CERQUEIRA, João Victor Abreu. Fotoproteção do PSI induzida por estresses abióticos que restringem a assimilação de CO2 é contrastante em J. curcas e R. communis. 2016. 105 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza, 2016. / Submitted by Aline Mendes (alinemendes.ufc@gmail.com) on 2016-08-22T18:46:26Z No. of bitstreams: 1 2016_dis_jvacerqueira.pdf: 2430427 bytes, checksum: a4b8e8c95163077e4dce7e0363493b4e (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2016-08-23T18:26:55Z (GMT) No. of bitstreams: 1 2016_dis_jvacerqueira.pdf: 2430427 bytes, checksum: a4b8e8c95163077e4dce7e0363493b4e (MD5) / Made available in DSpace on 2016-08-23T18:26:55Z (GMT). No. of bitstreams: 1 2016_dis_jvacerqueira.pdf: 2430427 bytes, checksum: a4b8e8c95163077e4dce7e0363493b4e (MD5) Previous issue date: 2016 / Several abiotic stresses induced photodamage to photosystem II (PSII). This photodamage is generated by an imbalance between the reducing power generated by the reductive stage of photosynthesis and limitations in consumption of the Calvin cycle. Stresses that promote stomatal closure with salinity and water deficiency are well known for this purpose. When the stress is prolonged, damage can induce photoinhibition of PSII. However, PSI can also be inhibited. In vivo, studies have revealed that the PSI is more resistant than PSII. Moreover, in vitro studies show that PSI in isolated thylakoid membranes suffers inhibition even at low light levels (50 μmol.m-2s-1). This suggests that PSI in vivo requires photoprotective mechanism to prevent a collapse of the plant. Also, due to the low turnover rate of PSI protein complex, photoinhibition is more severe in PSI than in PSII, because this phenomenon is irreversible. On the other hand, there have been few studies of the photoinhibition of PSI in plants due to three factors: the difficulty of measuring the activity of PSI, the limited combination of plant species and environmental conditions in which the phenomenon could occur, and the non-regulatory aspect of photoinhibition of PSI. However, new methodologies capable of measuring in vivo the PSI and capable of inducing the PSI photoinhibition techniques have been developed and used to better understand how plants avoid this phenomenon. Ricinus communis and the Jatropha curcas are two said species adapted to arid and semi-arid regions. Both species have developed mechanisms to deal with the energy imbalance generated by drought and salinity. For this reason, this two species were selected. In this study, both species were subjected to salt stress and the answers were analyzed using biophysical methods such as gas exchange, chlorophyll fluorescence, absorbance of P700. Surprisingly, the plant Ricinus communis had a larger pool of oxidation potential of P700 under stress even having reduced the cyclic electron flow (CEF). Jatropha curcas showed a reduction in the pool P700 and an increase in the EFC. The oxidation of P700 is displayed in the literature as a way of avoiding photoinhibition PSI, however this mechanism is generally attributed to the increase in proton motive force (PMF) generated by an increase in the EFC. The pmf actually increase because there was an increase in power dissipation as heat measured by the non-photochemical quenching (NPQ). The plant Ricinus communis had the PSI inhibited artificially. The plants under salt stress, better able to tolerate the photoinhibition of PSI despite having increased the hydrogen peroxide synthesis (H2O2). Subsequently, the experiment was repeated in Ricinus communis changing treated water deficit. The results presented by plants in salt stress and inhibited PSI were similar to stressed plants by water deficit stress and inhibited PSI. In conclusion, the study suggests that plant Ricinus communis under stresses which limit the Calvin cycle activate photoprotective mechanisms possibly increase the pmf oxidize P700, but this mechanism is not dependent on CEF. / Diversos estresses abióticos induzem o fotodano ao fotossistema II (PSII). Esse fotodano é gerado por um desbalanço entre o poder redutor gerado pela fase redutora da fotossíntese e o consumo desses redutores pelo ciclo de Calvin promovendo dano aos fotossistemas. Caso esses estresse se prolonguem, o dano pode induzir a fotoinibição. Ambos os fotossistemas podem sofre fotoinibição. Apesar disso, poucos grupos estudaram a fotoinibição do PSI em plantas devido a uma série de dificuldades para estudar esse complexo. Por outro lado, analises in vitro sugerem que a fotoinibição do PSI é mais severa do que a fotoinibição do PSII, pois esse fenômeno é irreversível. Ricinus communis e Jatropha curcas são duas espécies ditas adaptadas a regiões áridas e semiáridas. Ambas as plantas desenvolveram mecanismos para lidar com o desbalanço de energia gerado pela deficiência hídrica e pela salinidade. Por essa razão, essas plantas foram selecionadas como modelos. Nesse estudo, ambas as espécies foram submetidas ao estresse salino e as repostas foram analisadas por meio de métodos biofísicos como trocas gasosas, fluorescência da clorofila a e absorbância do P700. Surpreendentemente, as plantas de Ricinus communis apresentaram um maior potencial de oxidação do pool de P700 em estresse mesmo tendo reduzido o fluxo cíclico de elétrons (CEF) enquanto a Jatropha curcas apresentou uma redução no pool de P700 e um aumento no CEF. A oxidação do P700 tem sido apresentada pela literatura como uma forma de evitar a fotoinibição do PSI, no entanto esse mecanismo é geralmente atribuído ao aumento no ΔpH gerado por um aumento na CEF. Posteriormente, as plantas de Ricinus communis de ambos os tratamentos tiveram o PSI inibido artificialmente. As plantas que previamente sofreram com estresse salino, conseguiram tolerar melhor a fotoinibição do PSI apesar de terem aumentado a síntese de peróxido de hidrogênio (H2O2). Outro estudo foi realizado apenas com plantas de Ricinus communis com deficiência hídrica. Os resultados apresentados pelas plantas em estresse salino e com o PSI inibido foram similares aos de plantas estressadas pelo estresse de deficiência hídrica e com o PSI inibido. Em conclusão, o trabalho sugere que plantas de Ricinus communis expostas a estresses que limitam o ciclo de Calvin ativam mecanismos fotoprotetores que possivelmente aumentam a pmf e oxidam o P700, mas esse mecanismo não é dependente do CEF.

The photosynthetic process has the unique ability to capture energy from sunlight and accumulate that energy in sugars and starch. This thesis deals with the light driven part of photosynthesis. The aim has been to investigate how the light-absorbing protein complexes Photosystem I (PS I) and Photosystem II (PS II), react upon illumination of light with lower energy (far-red light; 700-850 nm) than the absorption peak at respective primary donor, P700 and P680.  The results were unexpected. At 295 K, we showed that both PS I and PS II were able to perform photochemistry with light up to 130 nm above its respective primary donor absorption maxima. As such, it was found that the primary donors’ action spectra extended approximately 80 nm further out into the red-region of the spectrum than previously reported.  The ability to perform photochemistry with far-red light was conserved at cryogenic temperatures (< 77 K) in both photosystems. By performing EPR measurements on various photosystem preparations, under different illumination conditions the origin of the effect was localized to their respective reaction center. It is also likely that underlying mechanism is analogous for PS I and PS II, given the similarities in spatial coordination of the reaction center pigments. For PS II, the results obtained allowed us to suggest a model involving a previously unknown electron transfer pathway. This model is based upon the conclusion that the primary cation from primary charge separation induced by far-red light resides primarily on ChlD1 in P680. This is in contrast to the cation being located on PD1, as has been suggested as for visible light illumination. The property to drive photochemistry with far-red wavelengths implies a hither to unknown absorption band, probably originating from the pigments that compose P700 and P680. The results presented here might clarify how the pigments inside P680 are coupled and also how the complex charge separation processes within the first picoseconds that initiate photosynthetic reactions occur.

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.

This thesis describes an investigation of the molecular mechanism underlying solar conversion processes that occur in Type I photosynthetic reaction centers, in which P700 plays a central role. Static Fourier transform infrared (FTIR) difference spectroscopy (DS) was used to probe the electronic and structural organization of P700 and P700+. In combination with isotope labeling and site directed mutagenesis we have investigated how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. Comparison of (P700+-P700) FTIR difference spectra (DS) obtained using wild type and mutant PS I led us to suggest that the 131 keto carbonyl group of PA is essentially free from hydrogen bonding in the ground state. Upon cation formation, this hydrogen bonding becomes stronger, probably because of a cation induced reorientation of the hydroxyl group of a nearby threonine residue. We also tentatively suggested that a difference band at 1639(-)/1660(+) cm-1 in (P700+-P700) FTIR DS might be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Most of this thesis is geared towards investigating the validity of this interpretation. (P700+-P700) FTIR DS obtained using mutant PS I particles in which hydrogen bonding to P700 is altered can be reconciled within the context of our new interpretation. (P700+-P700) FTIR DS obtained using uniformly 2H, 15N, and 13C labeled PS I particles also support our new interpretation, and indicate that the difference band at 1639(-)/ 1660(+) cm-1 cannot be associated with a strongly hydrogen bonded keto carbonyl group of PA. To investigate if the imidazole side-chain of ligating histidine residues could contribute to bands in (P700+-P700) FTIR DS vibrational mode frequencies and intensities for several protonation forms of 4-methylimidazole were calculated. The calculations suggest that the 1639(-)/1660(+) cm-1 band in (P700+-P700) FTIR DS may not be due to a C=C mode of the imidazole side chain of the ligating histidine residues. Thus we have produced data that suggests neither of the proposed interpretations alone can adequately explain the origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS. The origin of the 1639(-)/1660(+) cm-1 difference band in (P700+-P700) FTIR DS is therefore still an open question.

This dissertation presents a study of the molecular mechanism underlying the highly efficient solar energy conversion processes that occur in the Photosystem I (PS I) reaction centers in plants and bacteria. The primary electron donor P700 is at the heart of solar energy conversion process in PS I and the aim is to obtain a better understanding of the electronic and structural organization of P700 in the ground and excited states. Static Fourier Transform Infra-Red (FTIR) difference spectroscopy (DS) in combination with site directed mutagenesis and Density Functional Theory (DFT) based vibrational frequency simulations were used to investigate how protein interactions such as histidine ligation and hydrogen bonding modulate this organization. (P700+-P700) FTIR DS at 77K were obtained from a series of mutants from the cyanobacterium Synechocystis sp. 6803 (S. 6803) where the amino acid residues near the C=O groups of the two chlorophylls of P700 where specifically changed. (P700+-P700) FTIR DS was also obtained for a set of mutants from C. reinhardtii where the axial ligand to A0-, the primary electron acceptor in PS I was modified. The FTIR DS obtained from these mutants provides information on the axial ligands, the hydrogen bonding status as well as the polarity of the environment of specific functional groups that are part of the chlorophyll molecules that constitute P700. Assignment of the FTIR bands to vibrational modes in specific types of environment is very difficult. In order to assist the assignment of the difference bands in experimental spectra DFT based vibrational mode frequency calculations were undertaken for Chl-a and Chl-a+ model molecular systems under different set of conditions; in the gas phase, in solvents using the Polarizable Continuum Model (PCM), in the presence of explicit solvent molecules using QM/MM methods, and in the presence of axial ligands and hydrogen bonds. DFT methods were also used to calculate the charge, spin and redox properties of Chl-a/Chl-a’ dimer models that are representative of P700, the primary electron donor in PS I.