Formation of the early photoproduct lumi-R of cyanobacterial phytochrome cph1 observed by ultrafast mid-infrared spectroscopy

The photoreactions of the Pr ground state of cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 have been investigated by picosecond time-resolved mid-infrared spectroscopy at ambient temperature. With femtosecond excitation of the Pr state at 640 nm, the photoisomerized Lumi-R product state is generated with kinetics and associated difference spectra indicative of vibrational cooling with tau(1) = 3 ps time constant and excited state decay with tau(1) = 3 ps, tau(2) = 14 ps, and tau(3) = 134 ps time constants. The Lumi-R state is characterized by downshifted absorption of three C=C modes assigned to C(15)=C(16), C(4)=C(6), and a delocalized C=C mode, in addition to the downshifted C(19)=O mode. The Lumi-R minus Pr difference spectrum is indicative of global restructuring of the chromophore on the ultrafast timescale, which is discussed in light of C(15) Z/E photoisomerization in addition to changes near C(5), which could be low bond order torsional angle changes.     

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

Photoisomerization of a protein‐bound chromophore is the basis of light sensing and signaling in many photoreceptors. Phytochrome photoreceptors can be photoconverted reversibly between the Pr and Pfr states through photoisomerization of the methine bridge between rings C and D. Ground‐state heterogeneity of the chromophore has been reported for both Pr and Pfr. Here, we report ultrafast visible (Vis) pump–probe and femtosecond polarization‐resolved Vis pump–infrared (IR) probe studies of the Pfr photoreaction in native and ¹³C/¹⁵N‐labeled Cph1 phytochrome with unlabeled PCB chromophore, demonstrating different S₀ substates, Pfr‐I and Pfr‐II, with distinct IR absorptions, orientations and dynamics of the carbonyl vibration of ring D. We derived time constants of 0.24 ps, 0.7 ps and 6 ps, describing the complete initial photoreaction. We identified an isomerizing pathway with 0.7 ps for Pfr‐I, and silent dynamics with 6 ps for Pfr‐II. We discuss different origins of the Pfr substates, and favor different facial orientations of ring D. The model provides a quantum yield for Pfr‐I of 38%, in line with ~35% ring D rotation in the electronic excited state. We tentatively assign the silent form Pfr‐II to a dark‐adapted state that can convert to Pfr‐I upon light absorption.     

Time-resolved IR spectroscopy of N-methylthioacetamide: trans-->cis isomerization upon n-pi and pi-pi excitation and cis-->trans photoreaction

Time-resolved infrared spectroscopy was used to study the photoisomerization of N-Methylthioacetamide (NMTAA) in D2O in both the cis-->trans and the trans-->cis direction upon selective excitation of the n-pi (S1) and pi-pi (S2) electronic transitions. While isomerization and the return to the ground state takes place on two distinct time scales (cis isomerization is 30-40%, independent of the electronic state excited, while the cis-->trans isomerization proceeds with a 60-70% quantum efficiency. These results support a mechanism by which isomerization takes place via one common intermediate state independent of electronic excitation energy and initial conformation.     

Relative ground and excited-state pKa values of phytochromobilin in the photoactivation of phytochrome: a computational study

The conversion of the plant photoreceptor phytochrome from an inactive (Pr) to an active form (Pfr) is accomplished by a red-light induced Z --> E photoisomerization of its phytochromobilin chromophore. In recent years, the question whether the photoactivation involves a change in chromophore protonation state has been the subject of many experimental studies. Here, we have used quantum chemical methods to calculate relative ground and excited-state pKa values of the different pyrrole moieties of phytochromobilin in a protein-like environment. Assuming (based on experimental data) a Pr ZaZsZa chromophore and considering isomerizations at C15 and C5, it is found that moieties B and C are the strongest acids both in the ground state and in the bright first singlet excited state, which is rationalized in simple geometric and electronic terms. It is also shown that neither light absorption nor isomerization increases the acidity of phytochromobilin relative to the reference Pr state with all pyrrolenic nitrogens protonated. Hence, provided that the subset of chromophore geometries under investigation is biologically relevant, there appears to be no intrinsic driving force for a proton-transfer event. In a series of benchmark calculations, the performance of ab initio and time-dependent density functional theory methods for excited-state studies of phytochromobilin is evaluated in light of available experimental data.     

Photoisomerization of disperse red 1 studied with transient absorption spectroscopy and quantum chemical calculations

The photoisomerization of the push-pull substituted azo dye Disperse Red 1 is studied using femtosecond time-resolved absorption spectroscopy and other spectroscopic and computational techniques. In comparison with azobenzene, the pipi* state is more stabilized by the effects of push-pull substitution than the npi* state, but the latter is probably still the lowest in energy. This conclusion is based on the kinetics, anisotropy of the excited state absorption spectrum, the spectra of the ground states, and quantum chemical calculations. The S(1)(npi*) state is formed from the initially excited pipi* state in <0.2 ps, and decays to the ground state with time constants of 0.9 ps in toluene, 0.5 ps in acetonitrile, and 1.4 ps in ethylene glycol. Thermal isomerization transforms the Z isomer produced to the more stable E isomer with time constants of 29 s (toluene), 28 ms (acetonitrile), and 2.7 ms (ethylene glycol). The pathway of photoisomerization is likely to be rotation about the N=N bond. Quantum chemical calculations indicate that along the inversion pathway ground and excited state energy surfaces remain well separated, whereas rotation leads to a region where conical intersections can occur. For the ground-state Z to E isomerization, conclusive evidence is lacking, but inversion is more probably the favored pathway in the push-pull substituted systems than in the parent azobenzene.     

Femtosecond Spectroscopic Studies on the Red Light-Absorbing Form of Oat Phytochrome and 2,3-Dihydrobiliverdin

Abstract— The excited state behavior of the red light-absorbing form of phytochrome (Pr) was studied on the femtosecond time scale. After excitation of Pr with 75 fs laser pulses at 616 nm the kinetics of the transient absorption changes was recorded at selected wavelengths probing mainly the bleaching of the Pr ground-state absorption and the stimulated emission. The kinetic data obtained indicate the population of an excited state with a 3 ps lifetime immediately after excitation. This state precedes the formation of another excited state with a 32 ps lifetime. The decay of the latter state is followed by the appearance of a first product state that is assumed to represent lunii-R. In addition, 2,3-dihydrobiliverdin, which is considered to be an adequate model of the Pr chro-mophore, was included in the femtosecond studies. The absorption difference spectra recorded at various delay times show an immediate bleaching of the ground-state absorption. Simultaneously with bleaching a broad transient absorption appears between 410 and 525 nm. The data analysis yields similar kinetic components as they were observed in the decay of Pr. It is suggested from this finding that within the first tens of picoseconds after excitation the excited-state properties of Pr are mainly determined by the properties of the chromophore itself.     

Primary reactions of bacteriophytochrome observed with ultrafast mid-infrared spectroscopy

Phytochromes are red-light photoreceptor proteins that regulate a variety of responses and cellular processes in plants, bacteria, and fungi. The phytochrome light activation mechanism involves isomerization around the C(15)═C(16) double bond of an open-chain tetrapyrrole chromophore, resulting in a flip of its D-ring. In an important recent development, bacteriophytochrome (Bph) has been engineered for use as a fluorescent marker in mammalian tissues. Bphs covalently bind a biliverdin (BV) chromophore, naturally abundant in mammalian cells. Here, we report an ultrafast time-resolved mid-infrared spectroscopic study on the Pr state of two highly related Bphs from Rps. palustris , RpBphP2 (P2) and RpBphP3 (P3) with distinct photoconversion and fluorescence properties. We observed that the BV excited state of P2 decays in 58 ps, while the BV excited state of P3 decays in 362 ps. By combining ultrafast mid-IR spectroscopy with FTIR spectroscopy on P2 and P3 wild type and mutant proteins, we demonstrate that the hydrogen bond strength at the ring D carbonyl of the BV chromophore is significantly stronger in P3 as compared to P2. This result is consistent with the X-ray structures of Bph, which indicate one hydrogen bond from a conserved histidine to the BV ring D carbonyl for classical bacteriophytochromes such as P2, and one or two additional hydrogen bonds from a serine and a lysine side chain to the BV ring D carbonyl for P3. We conclude that the hydrogen-bond strength at BV ring D is a key determinant of excited-state lifetime and fluorescence quantum yield. Excited-state decay is followed by the formation of a primary intermediate that does not decay on the nanosecond time scale of the experiment, which shows a narrow absorption band at ～1540 cm(-1). Possible origins of this product band are discussed. This work may aid in rational structure- and mechanism-based conversion of BPh into an efficient near-IR fluorescent marker.     

Transient absorption studies of vibrational relaxation and photophysics of Prussian blue and ruthenium purple nanoparticles

Transient infrared and visible absorption studies have been used to characterize vibrational and electronic dynamics of Prussian blue (PB) and ruthenium purple (RP) nanoparticles produced and characterized in AOT reverse micelles. Studies include excitation and probing with both infrared (near 2000 cm(-1)) and visible (800 nm) pulses. From IR pump-IR probe measurements of the CN stretching bands, vibrational population lifetimes are determined to be 32 ± 4 ps for PB and 44 ± 14 ps for RP. These times are longer than those for ferrocyanide (4 ps) and ruthenocyanide (4 ps) in normal water and are closer to the times for these species in heavy water (25-30 ps) and for ferrocyanide in formamide (43 ps). The PB and RP lifetimes are also longer than those (<15 ps) previously measured for CN stretching bands following intervalence excitation and back-electron transfer (BET) for dinuclear mixed-valence compounds containing Fe, Ru, and Os in heavy water and formamide [A. V. Tivansky, C. F. Wang, and G. C. Walker, J. Phys. Chem. A 107, 9051 (2003)]. In 800 nm pump-IR probe experiments on RP and PB, transient IR spectra and decay curves are similar to those with IR excitation; a ground state bleach and a red shifted (by ~40 cm(-1)) excited state decay are observed. These results for the visible pumping are consistent with rapid (<1 ps) BET resulting in population in the ground electronic state with vibrational excitation in the CN mode. In addition, transient absorption studies were performed for PB and RP probing with visible light using both visible and IR excitation. The early time response for the 800 nm pump-800 nm probe of PB exhibits an instrument-limited, subpicosecond bleach followed by an absorption, which is consistent with the previously reported results [D. C. Arnett, P. Vohringer, and N. F. Scherer, J. Am. Chem. Soc. 117, 12262 (1995)]. The absorption exhibits a biexponential decay with decay times of 9 and 185 ps, which could have been attributed to the CN band excitation indicated from 800 pump-IR probe results. However, IR pump-800 nm probe studies reveal that excitation of the CN band directly results in a decreased visible absorption that decays in 18 ps rather than an induced absorption at 800 nm. Characteristics of the IR pump-800 nm probe, especially that it induces a bleach instead of an absorption, unambiguously indicate that the transient absorption from the 800 nm pump-800 nm probe is dominated by states other than the CN bands and is attributed to population in other, probably lower frequency, vibrational modes excited following visible excitation and rapid BET.     

Dual photochemistry of anthracene-9,10-endoperoxide studied by femtosecond spectroscopy

The dual photochemistry of anthracene-9,10-endoperoxide (APO) was investigated in a fs UV pump-supercontinuum probe experiment, along with anthracene (AC) and anthraquinone (AQ) for comparison. Excitation of APO at 282 nm leads to 100% product formation by two competing photoreaction channels. Cycloreversion generates with a ～25% quantum yield (QY) (1)O(2) and AC vibrationally excited in the singlet electronic ground state (hot AC). 1-2% of the AC is generated in the lowest triplet state, but no AC is generated in electronically excited singlet states. Generation and cooling of hot AC are modeled using solution phase and broadened gas-phase AC absorption spectra at various temperatures. Results indicate ultrafast generation of hot AC within 3 ps, much faster than reported before for derivatives of anthracene endoperoxide, and subsequent cooling with an 18 ps time constant. The homolytic O-O cleavage pathway generates a biradical, which converts into electronically excited diepoxide (DE). Our data indicate a 1.5 ps time constant that we tentatively assign to the biradical decay and DE formation. Cooling of DE in this electronically excited state takes place with a ～21 ps time constant. Excitation of AQ at 266 nm is followed by an ultrafast population of the T(1)(nπ*) triplet state of AQ with a time constant of (160 ± 60) fs.     

Characterization of hemicyanine Langmuir-Blodgett films by picosecond time-resolved fluorescence

Hemicyanine Langmuir-Blodgett films have been elaborated and characterized using stationary and time-resolved spectroscopic techniques. Depending on the experimental conditions, especially the pH of the water subphase, the absorption spectra of the films indicate the presence of non-fluorescent H-aggregates in the monolayer. Time-resolved fluorescence measurements revealed three mono-exponential decay times: a very short one (20-23 ps) attributed to an excited intramolecular charge transfer state and two longer ones (100-120 ps and 400-590 ps) attributed to the photoisomerization of the chromophores.     

Ultrafast Decay Dynamics of N-Ethylpyrrole Excited to the S1 Electronic State: A Femtosecond Time-Resolved Photoelectron Imaging Study

N-ethylpyrrole is one of ethyl-substituted derivatives of pyrrole and its excited-state decay dynamics has never been explored. In this work, we investigate ultrafast decay dynamics of N-ethylpyrrole excited to the S₁ electronic state using a femtosecond time-resolved photoelectron imaging method. Two pump wavelengths of 241.9 and 237.7 nm are employed. At 241.9 nm, three time constants, 5.0±0.7 ps, 66.4±15.6 ps and 1.3±0.1 ns, are derived. For 237.7 nm, two time constants of 2.1±0.1 ps and 13.1±1.2 ps are derived. We assign all these time constants to be associated with different vibrational states in the S₁ state. The possible decay mechanisms of different S₁ vibrational states are briefly discussed.     

Photochromic control of photoinduced electron transfer. Molecular double-throw switch

A molecular double-throw switch that employs a photochromic moiety to direct photoinduced electron transfer from an excited state donor down either of two pathways has been prepared. The molecular triad consists of a free base porphyrin (P) linked to both a C(60) electron acceptor and a dihydroindolizine (DHI) photochrome. Excitation of the porphyrin moiety of DHI-P-C(60) results in photoinduced electron transfer with a time constant of 2.3 ns to give the DHI-P(*)(+)-C(60)(*)(-) charge-separated state with a quantum yield of 82%. UV (366 nm) light photoisomerizes the DHI moiety to the betaine (BT) form, which has a higher reduction potential than DHI. Excitation of the porphyrin of BT-P-C(60) is followed by photoinduced electron transfer with a time constant of 56 ps to produce BT(*)(-)-P(*)(+)-C(60) in 99% yield. Isomerization of BT-P-C(60) back to DHI-P-C(60) may be achieved with visible light, or thermally. Thus, photoinduced charge separation originating from the porphyrin is reversibly directed down either of two different pathways by photoisomerization of the dihydroindolizine. The switch may be cycled many times.     

Excited state intramolecular proton transfer in Schiff bases. Decay of the locally excited enol state observed by femtosecond resolved fluorescence

Although the late (t>1 ps) photoisomerization steps in Schiff bases have been described in good detail, some aspects of the ultrafast (sub-100 fs) proton transfer process, including the possible existence of an energy barrier, still require experimental assessment. In this contribution we present femtosecond fluorescence up-conversion studies to characterize the excited state enol to cis-keto tautomerization through measurements of the transient molecular emission. Salicylideneaniline and salicylidene-1-naphthylamine were examined in acetonitrile solutions. We have resolved sub-100 fs and sub-0.5 ps emission components which are attributed to the decay of the locally excited enol form and to vibrationally excited states as they transit to the relaxed cis-keto species in the first electronically excited state. From the early spectral evolution, the lack of a deuterium isotope effect, and the kinetics measured with different amounts of excess vibrational energy, it is concluded that the intramolecular proton transfer in the S1 surface occurs as a barrierless process where the initial wave packet evolves in a repulsive potential toward the cis-keto form in a time scale of about 50 fs. The absence of an energy barrier suggests the participation of normal modes which modulate the donor to acceptor distance, thus reducing the potential energy during the intramolecular proton transfer.     

Fast intramolecular charge transfer with a planar rigidized electron donor/acceptor molecule

The planar rigidized molecule fluorazene (FPP) undergoes fast reversible intramolecular charge transfer (ICT) in the excited state, with a reaction time of 12 ps in the polar solvent ethyl cyanide at -45 degrees C. The ICT state of FPP has a dipole moment mu(e)(ICT) of 13 D, much larger than that of the locally excited state LE (1 D). The ICT behavior of FPP is similar to that of its flexible counterpart N-phenylpyrrole (PP), for which mu(e)(ICT) = 12 D. These results show that intramolecular charge transfer to a planar ICT state can occur efficiently. In designing ICT systems capable of rapid switching, it is therefore important to realize that large amplitude motions such as those necessary for the formation of a twisted intramolecular charge transfer (TICT) state are not required.     

Ligand-Localized Triplet-State Photophysics in a Platinum(II) Terpyridyl Perylenediimideacetylide

The synthesis, electrochemistry, and photophysical behavior of a Pt(II) terpyridyl perylenediimide (PDI) acetylide (1) charge-transfer complex is reported. The title compound exhibits strong (ε ≈ 5 × 10(4) M(-1)cm(-1)) low-energy PDI acetylide-based π-π* absorption bands in the visible range extending to 600 nm, producing highly quenched singlet fluorescence (Φ = 0.014 ± 0.001, τ = 109 ps) with respect to a nonmetalated PDI model chromophore. Nanosecond transient absorption spectroscopy revealed the presence of a long excited-state lifetime (372 ns in 2-methyltetrahydrofuran) with transient features consistent with the PDI-acetylide triplet state, ascertained by direct comparison to a model Pt(II) PDI-acetylide complex lacking low-energy charge-transfer transitions. For the first time, time-resolved step-scan FT-IR spectroscopy was used to characterize the triplet excited state of the PDI-acetylide sensitized in the title compound and its associated model complex. The observed red shifts (～30-50 cm(-1)) in the C═O and C≡C vibrations of the two Pt(II) complexes in the long-lived excited state are consistent with formation of the (3)PDI acetylide state and found to be in excellent agreement with the expected change in the relevant DFT-calculated IR frequencies in the nonmetalated PDI model chromophore (ground singlet state and lowest triplet excited state). Formation of the PDI triplet excited state in the title chromophore was also supported by sensitization of the singlet oxygen photoluminescence centered at ～1275 nm in air-saturated acetonitrile solution, Φ((1)O(2)) = 0.52. In terms of light emission, only residual PDI-based red fluorescence could be detected and no corresponding PDI-based phosphorescence was observed in the visible or NIR region at 298 or 77 K in the Pt(II) terpyridyl perylenediimideacetylide.     

Picosecond IR-UV pump-probe spectroscopic study of the dynamics of the vibrational relaxation of jet-cooled phenol. I. Intramolecular vibrational energy redistribution of the OH and CH stretching vibrations of bare phenol

The intramolecular vibrational energy redistribution (IVR) of the OH stretching vibration of jet-cooled phenol-h6 (C6H8OH) and phenol-d8 (C6D8OH) in the electronic ground state has been investigated by picosecond time-resolved IR-UV pump-probe spectroscopy. The OH stretching vibration of phenol was excited with a picosecond IR laser pulse, and the subsequent temporal evolutions of the initially excited level and the redistributed ones due to the IVR were observed by multiphoton ionization detection with a picosecond UV pulse. The IVR lifetime for the OH stretch vibration of phenol-h6 was determined to be 14 ps, while that of the OH stretch for phenol-d8 was found to be 80 ps. This remarkable change of the IVR rate constant upon the dueteration of the CH groups strongly suggests that the "doorway states" for the IVR from the OH level would be the vibrational states involving the CH stretching modes. We also investigated the IVR rate of the CH stretching vibration for phenol-h6. It was found that the IVR lifetime of the CH stretch is less than 5 ps. The fast IVR is described by the strong anharmonic resonance of the CH stretch with many other combinations or overtone bands.     

Ultrafast electronic and vibrational energy relaxation of Fe(acetylacetonate)3 in solution

Transient mid-infrared spectroscopy is used to probe the dynamics initiated by excitation of ligand-to-metal (400 nm) and metal-to-ligand (345 nm) charge transfer states of FeIII complexed with acetylacetonate (Fe(acac)3, where acac stands for deprotonated anion of acetylacetone) in solution. Transient spectra in the 1500-1600 cm-1 range show two broad absorptions red-shifted from the bleach of the nu(CO) (approximately 1575 cm-1) and nu(C=C) (approximately 1525 cm-1) ground state absorptions. Bleach recovery kinetics has a time constant of 12-19 ps in chloroform and tetrachloroethylene and it decreases by 30-40% in a 10% mixture of methanol in tetrachloroethylene. The transient absorptions experience band narrowing simultaneously with blue-shifting of the absorption maxima. Both phenomena have time constants of 3-9 ps with no evident dependence on the solvent. The experimental observations are ascribed to fast conversion of the initially excited charge transfer states to the ligand field manifold, and subsequent vibrational cooling on the lowest ligand field excited state prior to electronic conversion to the ground state. The analysis of time dependent bandwidths and positions of the transient absorptions provides some evidence of mode specific vibrational cooling.     

Steric Effects Govern the Photoactivation of Phytochromes

Phytochromes constitute a superfamily of photoreceptor proteins existing in two forms that absorb red (Pr) and far‐red (Pfr) light. Although it is well‐known that the conversion of Pr into Pfr (the biologically active form) is triggered by a Z→E photoisomerization of the linear tetrapyrrole chromophore, direct evidence is scarce as to why this reaction always occurs at the methine bridge between pyrrole rings C and D. Here, we present hybrid quantum mechanics/molecular mechanics calculations based on a high‐resolution Pr crystal structure of Deinococcus radiodurans bacteriophytochrome to investigate the competition between all possible photoisomerizations at the three different (AB, BC and CD) methine bridges. The results demonstrate that steric interactions with the protein are a key discriminator between the different reaction channels. In particular, it is found that such interactions render photoisomerizations at the AB and BC bridges much less probable than photoisomerization at the CD bridge.     

Solid‐State NMR Spectroscopy to Probe Photoactivation in Canonical Phytochromes

The photoreceptor phytochrome switches photochromically between two thermally stable states called Pr and Pfr. Here, we summarize recent solid‐state magic‐angle spinning (MAS) NMR work on this conversion process and interpret the functional mechanism in terms of a nano‐machine. The process is initiated by a double‐bond photoisomerization of the open‐chain tetrapyrrole chromophore at the methine bridge connecting pyrrole rings C and D. The Pr‐state chromophore and its surrounding pocket in canonical cyanobacterial and plant phytochromes has significantly less order, tends to form isoforms and is soft. Conversely, Pfr shows significantly harder chromophore–protein interactions, a well‐defined protonic and charge distribution with a clear classical counterion for the positively charged tetrapyrrole system. The soft‐to‐hard/disorder‐to‐order transition involves the chromophore and its protein surroundings within a sphere of at least 5.5 Å. The relevance of this collective event for signaling is discussed. Measurement of the intermediates during the Pfr → Pr back‐reaction provides insight into the well‐adjusted mechanics of a two‐step transformation. As both Pr → Pfr and Pfr → Pr reaction pathways are different in ground and excited states, a photochemically controlled hyper‐landscape is proposed allowing for ratchet‐type reaction dynamics regulating signaling activity.     

Structural evolution of the chromophore in the primary stages of trans/cis isomerization in photoactive yellow protein

We have studied the structural changes induced by optical excitation of the chromophore in wild-type photoactive yellow protein (PYP) in liquid solution with a combined approach of polarization-sensitive ultrafast infrared spectroscopy and density functional theory calculations. We identify the nuC8-C9 marker modes for solution phase PYP in the P and I0 states, from which we derive that the first intermediate state I0 that appears with a 3 ps time constant can be characterized to have a cis geometry. This is the first unequivocal demonstration that the formation of I0 correlates with the conversion from the trans to the cis state. For the P and I0 states we compare the experimentally measured vibrational band patterns and anisotropies with calculations and find that for both trans and cis configurations the planarity of the chromophore has a strong influence. The C7=C8-(C9=O)-S moiety of the chromophore in the dark P state has a trans geometry with the C=O group slightly tilted out-of-plane, in accordance with the earlier reported structure obtained in an X-ray diffraction study of PYP crystals. In the case of I0, experiment and theory are only in agreement when the C7=C8-(C9=O)-S moiety has a planar configuration. We find that the carboxylic side group of Glu46 that is hydrogen-bonded to the chromophore phenolate oxygen does not alter its orientation on going from the electronic ground P state, via the electronic excited P state to the intermediate I0 state, providing conclusive experimental evidence that the primary stages of PYP photoisomerization involve flipping of the enone thioester linkage without significant relocation of the phenolate moiety.