Time Resolved Absorption Spectroscopy for the Study of Electron Transfer Processes in Photosynthetic Systems

Transient absorption spectroscopy was used to study light induced electron transfer processes in Type 1 photosynthetic reaction centers. Flash induced absorption changes were probed at 800, 703 and 487 nm, and on multiple timescales from nanoseconds to tens of milliseconds. Both wild type and menB mutant photosystem I reaction centers from the cyanobacterium Synechocystis sp. PCC 6803 were studied. Photosystem I reaction centers from the green algae Chlamydomonas reinhardtii, and the newly discovered chlorophyll-d containing organism Acaryochloris marina, were also studied. The flash induced absorption changes obtained for menB mutant photosystem I reaction centers are distinguishable from wild type at 800 nm. MenB mutant photosystem I reaction centers displays a large amplitude decay phase with lifetime of ~50 ns which is absent in wild type photosystem I reaction centers. It is hypothesized that this ~50 ns phase is due to the formation of the triplet state of primary electron donor

Bioenergetics in Photosystem I: Time-Resolved Step-Scan FTIR and Visible Spectroscopic Studies of the Secondary Electron Acceptor A1

Time-resolved infrared and visible absorption difference spectroscopy was applied for the study of electron transfer (ET) reactions involving A1, the secondary electron acceptor in photosystem I (PSI). In PSI, the secondary electron acceptor A1 is a phylloquinone (PhQ) molecule. Flash-induced absorption changes at room and cryogenic temperatures in the infrared and visible spectral ranges were probed for PSI with a series of native and non-native quinones in the A1 binding site. Obtained kinetic and spectral data were analyzed for the functional and structural properties of A1 and PSI. Using transient absorption spectroscopy in the visible spectral range, the rates and directionality of ET processes in PSI with modified A1 were determined. A detailed kinetic simulation model was constructed and solved in the context of Marcus ET theory, and midpoint redox potentials of A1 was predicted within a tight range. The transient absorption kinetics for ten different quinones and the kinetic simulation revealed that the wasteful charge recombination process in native PSI occurs in the inverted region. Although inverted-region ET had been widely suggested to be an important mechanism contributing to photosynthetic efficiency, the mechanism had never been demonstrated in any native photosynthetic system. The result presented here is the first demonstration of inverted-region ET in a native photosynthetic reaction center in physiological conditions. Through Marcus theory-based simulation, inverted-region ET is quantitatively shown to be an important mechanism underlying the high efficiency in PSI ET. Time-resolved infrared difference spectroscopy was undertaken using step-scan FTIR technique with a microsecond temporal resolution. Highly-resolved double difference spectrum was constructed to identify infrared bands due to PhQ in the A1 binding site. Assisted by the DFT-based vibrational frequency calculations, vibrational modes due to anionic PhQ– were identified. The calculations suggest that PhQ is asymmetrically H-bonded, and that this interaction is especially strong for PhQ–, but not for PhQ. Additionally, discrepancies that previously existed between FTIR and EPR studies on PSI with plastoquinone-9 in the A1 site were resolved. A method to incorporate a benzoquinone was established, and (A1– – A1) FTIR difference spectra for a series of benzoquinones were produced for the first time

Room temperature crystallography and X-ray spectroscopy of metalloenzymes.

The ultrashort (10s of femtoseconds) X-ray pulses generated by X-ray free electron lasers enable the measurement of X-ray diffraction and spectroscopic data from radiation-sensitive metalloenzymes at room temperature while mostly avoiding the effects of radiation damage usually encountered when performing such experiments at synchrotron sources. Here we discuss an approach to measure both X-ray emission and X-ray crystallographic data at the same time from the same sample volume. The droplet-on-tape setup described allows for efficient sample use and the integration of different reaction triggering options in order to conduct time-resolved studies with limited sample amounts. The approach is illustrated by two examples, photosystem II that catalyzes the light-driven oxidation of water to oxygen, and isopenicillin N synthase, an enzyme that catalyzes the double ring cyclization of a tripeptide precursor into the β-lactam isopenicillin and can be activated by oxygen exposure. We describe the necessary steps to obtain microcrystals of both proteins as well as the operation procedure for the drop-on-tape setup and details of the data acquisition and processing involved in this experiment. At the end, we present how the combination of time-resolved X-ray emission spectra and diffraction data can be used to improve the knowledge about the enzyme reaction mechanism

Melina II: a web tool for comparisons among several predictive algorithms to find potential motifs from promoter regions

We present the second version of Melina, a web-based tool for promoter analysis. Melina II shows potential DNA motifs in promoter regions with a combination of several available programs, Consensus, MEME, Gibbs sampler, MDscan and Weeder, as well as several parameter settings. It allows running a maximum of four programs simultaneously, and comparing their results with graphical representations. In addition, users can build a weight matrix from a predicted motif and apply it to upstream sequences of several typical genomes (human, mouse, S. cerevisiae, E. coli, B. subtilis or A. thaliana) or to public motif databases (JASPAR or DBTBS) in order to find similar motifs. Melina II is a client/server system developed by using Adobe (Macromedia) Flash and is accessible over the web at http://melina.hgc.jp

Pectin and high-amylose maize starch increase caecal hydrogen production and relieve hepatic ischaemia-reperfusion injury in rats.

We investigated whether the feeding of high H2-generating dietary fibre and resistant starch (RS) could suppress hepatic ischaemia-reperfusion (IR) injury, which results from oxidative stress, in rats fed a pectin (Pec) or high-amylose maize starch (HAS) diet. Male Sprague-Dawley rats were fed a control (C) diet, with or without Pec (0-5 % Pec) or HAS (0-30 % HAS) supplementation for 7 d. Portal H2 concentration showed a significant dose-dependent increase with the amount of Pec or HAS supplementation. Plasma alanine and aspartate aminotransferase activities remarkably increased in the C rats (5 % cellulose) due to IR treatment, while it decreased significantly or showed tendencies to decrease in 5 % Pec and 20 % HAS diet-fed rats. The hepatic oxidised glutathione (GSSG):total glutathione ratio increased significantly in IR rats maintained on the C diet compared with sham-operated rats. On the other hand, reduced glutathione (GSH):total glutathione and GSH:GSSG ratios decreased significantly. The GSSG:total glutathione ratio that increased due to IR treatment decreased significantly on HAS and Pec intake, while GSH:total glutathione and GSH:GSSG ratios increased significantly. Hepatic sinusoids of IR rats fed the C diet were occluded, but those of IR rats fed the Pec diet were similar to those in the sham-operated rats. In conclusion, we found that Pec or HAS, which enhance H2 generation in the large intestine, alleviated hepatic IR injury. The present study demonstrates another physiological significance of dietary fibre and RS.We investigated whether the feeding of high H2-generating dietary fibre and resistant starch (RS) could suppress hepatic ischaemia-reperfusion (IR) injury, which results from oxidative stress, in rats fed a pectin (Pec) or high-amylose maize starch (HAS) diet. Male Sprague-Dawley rats were fed a control (C) diet, with or without Pec (0-5 % Pec) or HAS (0-30 % HAS) supplementation for 7 d. Portal H2 concentration showed a significant dose-dependent increase with the amount of Pec or HAS supplementation. Plasma alanine and aspartate aminotransferase activities remarkably increased in the C rats (5 % cellulose) due to IR treatment, while it decreased significantly or showed tendencies to decrease in 5 % Pec and 20 % HAS diet-fed rats. The hepatic oxidised glutathione (GSSG):total glutathione ratio increased significantly in IR rats maintained on the C diet compared with sham-operated rats. On the other hand, reduced glutathione (GSH):total glutathione and GSH:GSSG ratios decreased significantly. The GSSG:total glutathione ratio that increased due to IR treatment decreased significantly on HAS and Pec intake, while GSH:total glutathione and GSH:GSSG ratios increased significantly. Hepatic sinusoids of IR rats fed the C diet were occluded, but those of IR rats fed the Pec diet were similar to those in the sham-operated rats. In conclusion, we found that Pec or HAS, which enhance H2 generation in the large intestine, alleviated hepatic IR injury. The present study demonstrates another physiological significance of dietary fibre and RS