Wild-type Photosystem Research Articles

Electron transfer from A−0 to A1 in Photosystem I from Chlamydomonas reinhardtii occurs in both the A and B branch with 25–30-ps lifetime

We have recorded transient absorption kinetics at 390 nm with picosecond resolution in order to observe electron transfer from the reduced primary acceptor, A, to the secondary acceptor, A(1), in wild type and mutated Photosystem I from Chlamydomonas reinhardtii. In the mutants, the methionine axial ligand to the primary electron acceptor in either the A- or B-branch of electron transfer cofactors, was replaced with histidine. Both of the mutations reduced the formation of a positive signal at 390 nm, characteristic of A to a level approximately half of that observed in wild type Photosystem I. It is concluded that in the mutated branch of Photosystem I, electron transfer from A to A(1) does not occur. The absorption kinetics resulting from subtraction of either of the mutants' traces from that of wild type is interpreted to reflect the kinetics of A- or B-side electron transfer from A to A(1) in the the wild type Photosystem I. Each of these traces could be fitted with a monoexpoenential decay characterized by the same amplitude and 25-30-ps lifetime. The almost identical effect of both mutations on A formation confirm a similar engagement of both the A- ad B-branches in electron transfer to A(1) in Photosystem I from C. reinhardtii. This observation is in contrast to the unidirectional electron transfer concluded from the studies on similar mutants of cyanobacterial Photosystem I.(1) Thus, this contribution provides further evidence for functional differences between these two model Photosystems.

Time-Resolved FTIR Difference Spectroscopy for the Study of Photosystem I Particles with Plastoquinone-9 Occupying the A1Binding Site

In photosystem I from plants and cyanobacteria a phylloquinone molecule, called A1, functions as the secondary electron acceptor. In cyanobacteria, genes that encode for proteins involved in phylloquinone biosynthesis can be deleted. Here, we have studied three different gene deletion mutants called menB, menD, and menE mutants. In these mutants, plastoquinone-9 occupies the A1 binding site. Using time-resolved, step-scan FTIR difference spectroscopy we have produced A1(-)/A1 FTIR difference spectra for menB, menD, and menE photosystem I particles at 77 K. These difference spectra show that the P700 triplet state ((3)P700) is formed in a large fraction of the particles. Infrared spectral signatures that are not due to (3)P700 are also observed in the spectra and are suggested to be associated with plastoquinone-9 anion formation in a portion of the particles. By subtracting the known (3)P700 spectral signatures, we produce an A1(-)/A1 FTIR difference spectrum for PS I particles with plastoquinone-9 occupying the binding site. This spectrum shows that a band that we have previously assigned to a C:-O mode of the phylloquinone anion in WT A1(-)/A1 FTIR DS down-shifts approximately 8 cm(-1) when plastoquinone-9 occupies the A1 binding site. Using density functional theory type calculations to produce anion minus neutral infrared difference spectra for both phylloquinone and plastoquinone-9, it is shown that such a downshift is reasonable. A1(-)/A1 FTIR difference spectra, obtained using menB mutant photosystem I particles that were incubated in the presence of phylloquinone, are found to be very similar to those obtained using normal WT photosystem I particles. This result indicates that we were able to reincorporate phylloquinone back into the A1 binding site and that the reincorporated phylloquinone and its immediate protein environment, in both the neutral and anion state, are very similar to that found in wild type photosystem I particles. For the reconstituted menB mutant photosystem I particles, no spectral signatures associated with (3)P700 are observed, indicating that phylloquinone occupies the A1 site in all of the reconstituted menB particles.

Insertion of the plant photosystem I subunit G into the thylakoid membrane

Subunit G of photosystem I is a nuclear-encoded protein, predicted to form two transmembrane alpha-helices separated by a loop region. We use in vitro import assays to show that the positively charged loop domain faces the stroma, whilst the N- and C-termini most likely face the lumen. PSI-G constructs in which a His- or Strep-tag is placed at the C-terminus or in the loop region insert with the same topology as wild-type photosystem I subunit G (PSI-G). However, the presence of the tags in the loop make the membrane-inserted protein significantly more sensitive to trypsin, apparently by disrupting the interaction between the loop and the PSI core. Knock-out plants lacking PSI-G were transformed with constructs encoding the C-terminal and loop-tagged PSI-G proteins. Experiments on thylakoids from the transgenic lines show that the C-terminally tagged versions of PSI-G adopt the same topology as wild-type PSI-G, whereas the loop-tagged versions affect the sensitivity of the loop region to trypsin, thus confirming the in vitro observations. Furthermore, purification of PSI complexes from transgenic plants revealed that all the tagged versions of PSI-G are incorporated and retained in the PSI complex, although the C-terminally tagged variants of PSI-G were preferentially retained. This suggests that the loop region of PSI-G is important for proper integration into the PSI core. Our experiments demonstrate that it is possible to produce His- and Strep-tagged PSI in plants, and provide further evidence that the topology of membrane proteins is dictated by the distribution of positive charges, which resist translocation across membranes.

Ferredoxin-NADP+ Reductase: KINETICS OF ELECTRON TRANSFER, TRANSIENT INTERMEDIATES, AND CATALYTIC ACTIVITIES STUDIED BY FLASH-ABSORPTION SPECTROSCOPY WITH ISOLATED PHOTOSYSTEM I AND FERREDOXIN

The electron transfer cascade from photosystem I to NADP+ was studied at physiological pH by flash-absorption spectroscopy in a Synechocystis PCC6803 reconstituted system comprised of purified photosystem I, ferredoxin, and ferredoxin-NADP+ reductase. Experiments were conducted with a 34-kDa ferredoxin-NADP+ reductase homologous to the chloroplast enzyme and a 38-kDa N-terminal extended form. Small differences in kinetic and catalytic properties were found for these two forms, although the largest one has a 3-fold decreased affinity for ferredoxin. The dissociation rate of reduced ferredoxin from photosystem I (800 s(-1)) and the redox potential of the first reduction of ferredoxin-NADP+ reductase (-380 mV) were determined. In the absence of NADP+, differential absorption spectra support the existence of a high affinity complex between oxidized ferredoxin and semireduced ferredoxin-NADP+ reductase. An effective rate of 140-170 s(-1) was also measured for the second reduction of ferredoxin-NADP+ reductase, this process having a rate constant similar to that of the first reduction. In the presence of NADP+, the second-order rate constant for the first reduction of ferredoxin-NADP+ reductase was 20% slower than in its absence, in line with the existence of ternary complexes (ferredoxin-NADP+ reductase)-NADP+-ferredoxin. A single catalytic turnover was monitored, with 50% NADP+ being reduced in 8-10 ms using 1.6 microM photosystem I. In conditions of multiple turnover, we determined initial rates of 360-410 electrons per s and per ferredox-in-NADP+ reductase for the reoxidation of 3.5 microM photoreduced ferredoxin. Identical rates were found with photosystem I lacking the PsaE subunit and wild type photosystem I. This suggests that, in contrast with previous proposals, the PsaE subunit is not involved in NADP+ photoreduction.

Electron transfer from plastocyanin to the photosystem I reaction center in mutants with increased potential of the primary donor in Chlamydomonas reinhardtii.

The dependence of the P(700)(+)/P(700) midpoint potential on kinetics of reduction of P(700)(+) in vivo has been examined in a series of site-directed mutants of Chlamydomonas reinhardtii in which the histidyl axial ligand to the Mg(2+) of the P(700) chlorophyll a has been changed to several different amino acids. In wild-type photosystem I, the potential of P(700)(+)/P(700) is 447 mV and the in vivo half-time of P(700)(+) reduction by its natural donor, plastocyanin, is 4 micros. Substitution of the axial histidine ligand with cysteine increases the potential of P(700)(+)/P(700) to 583 mV and changes the rate of P(700)(+) reduction to 0.8 micros. Mutants with a range of potentials between 447 and 583 mV show a strong correlation of the P(700)(+)/P(700) potential to the rate of reduction of P(700)(+) by plastocyanin. There is also an increase in the rate of photosystem I-mediated electron transfer from the artificial electron donor DCPIP to methyl viologen in thylakoid membranes. The results indicate that the overall rate constant of P(700)(+) reduction is determined by the rate of electron transfer between the copper and P(700)(+) and confirmed that in vivo there is a preformed complex between plastocyanin and photosystem I. Using approximations of the Marcus electron transfer theory, it is possible to estimate that the distance between the copper of plastocyanin and P(700)(+) is approximately 15 A. On the basis of this distance, the plastocyanin docking site should lie in a 10 A hollow formed by the lumenal exposed loops between transmembrane helices i and j of PsaA and PsaB.

Assembly of Photosystem I: I. INACTIVATION OF THE rubA GENE ENCODING A MEMBRANE-ASSOCIATED RUBREDOXIN IN THE CYANOBACTERIUM SYNECHOCOCCUS SP. PCC 7002 CAUSES A LOSS OF PHOTOSYSTEM I ACTIVITY

A 4.4-kb HindIII fragment, encoding an unusual rubredoxin (denoted RubA), a homolog of the Synechocystis sp. PCC 6803 gene slr2034 and Arabidopsis thaliana HCF136, and the psbEFLJ operon, was cloned from the cyanobacterium Synechococcus sp. PCC 7002. Inactivation of the slr2034 homolog produced a mutant with no detectable phenotype and wild-type photosystem (PS) II levels. Inactivation of the rubA gene of Synechococcus sp. PCC 7002 produced a mutant unable to grow photoautotrophically. RubA and PS I electron transport activity were completely absent in the mutant, although PS II activity was approximately 80% of the wild-type level. RubA contains a domain of approximately 50 amino acids with very high similarity to the rubredoxins of anaerobic bacteria and archaea, but it also contains a region of about 50 amino acids that is predicted to form a flexible hinge and a transmembrane alpha-helix at its C terminus. Overproduction of the water-soluble rubredoxin domain in Escherichia coli led to a product with the absorption and EPR spectra of typical rubredoxins. RubA was present in thylakoid but not plasma membranes of cyanobacteria and in chloroplast thylakoids isolated from spinach and Chlamydomonas reinhardtii. Fractionation studies suggest that RubA might transiently associate with PS I monomers, but no evidence for an association with PS I trimers or PS II was observed. PS I levels were significantly lower than in the wild type ( approximately 40%), but trimeric PS I complexes could be isolated from the rubA mutant. These PS I complexes completely lacked the stromal subunits PsaC, PsaD, and PsaE but contained all membrane-intrinsic subunits. The three missing proteins could be detected immunologically in whole cells, but their levels were greatly reduced, and degradation products were also detected. Our results indicate that RubA plays a specific role in the biogenesis of PS I.

Function and organization of photosystem I in a cyanobacterial mutant strain that lacks PsaF and PsaJ subunits.

Photosystem I functions as a light-driven plastocyanin-ferredoxin oxidoreductase in the photosynthetic membranes of cyanobacteria and chloroplasts. A mutant strain of the cyanobacterium Synechocystis sp. PCC 6803 that contains a deletion of the psaF gene and a transcriptionally inactive psaJ gene has assembled photosystem I complexes that lack PsaF, a lumenal protein and PsaJ, a 4-kDa hydrophobic protein. The cells of the mutant and wild type strains have similar rates of photosynthetic electron transfer and P700+ rereduction under linear and cyclic electron transfer conditions. Analysis of flash-induced absorption transients at 700 nm demonstrate that the absence of PsaF in purified mutant photosystem I did not affect the rate of P700 rereduction by cytochrome c553. Therefore, PsaF is not essential for docking of cytochrome c553. We also studied the organization of the proteins of mutant and wild type photosystem I by comparing their accessibility to digestion by thermolysin or to removal by 1 M NaI. The PsaA-PsaB subunits were more easily degraded by thermolysin in the mutant photosystem I. Thermolysin cleavage of PsaB yielded two major fragments that were immunoreactive with an antibody raised against the C terminus of PsaB. The N termini of these PsaB peptides mapped at Ile482 and Ile498 residues, thus identifying a surface-exposed domain of the core of photosystem I. The PsaE subunit could be removed by 1 M NaI and was rapidly digested by thermolysin in the mutant but not in the wild type photosystem I. Therefore, PsaF and PsaJ subunits of photosystem I have dispensable accessory roles in the function and organization of the complex.

Further investigations on structural and catalytic properties of O2 evolving preparations from tobacco and two chlorophyll deficient tobacco mutants

Previous investigations (Specht, S., Pistorius, E.K. and Schmid, G.H.: Photosynthesis Res. 13, 47-56, 1987) of Photosystem II membranes from tobacco (Nicotiana tabacum L. cv. John William's Broadleaf) which contain normally stacked thylakoid membranes and from two chlorophyll deficient tobacco mutants (Su/su and Su/su var. Aurea) which have low stacked or essentially unstacked thylakoids with occasional membrane doublings, have been extended by using monospecific antisera raised against the three extrinsic polypeptides of 33,21 and 16 kDa. The results show that all three peptides are synthesized as well in wild type tobacco as in the two mutants to about the same level and that they are present in thylakoid membranes of all three plants. However, in the mutants the 16 and 21 kDa peptides (but not the 33 kDa peptide) are easily lost during solubilization of Photosystem II membranes. In the absence of the 16 and 21 kDa peptide Photosystem II membranes from the mutants have a higher O2 evolving activity without addition of CaCl2 than the wild type Photosystem II membranes. On the other hand, after removal of the 33 kDa peptide no significant differences in the binding of Mn could be detected among the three plants. The results also show that reaction center complexes from wild type tobacco and the mutant Su/su are almost identical to the Triton-solubilized Photosystem II membranes from the mutant Su/su var. Aurea.