Staffan E. Tjus

PHOTOINHIBITION OF PHOTOSYSTEM I DAMAGES BOTH REACTION CENTRE PROTEINS PSI-A AND PSI-B AND ACCEPTOR-SIDE LOCATED SMALL PHOTOSYSTEM I POLYPEPTIDES

Photoinhibition of Photosystem I at chilling temperatures was investigated. Illumination of barley and cucumber leaves at 4°C induced a lowered Photosystem I activity. In barley, the reaction centre proteins PSI-A and PSI-B were both partially degraded as was the nuclear-encoded PSI-D polypeptide. Barley leaves infiltrated with KCN to increase oxidative stress, showed increased photoinhibition of Photosystem I, including reduced photochemical activity and marked degradation of several Photosystem I polypeptides. The most rapid and pronounced degradation was found in the PSI-D and PSI-E polypeptides exposed at the Photosystem I acceptor side. The PSI-A, -B, -C, -G, -H, -K and -L polypeptides were less extensively damaged. No damage of the lumenally oriented PSI-F and -N polypeptides was detected. The elevated photoinhibition of Photosystem I seen in KCN treated barley is most likely induced by a combination of increased active oxygen due to inhibited scavenging and increased accumulation of reducing power due to inhibition of the Calvin cycle. In barley, photo-inactivation of Photosystem I closely followed the degradation of PSI-A and PSI-B. Illumination of cucumber resulted in a pronounced loss of activity and appearance of specific PSI-A and PSI-B degradation products whereas the total PSI-A/B degradation was small. The PSI-A/B degradation identified in barley is interpreted to reflect a physiologically relevant process being part of a repair cycle, whereas the much smaller PSI-A/B degradation observed in cucumber is interpreted to represent an irreversible damage induced far below the temperature tolerance for cucumber.

Rapid isolation of photosystem I chlorophyll-binding proteins by anion exchange perfusion chromatography

With the new method of anion exchange perfusion chromatography we have devised an extremely rapid technique to subfractionate spinach Photosystem I into its chlorophyll a containing core complex and various components of the Photosystem I light-harvesting antenna (LHC I). The isolation time for the LHC I subcomplexes following solubilisation of native Photosystem I was reduced from 50 h using traditional density centrifugation procedures down to only 10–25 min by perfusion chromatography. Within this very short period of isolation, LHC I has been obtained as subfractions highly enriched in Lhca2+3 (LHC I-680) and Lhca1+4 (LHC I-730). Moreover, other highly enriched subfractions of LHC I such as Lhca2, Lhca3 and Lhca1+2+4 were obtained where the later two populations have not previously been obtained in a soluble form and without the use of SDS. These various subfractions of the LHC I antenna have been characterised by absorption spectroscopy, 77 K fluorescence-spectroscopy and SDS-PAGE demonstrating their identities, functional intactness and purity. Furthermore, the analyses located a chlorophyll b pool to preferentially transfer its excitation energy to the low energy F735 chromophore, and located specifically the origin of the 730 nm fluorescence to the Lhca4 component. It was also revealed that Lhca2 and Lhca3 have identical light-harvesting properties. The isolated Photosystem I core complex showed high electron transport capacity (1535 μmoles O2 mg Chl−1 h−1) and low fluorescence yield (0.4%) demonstrating its high functional integrity. The very rapid isolation procedure based upon perfusion chromatography should in a significant way facilitate the subfractionation of Photosystem I proteins and thereby allow more accurate functional and structural studies of individual components.

Energy transfer in photosystem I. Time resolved fluorescence of the native photosystem I complex and its core complex

Energy transfer within isolated spinach photosystem I (PS I) complexes with different antenna size were studied using time-resolved picosecond and steady-state fluorescence spectroscopy. In both the native PS I complexes and the PS I core complexes lacking the outer chlorophyll a/b antenna we observed a fast dominating emission component ≈ 35 ps at room temperature which is associated with the trapping process by the reaction centre. In the native PS I complex there also appears a 120 ps component which was not observed in the PS I core complex. This component most likely represents an energy transfer from low energy pigments in the light-harvesting complex I antenna and into the core. Due to a very fast energy equilibration (< 10 ps) it was not possible to resolve the energy transfer at room temperature. At 77 K, however, it was possible to follow the energy transfer from F690 to F720 with a transfer time of ≈ 35 ps within the native PS I complex and slightly longer, 78 ps, in the PS I core complex. The native PS I complex also exhibited in the region 700–740 nm a 102 ps component which originates from F720 and represents energy transfer from F720 to P700 at 77 K. At low temperatures the PS I core complex exhibited a component of 161 ps which is associated with F720 and has the same function as the 102 ps component of the native PS I complex. We conclude that the F720 emission originates from pigments in the core antenna system. This emission also increases at low temperature. In the native PS I complex there is an initial increase in the F720 emission as the temperature is lowered but at 77 K the F735 emission originating from LHC I dominates.

ULTRAFAST ENERGY TRANSFER DYNAMICS RESOLVED IN ISOLATED SPINACH LIGHT-HARVESTING COMPLEX I AND THE LHC I-730 SUBPOPULATION

Abstract The energy migration in isolated bulk LHC I and its subpopulations LHC I-680 and LHC I-730 have been studied by fluorescence spectroscopy and femtosecond absorption spectroscopy. LHC I-680 and LHC I-730 display significant differences in both their steady-state and time-resolved fluorescence properties. The steady-state emission maximum is located at 680 nm for LHC I-680 and at 730 nm for LHC I-730. It was also found that different kinetic models were required to fit the time-resolved fluorescence data of the two subpopulations, two components of 374 ps and 3.9 ns were required for LHC I-680 and three components of 157 ps, 510 ps and 2.8 ns for LHC I-730, respectively. Using femtosecond absorption recovery spectroscopy we were able to resolve a fast 200–400 fs depolarization in bulk LHC I at 655, 665 and 670 nm. The results indicate a fastest hopping time between individual chlorophylls of 200–400 fs in bulk LHC I. In the subpopulation LHC I-730, on the other hand, both the isotropic absorption recovery decay and the depolarization process occurred with a 15 ps lifetime, while in the subpopulation LHC I-680 we could not resolve any ultrafast relaxation process. The 15 ps phase is assigned to a transfer of excitation energy to the pigment giving rise to far-red emission component F735 of PS I.

Loss of the trans-thylakoid proton gradient is an early event during photoinhibitory illumination of chloroplast preparations

Abstract During photoinhibitory illumination of isolated thylakoids or intact chloroplasts from spinach, an initial increase of Photosystem I electron transport was observed while proton uptake associated with PMS-mediated cyclic electron transport was rapidly lost. The latter reaction was at least as light-sensitive as Photosystem II electron transport, normally considered to be the primary target for light stress. Thus under both moderate and extreme light stress, loss of the proton gradient associated with cyclic electron transport around Photosystem I was an early event. In accordance with this observation, photoinhibitory light very rapidly caused inactivation of cyclic photophosphorylation. There was no kinetic correlation between light-induced degradation of the D1 protein and collapse of the proton gradient. Notably, under anaerobic conditions when D1 protein degradation does not occur, loss of proton uptake still occurred. Low temperatures (3°C) provided partial protection against the photodamage, but a subsequent increase of the temperature to 25°C resulted in a total loss of the proton uptake in total darkness. The proton gradient could not be re-established by addition of DCCD. Moreover, there were no changes in the polypeptide composition of CF 1 or any impairment of the ATPase activity during photoinhibitory illumination. The mechanism of the light-induced loss of the proton gradient and its correlation to other effects of light stress at the molecular and physiological levels are discussed.

Extrinsic polypeptides of spinach photosystem I

By combining Triton X-114 partitioning with alkaline-salt and chaotropic washings of thylakoid membrane vesicles and photosystem I particles, we have studied the protein subunit composition and organization of spinach photosystem I. Upon fractionation of photosystem I particles with Triton X-114, 6 polypeptides of 5.0, 8.2 (psaE), 10.5, 16.6 (psaG), 19.3 and 22.1 kDa (psaD) were considered to be extrinsic membrane proteins. By combining this partitioning with salt washes of thylakoid membranes, the polypeptides of 8.2, 11.6 (psaH), 19.3 and 22.1 kDa were directly shown to be stromally oriented and extrinsic while no extrinsic subunits were identified at the inner thylakoid surface. The 5.0, 8.2, 10.5, 17.2, 19.3 and 22.1 kDa polypeptides appear to have regulatory rather than catalytic functions as their release from photosystem I particles upon high salt-alkali treatment does not affect photosystem I-mediated electron transport.

Perfusion chromatography-a new procedure for very rapid isolation of integral photosynthetic membrane proteins.

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.

Photoinhibitory Damage of Barley Photosystem I at Chilling Temperatures Induced under Controlled Illumination is also Identified in the Field

Light is needed for photosynthesis but may cause photoinhibition when energy input exceeds consumption (1, 2). Illumination at chilling temperatures further increases the photoinhibitory damage (1–3).

Extrinsic Membrane Proteins of Photosystem I

Photosystem I is a multiprotein complex that mediates light-induced electron transport between plastocyanin and NADP+. Depending on plant species and SDS-PAGE system, the number of polypeptides resolved in the photosystem I complex differs between 8–12 (1), excluding the polypeptides of the light harvesting complex I. The polypeptides have been given roman numbers I–VII. Subunits la, Ib and VII have been assigned direct electron transport functions. Subunit II has been shown to crosslink with ferredoxin and subunit III to interact with plastocyanin (for review see 1). The biochemical functions for the other subunits remain to be established although most of them have been cloned and sequenced. Here we have used biochemical approaches, involving alkaline salt washing and Triton X-114 fractionation, in order to identify extrinsic membrane proteins of photosystem I.