Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm ؊1 , with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6-to 0.8-ps time scale, whereas P ؉ , the precursor state for water oxidation, is formed after Ϸ6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the ''accessory chlorophyll'' ChlD1 and the pheophytin on the so-called active branch.electron transfer ͉ photosynthesis ͉ pump-probe T he primary steps of energy and electron transfer in green plants' photosynthesis occur in two large protein complexes called Photosystem I and Photosystem II (PSII). PSII is an aggregate of many individual pigment-protein complexes. The core of PSII consists of the chlorophyll (Chl)-binding antennaproteins CP43 and CP47, which feed excitation energy into the D 1 D 2 cytb559 reaction center (RC). Crystal structures of PSII cores from cyanobacteria have been resolved with increasingly high resolution (1-3); currently, the resolution is 3.2 Å (4). The structure of the PSII RC shows four Chls and two pheophytins (H) arranged in two branches very similar to the bacterial RC. In the heart of the PSII RC, there is a dimer of Chls, and in each branch there is one monomeric Chl and one H. Furthermore, there are two distant Chls bound to the periphery of the PSII RC. Although the structure suggests there may be a ''special pair'' of strongly electronically coupled pigments in the PSII RC, the visible absorption spectrum does not show a distinct band. This finding is in contrast to the bacterial RC, where the lowest energy absorption band fully originates from one of the exciton transitions of a special pair of bacteriochlorophylls.Since the first purification of the PSII RC in 1987 (5), it has been speculated that its way of operation would be similar to that of the bacterial RC, with a special pair that upon excitation drives a charge separation in Ϸ3 ps. This idea was based on the strong homology between the bacterial RC and the PSII RC, the strong similarity in the pigment composition, even details in the way the pigments interacted with the protein, and the near-identity of the electron transfer events at the acceptor side. Conversely, it was clear that major differences between the two RCs had to exist at the electron donor side where in the PSII RC charge separation eventually leads to the oxidation of water and the production of molecular oxygen, requiring a very large oxidation potential of the primary electron donor (Ͼ1.2 V vs. 0.45 V in the bacterial RC).In the mid-1990s, it was recognized that energy transfer and charge separation in the PSII RC most likely proceeded in a manner that is very different from that in the b...

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm(-1) was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q(A), were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P(L)/P(M))). The region between 1600 and 1800 cm(-1) encompasses absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state P(+)B(L)(-) (where B(L) is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P(+)B(L)(-); the difference spectrum indicates that the 9-keto C=O stretch of B(L) is located around 1670-1680 cm(-1). After subsequent electron transfer to the bacteriopheophytin H(L) in approximately 1 ps, the state P(+)H(L)(-) was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P(+)H(L)(-) radical pair on the approximately 20 ps timescale, accompanied by a change in the relative ratio of the P(L)(+) and P(M)(+) bands and by a minor change in the band amplitude at 1640 cm(-1) that may be tentatively ascribed to the response of an amide C=O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P(+)H(L)(-) is due to an increased localization of the electron hole on the P(L) half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C=O oscillators.

The core of photosystem II (PSII) of green plants contains the reaction center (RC) proteins D1D2-cytb559 and two core antennas CP43 and CP47. We have used time-resolved visible pump/midinfrared probe spectroscopy in the region between 1600 and 1800 cm(-1) to study the energy transfer and charge separation events within PSII cores. The absorption difference spectra in the region of the keto and ester chlorophyll modes show spectral evolution with time constants of 3 ps, 27 ps, 200 ps, and 2 ns. Comparison of infrared (IR) difference spectra obtained for the isolated antennas CP43 and CP47 and the D1D2-RC with those measured for the PSII core allowed us to identify the features specific for each of the PSII core components. From the presence of the CP43 and CP47 specific features in the spectra up to time delays of 20-30 ps, we conclude that the main part of the energy transfer from the antennas to the RC occurs on this timescale. Direct excitation of the pigments in the RC evolution associated difference spectra to radical pair formation of PD1+PheoD1- on the same timescale as multi-excitation annihilation and excited state equilibration within the antennas CP43 and CP47, which occur within approximately 1-3 ps. The formation of the earlier radical pair ChlD1+PheoD1-, as identified in isolated D1D2 complexes with time-resolved mid-IR spectroscopy is not observed in the current data, probably because of its relatively low concentration. Relaxation of the state PD1+PheoD1-, caused by a drop in free energy, occurs in 200 ps in closed cores. We conclude that the kinetic model proposed earlier for the energy and electron transfer dynamics within the D1D2-RC, plus two slowly energy-transferring antennas C43 and CP47 explain the complex excited state and charge separation dynamics in the PSII core very well. We further show that the time-resolved IR-difference spectrum of PD1+PheoD1- as observed in PSII cores is virtually identical to that observed in the isolated D1D2-RC complex of PSII, demonstrating that the local structure of the primary reactants has remained intact in the isolated D1D2 complex.

Electron transfer at the reaction center of the purple photosynthetic bacterium Rb. sphaeroides R-26 was measured at room temperature by the time-resolved transient absorption spectroscopy technique with 200 fs temporal resolution. The absorbance changes characteristic of the excited state of the primary donor and extending over the whole spectral range investigated from 350 nm up to 720 nm appeared after excitation with a laser pulse of about 100 fs duration at 800 nm. The time evolution of the spectra reflected the excitation of bacteriochlorophylls (BChl) M and L and the subsequent transfer of this excitation to the primary electron donor (P), with the time constant shorter than 1 ps. The decay time constant of the excited primary donor P was determined as about 3 ps, and it was faster than the rise of the reduced intermediary acceptor bacteriopheophytin (BPhe(L)). Photoreduction of BPhe(L) and its further reoxidation was clearly observed as an increase in its bleaching band intensity at around 540 nm in about 4 ps and its decrease in about 200 ps. Our findings support the theoretical model assuming the involvement of the intermediate state P(+)BChl- in the so-called "two-step" model. In this model an electron is transferred in a sequence from the excited special pair P* to bacteriochlorophyll, BChl(L), then to bacteriopheophytin, BPhe(L), and further on to quinone, Q(A). The branched charge separation, partially via P and partially via BChl(L), was also observed.

Energy and electron transfer in a tyrosine M210 to tryptophan (YM210W) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated through time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy at room temperature, with the aim to further characterize the primary charge separated states in the RC. This mutant is known to display slow and multi-exponential charge separation, and was used in earlier work to prove the existence of an alternative route for charge separation starting from the accessory bacteriochlorophyll in the active branch, B(L). The mutant RCs were excited at 860 nm (direct excitation of the primary donor (P) BChls (P(L)/P(M))), 600 nm (unselective excitation), 805 nm (direct excitation of both accessory bacteriochlorophyll cofactors B(L) and B(M)) and 795 nm (direct excitation of B(L)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein were recorded in the region between 1600 and 1775 cm(-1), and both a sequential analysis and simultaneous target analysis of the data were performed. The decay of P* in the YM210W mutant was multi-exponential with lifetimes of 29 and 63.5 ps. The decay of P(+)B(L)(-) state was approximately 10 times longer in the YM210W RC than in the R-26 RC (approximately 7 ps vs. approximately 0.7 ps), and in the mid-IR difference absorption spectrum of P(+)B(L)(-) the stretching frequency of the 9-keto C=O group of B(L) in the ground state was located around 1675-1680 cm(-1), consistent with the presence of a hydrogen bond donated by an adjacent water molecule. Excitation at 795 nm produced a small amount of B(L)*-driven charge separation, as assessed from the excitation wavelength dependence of the raw difference spectra recorded during the first few ps after excitation. This process led to the formation of P(+)B(L)(-). Only the relaxed form of the P(+)H(L)(-) radical pair was observed in the YM210W mutant, and the mid-IR difference absorption spectra of P(+)H(L)(-) and P(+)B(L)(-) showed a change in the relative amplitude of the P(L)(+) and P(M)(+) bands when compared to equivalent spectra for the R-26 RC. This indicates that the YM210W mutation causes an increased localization of the electron hole on the P(M) half of the dimer. The absorbance difference spectrum of P(+)H(L)(-) in the R-26 RC contains a feature attributable to a Stark shift of one or more amide C=O oscillators. This feature was shifted to lower frequency by approximately 5 cm(-1) in the YM210W RC, and consideration of the limited structural changes in this RC indicates that this feature arises from an amide C=O group in the immediate vicinity of the M210 residue, most probably that of the adjacent M209 amino acid.

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted betaL). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B(L) and B(M)), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P(L)/P(M))). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm(-1) and 1770 cm(-1), and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P* decayed on a timescale of approximately 6.3 ps to P+BL-. The decay of P+BL- occurred with a lifetime of approximately 2 ps, approximately 3 times slower than that observed in wild-type and R-26 RCs (approximately 0.7 ps). Further electron transfer to the betaL BChl resulted in formation of the P+betaL- state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P+BL- and P+betaL- showed clear differences related to the different environments of the two BChls in the mutant RC.

Background: Local anesthetic resistance is a clinical entity characterized by inadequate analgesia despite technically well-performed procedures. The exact etiology and pathogenesis of this condition are not yet fully understood. Case Presentation: A 36-year-old Caucasian female presented to labor and delivery for induction of labor. On admission, the patient reported failure of epidural anesthesia during the previous delivery. An epidural catheter was placed, and analgesia was reported only at high doses of local anesthetic. The patient’s maximum pain level during delivery never reached a score of 2 out of 10. Conclusion: The most common causes of regional anesthetic failure are technical or placement failure, failure related to the local anesthetic itself, or localized infection. This patient appeared to have a true local anesthetic resistance, which was overcome by doubling the customary concentration of local anesthetic. Atypical responses to local anesthetics observed in the patient may be due to incomplete penetrance mutations in sodium channels since local anesthetics work through blocking nerve conduction by acting on these channels.

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