. Complex I couples the transfer of two electrons from NADH to quinone and the translocation of four protons across the membrane2, but the coupling mechanism remains contentious. Here we present cryo-electron microscopy structures of Escherichia coli complex I (EcCI) in different redox states, including catalytic turnover. EcCI exists mostly in the open state, in which the quinone cavity is exposed to the cytosol, allowing access for water molecules, which enable quinone movements. Unlike the mammalian paralogues3, EcCI can convert to the closed state only during turnover, showing that closed and open states are genuine turnover intermediates. The open-to-closed transition results in the tightly engulfed quinone cavity being connected to the central axis of the membrane arm, a source of substrate protons. Consistently, the proportion of the closed state increases with increasing pH. We propose a detailed but straightforward and robust mechanism comprising a 'domino effect' series of proton transfers and electrostatic interactions: the forward wave ('dominoes stacking') primes the pump, and the reverse wave ('dominoes falling') results in the ejection of all pumped protons from the distal subunit NuoL. This mechanism explains why protons exit exclusively from the NuoL subunit and is supported by our mutagenesis data. We contend that this is a universal coupling mechanism of complex I and related enzymes.

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Respiratory complex I with charge symmetry in the membrane arm pumps protons.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, is essential for cellular energy metabolism coupling NADH oxidation to proton translocation. The mechanism of proton translocation by complex I is still under debate. Its membrane arm contains an unusual central axis of polar and charged amino acid residues connecting the quinone binding site with the antiporter-type subunits NuoL, NuoM, and NuoN, proposed to catalyze proton translocation. Quinone chemistry probably causes conformational changes and electrostatic interactions that are propagated through these subunits by a conserved pattern of predominantly lysine, histidine, and glutamate residues. These conserved residues are thought to transfer protons along and across the membrane arm. The distinct charge distribution in the membrane arm is a prerequisite for proton translocation. Remarkably, the central subunit NuoM contains a conserved glutamate residue in a position that is taken by a lysine residue in the two other antiporter-type subunits. It was proposed that this charge asymmetry is essential for proton translocation, as it should enable NuoM to operate asynchronously with NuoL and NuoN. Accordingly, we exchanged the conserved glutamate in NuoM for a lysine residue, introducing charge symmetry in the membrane arm. The stably assembled variant pumps protons across the membrane, but with a diminished H+/e- stoichiometry of 1.5. Thus, charge asymmetry is not essential for proton translocation by complex I, casting doubts on the suggestion of an asynchronous operation of NuoL, NuoM, and NuoN. Furthermore, our data emphasize the importance of a balanced charge distribution in the protein for directional proton transfer.

Structural insights into the action mechanisms of artificial electron acceptors in photosystem II.

Photosystem II (PSII) utilizes light energy to split water, and the electrons extracted from water are transferred to QB, a plastoquinone molecule bound to the D1 subunit of PSII. Many artificial electron acceptors (AEAs) with molecular structures similar to that of plastoquinone can accept electrons from PSII. However, the molecular mechanism by which AEAs act on PSII is unclear. Here, we solved the crystal structure of PSII treated with three different AEAs, 2,5-dibromo-1,4-benzoquinone, 2,6-dichloro-1,4-benzoquinone, and 2-phenyl-1,4-benzoquinone, at 1.95 to 2.10 Å resolution. Our results show that all AEAs substitute for QB and are bound to the QB-binding site (QB site) to receive electrons, but their binding strengths are different, resulting in differences in their efficiencies to accept electrons. The acceptor 2-phenyl-1,4-benzoquinone binds most weakly to the QB site and showed the highest oxygen-evolving activity, implying a reverse relationship between the binding strength and oxygen-evolving activity. In addition, a novel quinone-binding site, designated the QD site, was discovered, which is located in the vicinity of QB site and close to QC site, a binding site reported previously. This QD site is expected to play a role as a channel or a storage site for quinones to be transported to the QB site. These results provide the structural basis for elucidating the actions of AEAs and exchange mechanism of QB in PSII and also provide information for the design of more efficient electron acceptors.

Iron mobilization from intact ferritin: effect of differential redox activity of quinone derivatives with NADH/O2 and in situ-generated ROS.

Ferritins are multimeric nanocage proteins that sequester/concentrate excess of free iron and catalytically synthesize a hydrated ferric oxyhydroxide bio-mineral. Besides functioning as the primary intracellular iron storehouses, these supramolecular assemblies also oversee the controlled release of iron to meet physiologic demands. By virtue of the reducing nature of the cytosol, reductive dissolution of ferritin-iron bio-mineral by physiologic reducing agents might be a probable pathway operating in vivo. Herein, to explore this reductive iron-release pathway, a series of quinone analogs differing in size, position/nature of substituents and redox potentials were employed to relay electrons from physiologic reducing agent, NADH, to the ferritin core. Quinones are well known natural electron/proton mediators capable of facilitating both 1/2 electron transfer processes and have been implicated in iron/nutrient acquisition in plants and energy transduction. Our findings on the structure-reactivity of quinone mediators highlight that iron release from ferritin is dictated by electron-relay capability (dependent on E1/2 values) of quinones, their molecular structure (i.e., the presence of iron-chelation sites and the propensity for H-bonding) and the type/amount of reactive oxygen species (ROS) they generate in situ. Juglone/Plumbagin released maximum iron due to their intermediate E1/2 values, presence of iron chelation sites, the ability to inhibit in situ generation of H2O2 and form intramolecular H-bonding (possibly promotes semiquinone formation). This study may strengthen our understanding of the ferritin-iron-release process and their significance in bioenergetics/O2-based cellular metabolism/toxicity while providing insights on microbial/plant iron acquisition and the dynamic host-pathogen interactions.

A novel photosensitizer based on hypocrellin B activated by cysteine over-expressed in cancer cells.

L-Cysteine (Cys)-activatable photosensitizer 3 was designed and synthesized based on hypocrellin B (1). Cys is a novel tumor-associated biomarker. 3 exhibited negligible photosensitizing ability without Cys. However, when 1 was released from 3 by reaction with Cys, the photosensitizing activity was restored. Furthermore, 3 showed selective and effective photo-cytotoxicity against only cancer cells such as HeLa and A549 cells that highly express Cys when irradiated with 660 nm light, which is inside the phototherapeutic window.

Identifying Quinones in Complex Aqueous Environmental Media (Biochar Extracts) through Tagging with Cysteine and Cysteine-Contained Peptides and High Resolution Mass Spectrometry Analysis.

Quinones are among the most important components in natural organic matter (NOM) for redox reactions; however, no quinones in complex environmental media have been identified. To aid the identification of quinone-containing molecules in ultracomplex environmental samples, we developed a chemical tagging method that makes use of a Michael addition reaction between quinones and thiols (-SH) in cysteine (Cys) and cysteine-contained peptides (CCP). After the tagging, candidates of quinones in representative aqueous environmental samples (water extractions of biochar) were identified through high-resolution mass spectrometry (HRMS) analysis. The MS and UV spectra analysis showed rapid reactions between Cys/CCP and model quinones with ß-carbon from the same benzene ring available for Michael addition. The tagging efficiency was not influenced by other co-occurring nonquinone representative compounds, including caffeic acid, cinnamic acid, and coumaric acid. Cys and CCP were used to tag quinones in water extractions of biochars, and possible candidates of quinones (20 and 53 based on tagging with Cys and CCP, respectively) were identified based on the HRMS features for products of reactions with Cys/CCP. This study has successfully demonstrated that such a Michael addition reaction can be used to tag quinones in complex environmental media and potentially determine their identities. The method will enable an in-depth understanding of the redox chemistry of NOM and its critical chemical compositions and structures.

Deactivation blocks proton pathways in the mitochondrial complex I.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane-bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

Electrochemical Analysis for Total Alkalinity of Water by the Measurement of Cathodic Prepeak of Quinone Caused by Surplus Acid.

In this study, an electrochemical analysis, coupled with the concept of back neutralization titration and the voltammetric determination of surplus acid, is proposed for determining the total alkalinity of water samples. When linear sweep voltammetry of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ) with H2SO4 in a water and ethanol (44 : 56, v/v) mixture was carried out using a bare glassy carbon working electrode, a cathodic prepeak of DBBQ caused by H2SO4 was observed on the voltammogram at a more positive potential than when compared with the original cathodic peak of DBBQ. When similar voltammetry was carried out in the presence of Na2CO3 and H2SO4, the cathodic prepeak height of DBBQ was decreased with an increase in the Na2CO3 concentration. The decrease of the cathodic prepeak height of DBBQ was found to be linearly related to the Na2CO3 concentration ranging from 0.025 to 2.5 mM (r2 = 0.998). The total equivalent concentrations of inorganic bases in samples of mineral water and tap water were determined, and then the results were converted to the total alkalinities of the water samples (mg/L CaCO3). The total alkalinities of the water samples determined by the present electrochemical analysis were essentially the same compared with those by the neutralization titration method. From these results, we were able to demonstrate that the present electrochemical analysis with accuracy and precision could be applied to determine the total alkalinity, which is one of the indicators to examine water quality. The present electrochemical analysis would contribute to achieving the sustainable development goals (SDGs) of #6 and #14.

In Vitro and in Vivo Study of a Photostable Quinone Compound with Enhanced Therapeutic Efficacy against Chagas Disease.

Chagas disease, a neglected tropical disease caused by the protozoan Trypanosoma cruzi poses a significant health challenge in rural areas of Latin America. The current pharmacological options exhibit notable side effects, demand prolonged administration, and display limited efficacy. Consequently, there is an urgent need to develop drugs that are safe and clinically effective. Previously, we identified a quinone compound (designated as compound 2) with potent antiprotozoal activity, based on the chemical structure of komaroviquinone, a natural product renowned for its antitrypanosomal effects. However, compound 2 was demonstrated considerably unstable to light. In this study, we elucidated the structure of the light-induced degradation products of compound 2 and probed the correlation between the quinone ring's substituents and its susceptibility to light. Our findings led to the discovery of quinones with significantly enhanced light stability, some of which exhibiting antitrypanosomal activity. The most promising compound was evaluated for drug efficacy in a mouse model of Chagas disease, revealing where a notable reduction in blood parasitemia.

Further Polycyclic Quinones of Micromonospora sp.

The crude acetone extract of a marine Micromonospora sp. strain associated with Eudistoma vannnamei was fractioned with hexane and ethyl acetate. The crude extract and both soluble fractions were assayed against several bacteria strains. The new polycyclic quinones 12-hydroxy-9-propyltetracene-6,1-dione (1), 5,12-dihydroxy-4-methoxy-9-propyltetracene-5,12-dione (2), and 4,6-dihydroxy-3-methoxycarbonyl- methyl-6a-(oxobutyl)-5,12-anthraquinone (3), along with the known 4,6-dihydroxy-3-methoxycarbonyl-methyl-6a-(oxo-3-methyl-butyl)-5,12-anthraquinone (4) and 4,6-dihydroxy-3-methoxycarbonyl-methyl-6a-(oxopentyl)-5,12-anthraquinone (5) were isolated from the hexane-soluble fraction, while from the active ethyl acetate fraction were isolated the known 4,6,11-trihydroxy-9-propyltetracene-5,12-dione (6), 4-methoxy-9-propyltetracene-6,11-dione (7), 7,8,9,10-tetrahydro-9-hydroxy-4-methoxy-9-propyltetracene-6,11-dione (8), and 10ß-carbomethoxy-7,8,9,10-tetrahydro-4,6,7α,9α,11-pentahydroxy-9-propyltetracene-5,12-dione (9). The structures of the new compounds were established by interpretation of HRMS and NMR techniques. A study of molecular docking was performed with the compounds from the active ethyl acetate fraction to correlate tentatively with the antimicrobial activity. Molecular docking, RMSD, RMSF, and MM-GBSA evaluations were performed to investigate the inhibitory activity of 6-8 against the protein PDB-codex 1MWT, being considered a promising target for studying drug development responsible for inhibiting replication of Staphylococcus aureus. Penicillin G was used as the standard inhibitory. Anthracyclinones 6-8 were the best hydrolase inhibitor with affinity energy -8.1 to -7.9 kcal/mol compared to penicillin G, which presented -6.9 kcal/mol. Both 8 and 7 present potent inhibitory effects against hydrolase through molecular dynamics simulation and exhibit favorable drug-like properties, promising new hydrolase blockers to fight bacterial infections from Staphylococcus aureus.

Gold Nanoparticles-Based Colorimetric Immunoassay of Carcinoembryonic Antigen with Metal-Organic Framework to Load Quinones for Catalytic Oxidation of Cysteine.

This work reported gold nanoparticles (AuNPs)-based colorimetric immunoassay with the Cu-based metal-organic framework (MOF) to load pyrroloquinoline quinone (PQQ) for the catalytic oxidation of cysteine. In this method, both Cu2+ and PQQ in the MOF could promote the oxidation of inducer cysteine by redox cycling, thus limiting the cysteine-induced aggregation of AuNPs and achieving dual signal amplification. Specifically, the recombinant carcinoembryonic antigen (CEA) targets were anchored on the MOF through the metal coordination interactions between the hexahistidine (His6) tag in CEA and the unsaturated Cu2+ sites in MOF. The CEA/PQQ-loaded MOF could be captured by the antibody-coated ELISA plate to catalyze the oxidation of cysteine. However, once the target CEA in the samples bound to the antibody immobilized on the plate surface, the attachment of CEA/PQQ-loaded MOF would be limited. Cysteine remaining in the solution would trigger the aggregation of AuNPs and cause a color change from red to blue. The target concentration was positively related to the aggregation and color change of AuNPs. The signal-on competitive plasmonic immunoassay exhibited a low detection limit with a linear range of 0.01-1 ng/mL. Note that most of the proteins in commercial ELISA kits are recombinant with a His6 tag in the N- or C-terminal, so the work could provide a sensitive plasmonic platform for the detection of biomarkers.

Redox Properties of Bacillus subtilis Ferredoxin:NADP+ Oxidoreductase: Potentiometric Characteristics and Reactions with Pro-Oxidant Xenobiotics.

Bacillus subtilis ferredoxin:NADP+ oxidoreductase (BsFNR) is a thioredoxin reductase-type FNR whose redox properties and reactivity with nonphysiological electron acceptors have been scarcely characterized. On the basis of redox reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of the flavin adenine dinucleotide (FAD) cofactor was estimated to be -0.240 V. Photoreduction using 5-deazaflavin mononucleotide (5-deazaFMN) as a photosensitizer revealed that the difference in the redox potentials between the first and second single-electron transfer steps was 0.024 V. We examined the mechanisms of the reduction of several different groups of non-physiological electron acceptors catalyzed by BsFNR. The reactivity of quinones and aromatic N-oxides toward BsFNR increased when increasing their single-electron reduction midpoint redox potentials. The reactivity of nitroaromatic compounds was lower due to their lower electron self-exchange rate, but it exhibited the same trend. A mixed single- and two-electron reduction reaction was characteristic of quinones, whereas reactions involving nitroaromatics proceeded exclusively via the one-electron reduction reaction. The oxidation of FADH• to FAD is the rate-limiting step during the oxidation of fully reduced FAD. The calculated electron transfer distances in the reaction with nitroaromatics were close to those of other FNRs including the plant-type enzymes, thus demonstrating their similar active site accessibility to low-molecular-weight oxidants despite the fundamental differences in their structures.

Quantification of Quinones in Environmental Media by Chemical Tagging with Cysteine-Containing Peptides Coupled to Size Exclusionary Separation.

Quinones are one of the most important redox-reactive organic compounds in natural environments, such as soil, water, and sediment, playing an important role in regulating the environmental processes and biogeochemical cycles of critical elements under climate change, including the influences of extreme events such as wildfires. However, to date, no existing methods can quantify quinones in complex environmental media. To overcome this challenge, a quantification method was developed by coupling chemical tagging of quinones by cysteine-containing nonaromatic peptides (Cpep) through a Michael addition reaction with size exclusionary chromatography (SEC) separation and ultraviolet (UV) analysis─leveraging on the characteristic absorbance of aromatic rings at 254 nm and molecular size of peptide. The method was demonstrated using model quinones, including 1,4-benzoquinone (BQ), 1,4-naphthoquinone (NQ), and 1,4-anthraquinone (AQ), with a detection limit of 3.3, 0.7, and 0.2 µM, respectively. Concentrations of quinones in water extractions of biochars, soils, and wildfire-derived ashes were determined to range from 0.8 to 14 µM and were positively correlated with their redox reactivity determined by a chemical assay. This method provides a novel rapid quantification of quinones in complex environmental media as well as a quick assessment for redox reactivity and opens up new avenues for studying environmental transformation and remediation of contaminants.

Time-resolved FTIR difference spectroscopy for the study of photosystem I with high potential naphthoquinones incorporated into the A1 binding site 2: Identification of neutral state quinone bands.

Time-resolved step-scan FTIR difference spectroscopy at 77 K has been used to study photosystem I (PSI) from Synechocystis sp. PCC 6803 with four high-potential, 1,4-naphthoquinones (NQs) incorporated into the A1 binding site. The incorporated quinones are 2-chloro-NQ (2ClNQ), 2-bromo-NQ (2BrNQ), 2,3-dichloro-NQ (Cl2NQ), and 2,3-dibromo-NQ (Br2NQ). For completeness 2-methyl-NQ (2MNQ) was also incorporated and studied. Previously, PSI with the same quinones incorporated were studied in the, so-called, anion spectral region between 1550 and 1400 cm-1 (Agarwala et al. in Biochim Biophys Acta 1864(1):148918, 2023). Here we focus on spectra in the previously unexplored 1400-1200 cm-1 spectral region. In this region several bands are identified and assigned to the neutral state of the incorporated quinones. This is important as identification of neutral state quinone bands in the regular 1700-1600 cm-1 region has proven difficult in the past. For neutral PhQ in PSI a broad, intense band appears at ~ 1300 cm-1. For the symmetric di-substituted NQs (Cl2NQ/Br2NQ) a single intense neutral state band is found at ~ 1280/1269 cm-1, respectively. For both mono-substituted NQs, 2ClNQ and 2BrNQ, however, two neutral state bands are observed at ~ 1280 and ~ 1250 cm-1, respectively. These observations from time-resolved spectra agree well with conclusions drawn from absorption spectra of the NQs in THF, which are also presented here. Density functional theory based vibrational frequency calculations were undertaken allowing an identification of the normal modes associated with the neutral state quinone bands.

Organocatalytic Enantioselective Synthesis of Axially Chiral Molecules: Development of Strategies and Skeletons.

The growing importance of axially chiral architectures in different scientific domains has unveiled shortcomings in terms of efficient synthetic access and skeletal variety. This account describes our strategies in answering these challenges within the organocatalytic context where the emergence of bifunctional catalysts such as chiral phosphoric acids (CPAs) has proven invaluable in controlling the sense of axial chirality. The wide occurrence of bi(hetero)aryl skeletons in privileged structures constitutes a strong motivation to devise more effective arylation methods. Our design revolves around modulating the intrinsic nucleophilicity of aromatic amines and alcohols. The first approach involves the design of an electron-withdrawing activating group which could associate with the catalyst for reactivity enhancement and selectivity control. The resonance of arenes offers the unique mechanistic possibility to select between activating sites. C2-Azo- and nitroso-substituted naphthalenes undergo atroposelective ortho C- or N-arylation with (hetero)aromatic nucleophiles. For monocyclic benzenes, programmable charge localization leads to regioselective activation by catalytic control alone or aided by substrate design. For instance, selective addition to nitroso nitrogen enables successive annulation initiated by the amine to yield axially chiral N-arylbenzimidazoles. In a biomimetic manner, a finely tuned catalyst could direct a para-selective nucleophilic approach in the atroposelective arylation of azobenzenes. The second strategy employs electrophilic arene precursors for arylation which occurs via rearomatization with central-to-axial chirality transfer. This enabled the arylation of (imino)quinones with indoles to access phenylindole atropisomers. By adapting this chemistry with an additional oxidation event to liberate the carbonyl functionalities, aryl-o-naphthoquinone and aryl-p-quinone atropisomers were attained. Along with the development of new arylation strategies, deriving new axially chiral structures has been another consistent theme of our research program. The atroposelective functionalization of alkynes provides broad entry to atropisomeric alkenes. The monofunctionalization of alkynes through the interception of an electrophilic vinylidene-quinone-methide (VQM) intermediate with 2-naphthols yielded the new EBINOL scaffolds. By designing an internal directing group, the atroposelective dihalogenation of alkynes was realized using abundant alkali halides despite their weak nucleophilicities and poor solubilities. The atroposelective N-alkylation of alkenes was pursued to prepare multifunctionalized alkene atropisomers that could be converted into 2-arylpyrroles with chirality transfer. The synthesis of B-aryl-1,2-azaborines containing a C-B chiral axis was accomplished where the CPA catalyst effects the desymmetrization and defines the configuration of the distal C-B bond. Inspired by the axially chiral scaffold of allenes, we leveraged the developed arene activation strategy to achieve para-addition and dearomatization of judiciously designed azobenzenes, which led to structurally novel cyclohexadienylidene-based hydrazones. To complement these structures, axially chiral cyclohexadienyl oxime ethers were also attained through CPA-catalyzed condensation between hydroxylamines and spiro[4.5]trienones.

Simple Strategy for Benzo[g]chromene Synthesis via a Photochemical Intramolecular Cyclization Reaction.

Photochemical reactions are often a desirable strategy for organic synthesis because they do not require toxic and expensive reagents and produce less waste than thermal reactions. Herein, a facile photochemical strategy is described to synthesize benzo[g]chromene derivatives. This strategy utilizes the photoredox reaction of quinones, which allows C-H bond oxidation in the vicinity of the photoexcited quinone carbonyl group. The reaction mechanism was investigated using 1H NMR analysis. The intramolecular cyclization reaction proceeded via the formation of 1,3-dioxole compounds as intermediates by the photoredox reaction of p-quinone, followed by re-cyclization. The synthesized benzo[g]chromene derivatives are important heterocyclic skeletons with useful biological and pharmacological properties.

A trick of the tail: computing the entropic contribution to the energetics of quinone-protein unbindung.

We estimate the entropic contributions to the free energy of quinone unbinding in bacterial and mitochondrial respiratory chains using molecular dynamics (MD) and Monte Carlo (MC) computer simulations. For a varying length of the isoprenoid side chain, MD simulations in lipid bilayers and in unpolar solvents are used to assess the dihedral angle distributions along the chain. These form the basis of a MC estimate of the number of molecular structures that do not exhibit steric self-overlap and that are confined to the bilayer. We obtain an entropy drive of TΔS = 1.4 kcal mol-1 for each isoprene unit, which in sum is comparable to the redox potential differences involved in respiratory chain electron transfer. We postulate an entropy-driven zipper for quinone unbinding and discuss it in the context of the bioenergetics and the structure of complex I, and we indicate possible consequences of our findings for MD-based free energy computations.

Probing molecular interactions of semiquinone radicals at quinone reduction sites of cytochrome bc1 by X-band HYSCORE EPR spectroscopy and quantum mechanical calculations.

Quinone redox reactions involve a semiquinone (SQ) intermediate state. The catalytic sites in enzymes stabilize the SQ state via various molecular interactions, such as hydrogen bonding to oxygens of the two carbonyls of the benzoquinone ring. To understand how these interactions contribute to SQ stabilization, we examined SQ in the quinone reduction site (Qi) of cytochrome bc1 using electron paramagnetic resonance (ESEEM, HYSCORE) at the X-band and quantum mechanical (QM) calculations. We compared native enzyme (WT) with a H217R mutant (replacement of histidine that interacts with one carbonyl of the occupant of Qi to arginine) in which the SQ stability has previously been shown to markedly increase. The 14N region of the HYSCORE 2D spectrum for SQi in WT had a shape typical of histidine residue, while in H217R, the spectrum shape changed significantly and appeared similar to the pattern described for SQ liganded natively by arginine in cytochrome bo3. Parametrization of hyperfine and quadrupolar interactions of SQi with surrounding magnetic nuclei (1H, 14N) allowed us to assign specific nitrogens of H217 or R217 as ligands of SQi in WT and H217R, respectively. This was further substantiated by qualitative agreement between the experimental (EPR-derived) and theoretical (QM-derived) parameters. The proton (1H) region of the HYSCORE spectrum in both WT and H217R was very similar and indicative of interactions with two protons, which in view of the QM calculations, were identified as directly involved in the formation of a H-bond with the two carbonyl oxygens of SQ (interaction of H217 or R217 with O4 and D252 with O1). In view of these assignments, we explain how different SQ ligands effectively influence SQ stability. We also propose that the characteristic X-band HYSCORE pattern and parameters of H217R are highly specific to the interaction of SQ with the nitrogen of arginine. These features can thus be considered as potential markers of the interaction of arginine with SQ in other proteins.

Pseudoceranoids A-J, Sesquiterpene-Based Meroterpenoids with Cytotoxicity from the Sponge Pseudoceratina purpurea.

Pseudoceranoid A (1), a rare merosesquiterpene featuring a rearranged 4,9-friedodrimane-type core with a crotonolactone moiety, two new rearranged 4,9-friedodrimane-type sesquiterpene cyclopentanones (2 and 3), and three new rearranged 4,9-friedodrimane-type sesquiterpene hydroquinones (4-6), along with two new drimane-type sesquiterpene derivatives (7 and 8), as well as two new 4,9-friedodrimane-type sesquiterpene quinones (9 and 10), were isolated from the South China Sea sponge Pseudoceratina purpurea. The structures of compounds were established by analysis of spectroscopic data, as well as by single-crystal X-ray diffraction, DP4+ probability analyses, and calculated electronic circular dichroism. Compound 4 showed weak cytotoxicity against K562, H69AR, and MDAMB-231 cell lines with IC50 values of 3.01, 7.74, and 9.82 µM, respectively. Compound 5 exhibited cytotoxicity against the H69AR cell line with an IC50 value of 2.85 µM.