Panida Surawatanawong

Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer

Understanding how flavin-dependent enzymes activate oxygen for their oxidation and oxygenation reactions is one of the most challenging issues in flavoenzymology. Density functional calculations and transient kinetics were performed to investigate the mechanism of oxygen activation in the oxygenase component (C2) of p-hydroxyphenylacetate 3-hydroxylase (HPAH). We found that the protonation of dioxygen by His396 via a proton-coupled electron transfer mechanism is the key step in the formation of the triplet diradical complex of flavin semiquinone and (•)OOH. This complex undergoes intersystem crossing to form the open-shell singlet diradical complex before it forms the closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH). Notably, density functional calculations indicated that the formation of C4aOOH is nearly barrierless, possibly facilitated by the active site arrangement in which His396 positions the proximal oxygen of the (•)OOH in an optimum position to directly attack the C4a atom of the isoalloxazine ring. The nearly barrierless formation of C4aOOH agrees well with the experimental results; based on transient kinetics and Eyring plot analyses, the enthalpy of activation for the formation of C4aOOH is only 1.4 kcal/mol and the formation of C4aOOH by C2 is fast (∼10(6) M(-1) s(-1) at 4 °C). The calculations identified Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen bond from the H(N5) of the isoalloxazine ring. Both Ser171 and Trp112 facilitate H2O2 elimination by donating hydrogen bonds to the proximal oxygen of the OOH moiety during the proton transfer. According to our combined theoretical and experimental studies, the existence of a positively charged general acid at the position optimized for facilitating the proton-coupled electron transfer has emerged as an important catalytic feature for the oxygen activation process in flavin-dependent enzymes.

Proton-coupled electron transfer and adduct configuration are important for C4a-hydroperoxyflavin formation and stabilization in a flavoenzyme

Determination of the mechanism of dioxygen activation by flavoenzymes remains one of the most challenging problems in flavoenzymology for which the underlying theoretical basis is not well understood. Here, the reaction of reduced flavin and dioxygen catalyzed by pyranose 2-oxidase (P2O), a flavoenzyme oxidase that is unique in its formation of C4a-hydroperoxyflavin, was investigated by density functional calculations, transient kinetics, and site-directed mutagenesis. Based on work from the 1970s-1980s, the current understanding of the dioxygen activation process in flavoenzymes is believed to involve electron transfer from flavin to dioxygen and subsequent proton transfer to form C4a-hydroperoxyflavin. Our findings suggest that the first step of the P2O reaction is a single electron transfer coupled with a proton transfer from the conserved residue, His548. In fact, proton transfer enhances the electron acceptor ability of dioxygen. The resulting ·OOH of the open-shell diradical pair is placed in an optimal position for the formation of C4a-hydroperoxyflavin. Furthermore, the C4a-hydroperoxyflavin is stabilized by the side chains of Thr169, His548, and Asn593 in a face-on configuration where it can undergo a unimolecular reaction to generate H2O2 and oxidized flavin. The computational results are consistent with kinetic studies of variant forms of P2O altered at residues Thr169, His548, and Asn593, and kinetic isotope effects and pH-dependence studies of the wild-type enzyme. In addition, the calculated energy barrier is in agreement with the experimental enthalpy barrier obtained from Eyring plots. This work revealed new insights into the reaction of reduced flavin with dioxygen, demonstrating that the positively charged residue (His548) plays a significant role in catalysis by providing a proton for a proton-coupled electron transfer in dioxygen activation. The interaction around the N5-position of the C4a-hydroperoxyflavin is important for dictating the stability of the intermediate.

Density Functional Study of the Thermodynamics of Hydrogen Production by Tetrairon Hexathiolate, Fe4[MeC(CH2S)3]2(CO)8, a Hydrogenase Model

The tetranuclear iron complex Fe(4)[MeC(CH(2)S)(3)](2)(CO)(8) (1) functions like a hydrogenase to catalyze proton reduction to H(2) in the presence of 2,6-dimethylpyridinium acid (LutH(+)). Experimentally, at the first reduction potential (-1.22 V vs Fc/Fc(+)), the concentration of LutH(+) decreases slowly, while at the second reduction potential, which is sufficient to reduce 1(-) (-1.58 V vs Fc/Fc(+)), the concentration of LutH(+) decreases more rapidly. Here, density functional theory predicts both reduction potentials (E(0)) and proton-transfer free energies relative to LutH(+) for numerous intermediates and several important transition states as a basis for developing thermodynamics cycles for routes to hydrogen production by 1. At the less negative potential, one-electron reduction of 1 is followed by protonation to form a bridging hydride complex; then, a second one-electron reduction is followed by a second protonation, an ECEC process. This doubly reduced and doubly protonated species has a structure with bridging hydrides between both outer Fe-Fe pairs and can produce H(2) and regenerate 1 only by bringing the two hydrogens into proximity through a high-energy intermediate or transition state, a result consistent with the experimentally slow uptake of LutH(+) at this potential. In contrast, at the more negative (lower) reduction potential the two-electron-reduced species, 1(2-), which has bridging carbonyls between both Fe-Fe pairs, is protonated at a terminal Fe position to form a species that produces H(2) by rapidly picking up a second proton and regenerating 1 in an EECC process. Thus, the latter route avoids the high-energy intermediates and transition states necessarily accessed by the former route, a result that explains the more rapid uptake of LutH(+) at the second more negative potential. Although both routes arrive at a doubly reduced, singly protonated species in the third step of these processes, the calculations predict that a high barrier prevents the rapid interconversion of these two nearly isoenergetic species. The calculations confirm the importance of terminal metal hydrides, rather than bridging hydrides, for rapid H(2) production and show in detail how the bridging CO maintains the terminal hydride structure at the lower reduction potential even though the bridging hydride conformation is more stable. These results provide clues for designing new biomimic electrocatalysts and further evidence for the terminal Fe-H mechanism in [FeFe]-hydrogenase. The calculations also predict that at even lower reduction potentials, new more highly reduced intermediate species can be accessed that could lead to alternative routes to H(2).

Electronic Structures and Spectroscopy of the Electron Transfer Series [Fe(NO)L2]z (z = 1+, 0, 1–, 2–,3–; L = Dithiolene)

The electronic structures and spectroscopic parameters for the electron transfer series of [Fe(NO)(L)(2)](z) (z = 1+, 0, 1-, 2-, 3-; L = S(2)C(2)R(2); R = p-tolyl (1) and CN (2)) were calculated and compared to experiment. Some compounds in the series were isolated and characterized by spectroscopy. The calculations support the notion that all the monocation (S(t) = 0), neutral (S(t) = ½), and monoanion (S(t) = 0) complexes contain NO(+) (S(NO) = 0), in which the redox active fragment is either the bis-dithiolene (2 L) or the central iron. The calculated electronic structures give insight into how p-tolyl and CN substituents and the redox states of the 2 L ligand impact the spin density on the iron in the monocation and neutral species. The electronic structure of [1](0) has some [Fe(I)(NO(+))(L(2)(2-))](0) character in resonance with [Fe(II)(NO(+))(L(2)(2-))](0) whereas [2](0) has a smaller amount of a [Fe(I)(NO(+))(L(2)(2-))](0) description in its ground state wavefunction. Similarly, the electronic structure of [1](1+) also has some [Fe(I)(NO(+))(L(2)(1-))](1+) character in resonance with [Fe(II)(NO(+))(L(2)(2-))](1+) whereas [2](1+) is best described as [Fe(II)(NO(+))(L(•))(2)](1+). For the monoanion, the bis-dithiolene fragment is fully reduced and both [1](-) and [2](-) are best formulated as [Fe(II)(NO(+))(L(2)(4-))](-). The reduction of the monoanion to give dianions [1](2-) and [2](2-) results in {FeNO}(7) species. The calculated (57)Fe isomer shift and hyperfine couplings are in line with the experiment and support a description of the form [Fe(III)(NO(-))(L(2)(4-))](2-), in which Fe(III) S(Fe) = (3)/(2) is antiferromagnetically coupled to NO(-) (S(NO) = 1). Finally, the calculated redox potential and ν(NO) frequency for the {FeNO}(8) trianionic species [2](3-) is in agreement with experiment and consistent with a triplet ground state [Fe(II)(NO(-))(L(2)(4-))](3-), in which Fe(II) (S(Fe) = 2) is involved in antiferromagnetic coupling with NO(-) (S(NO) = 1).

Pyridine–triazole ligands for copper-catalyzed aerobic alcohol oxidation

A series of Cu(NN′)2(OTf)2 complexes containing pyridine–triazole ligands [OTf = OSO2CF3; NN′ = NN′Ph (1), NN′hex (2), NN′py (3)] with different substituents at the triazole N4 position or 2,2′-bipyridine (bpy; 4) have been synthesized. Crystal structures of 1 and 3 reveal a trans-isomer with strong preference for regular-type triazole coordination (for 3) whereas the Cu–bipyridine complex 4 is more stable in a cis-form. Cyclic voltammetry of 1–4 suggest that the electron-donating strength follows the trend: bpy > NN′py > NN′hex ∼ NN′Ph. The catalyst systems consisting of 5 mol% Cu(OTf)2/NN′/TEMPO (TEMPO = (2,2,6,6-tetramethylpiperidin-1-yl)oxy) in the presence of 2 × 2.0 cm2 Cu0 sheets as a reducing agent and 10 mol% N-methylimidazole (NMI) exhibit good activities for aerobic oxidation of benzyl alcohol to benzaldehyde. Catalytic studies have shown that the activities were higher with more electron-rich N-based ligands. Furthermore, oxidation of aliphatic alcohols such as 1-hexanol and 2-methyl-1-pentanol using the Cu catalyst system with the NN′py ligand at room temperature afforded the corresponding aldehydes in >99% and 46% yields, respectively after 24 h.

Electrophilic difluoro(phenylthio)methylation: generation, stability, and reactivity of α-fluorocarbocations.

Electrophilic difluoro(phenylthio)methylation of allylsilanes has been achieved using bromodifluoro(phenylthio)methane (PhSCF2Br) and silver hexafluoroantimonate (AgSbF6). The structural assignment and observation of α-fluorocarbocation were substantiated by NMR and theoretical calculations. Detailed mechanistic and electronic studies have provided a fundamental understanding of the reactivity and stability of the difluoro(phenylthio)methylium cation (PhSCF2(+)).

Flavans from Desmos cochinchinensis as potent aromatase inhibitors

Flavans from the roots of Desmos cochinchinensis exhibited potent aromatase inhibitory activity at nanomolar levels, and could be leads for the development of anti-aromatase drugs. In addition, these aromatase inhibitors did not show pronounced cytotoxic activity. Flavans exert their inhibitory activity through binding, as revealed by molecular docking studies, with aromatase at Arg115, Met374, and Leu477; the C-7 hydroxyl (or methoxyl) forms hydrogen bonds with Met374 and Arg115 of aromatase.

Polycyclic polyprenylated acylphloroglucinols and biphenyl derivatives from the roots of Garcinia nuntasaenii Ngerns. & Suddee

Seven previously undescribed compounds, including three polycyclic polyprenylated acylphloroglucinols (garcinuntins A-C), three biphenyl derivatives (garcinuntabiphenyls A-C) and a lanostane triterpene (garcinuntine), along with thirteen known compounds were isolated from the root of Garcinia nuntasaenii Ngerns. & Suddee. Their structures were elucidated on the basis of spectroscopic techniques. For garcinuntins A-C, the absolute configurations were confirmed by the combination of single X-ray crystallography and ECD calculations. Anti-HIV activity using anti-HIV-1 reverse transcriptase and syncytium inhibition assays, and cytotoxic activity against a panel of cultured mammalian cancer cell lines of isolated compounds were investigated.