Expression of the nuclear encoded OEE1 protein is required for oxygen evolution and stability of photosystem II particles in Chlamydomonas reinhardtii.

The 18 O exchange rates for the substrate water bound in the S 3 state were determined in different photosystem II sample types using time-resolved mass spectrometry. The samples included thylakoid membranes, saltwashed Triton X-100-prepared membrane fragments, and purified core complexes from spinach and cyanobacteria. For each sample type, two kinetically distinct isotopic exchange rates could be resolved, indicating that the biphasic exchange behavior for the substrate water is inherent to the O 2 -evolving catalytic site in the S 3 state. However, the fast phase of exchange became somewhat slower (by a factor of ϳ2) in NaClwashed membrane fragments and core complexes from spinach in which the 16-and 23-kDa extrinsic proteins have been removed, compared with the corresponding rate for the intact samples. For CaCl 2 -washed membrane fragments in which the 33-kDa manganese stabilizing protein (MSP) has also been removed, the fast phase of exchange slowed down even further (by a factor of ϳ3). Interestingly, the slow phase of exchange was little affected in the samples from spinach. For core complexes prepared from Synechocystis PCC 6803 and Synechococcus elongatus, the fast and slow exchange rates were variously affected. Nevertheless, within the experimental error, nearly the same exchange rates were measured for thylakoid samples made from wild type and an MSP-lacking mutant of Synechocystis PCC 6803. This result could indicate that the MSP has a slightly different function in eukaryotic organisms compared with prokaryotic organisms. In all samples, however, the differences in the exchange rates are relatively small. Such small differences are unlikely to arise from major changes in the metal-ligand structure at the catalytic site. Rather, the observed differences may reflect subtle long range effects in which the exchange reaction coordinates become slightly altered. We discuss the results in terms of solvent penetration into photosystem II and the regional dielectric around the catalytic site.The oxidation of water during photosynthesis is catalyzed by the membrane-bound pigment protein complex photosystem II (PSII).1 In the net reaction, four electrons, four protons, and one molecule of O 2 are liberated from the splitting of two water molecules. A key insight into the reaction mechanism was the observation that the O 2 produced in brief, saturating light flashes follows a periodicity of four (1). The period four phenomenon is now universally interpreted in terms of the S-state model, in which the catalytic site cycles through five intermediary S n states (n ϭ 0 -4) (2). Beginning in S 0 and traversing to S 4 , each S n state is driven forward by a single quantum event at the PSII reaction center. Upon reaching the S 4 state (which may be equivalent to S 3 Y Z ⅐ ) O 2 is released, S 0 is regenerated, and the cycle begins anew. The catalytic site for water oxidation involves an inorganic metal cluster consisting of four manganese ions, one Ca 2ϩ ion, possibly one Cl Ϫ ion, and the redox-active Tyr-161 resid...

Section: Resultsmentioning

confidence: 99%

“…This genetic deletion results in a strain (⌬psbO) that can evolve O 2 (17)(18)(19). However, genetic deletion of the MSP in the eukaryotic green alga Chlamydomonas results in an obligatory heterotroph (20). Thus, there appears to be some difference in the absolute requirement of the MSP for O 2 evolution between prokaryotic and eukaryotic organisms.…”

mentioning

confidence: 99%

Substrate Water Exchange in Photosystem II Depends on the Peripheral Proteins

Hillier¹,

Hendry²,

Burnap³

et al. 2001

“…MSP has also been removed by mutagenesis in the cyanobacterium, Synechocystis sp. PCC 6803 (7), and in a green algae, Chlamydomonas reinhardtii (8). In the presence of low concentrations of calcium and chloride, plant MSP was found to be required for photosynthetic oxygen evolution and for maintaining the stability of the manganese cluster (9,10).…”

Section: Labeling Of Msp Is Shown To Cause 30 -60 CMmentioning

confidence: 99%

Deprotonation of the 33-kDa, Extrinsic, Manganese-stabilizing Subunit Accompanies Photooxidation of Manganese in Photosystem II

Hutchison¹,

Steenhuis²,

Yocum³

et al. 1999

Photosystem II catalyzes photosynthetic water oxidation. The oxidation of water to molecular oxygen requires four sequential oxidations; the sequentially oxidized forms of the catalytic site are called the S states. An extrinsic subunit, the manganese-stabilizing protein (MSP), promotes the efficient turnover of the S states. MSP can be removed and rebound to the reaction center; removal and reconstitution is associated with a decrease in and then a restoration of enzymatic activity. We have isotopically edited MSP by uniform 13 C labeling of the Escherichia coli-expressed protein and have obtained the Fourier transform infrared spectrum associated with the S 1 to S 2 transition in the presence either of reconstituted 12 C or 13 C MSP. 13 C labeling of MSP is shown to cause 30 -60 cm ؊1 shifts in a subset of vibrational lines. The derived, isotope-edited vibrational spectrum is consistent with a deprotonation of glutamic/ aspartic acid residues on MSP during the S 1 to S 2 transition; the base, which accepts this proton(s), is not located on MSP. This finding suggests that this subunit plays a role as a stabilizer of a charged transition state and, perhaps, as a general acid/base catalyst of oxygen evolution. These results provide a molecular explanation for known MSP effects on oxygen evolution.In oxygenic photosynthesis, the multi-subunit protein complex, photosystem II (PSII), 1 uses light energy to oxidize water and to form molecular oxygen. Hydrophobic subunits, such as the D1 and D2 proteins, bind most of the prosthetic groups involved in charge separation. Water oxidation occurs at a catalytic site containing four manganese atoms. The catalytic site accumulates the four oxidizing equivalents required for water oxidation. The five sequentially oxidized states of the catalytic site are called the S states. Each oxidation of the catalytic site is associated with the reduction of quinone acceptor molecules, Q A , a single electron acceptor, and then Q B , a two-electron, two-proton acceptor (reviewed in Ref. 1).The 33-kDa, manganese-stabilizing protein (MSP) of PSII plays an important role in water oxidation (reviewed in Ref. 2). As an extrinsic subunit, MSP can be removed from the plant reaction center by several different types of biochemical treatments (3-6). MSP has also been removed by mutagenesis in the cyanobacterium, Synechocystis sp. PCC 6803 (7), and in a green algae, Chlamydomonas reinhardtii (8). In the presence of low concentrations of calcium and chloride, plant MSP was found to be required for photosynthetic oxygen evolution and for maintaining the stability of the manganese cluster (9, 10). In the presence of high concentrations of calcium and chloride, oxygen evolution occurs, but the steady state rate of enzymatic activity is impaired upon removal of MSP (7,9,(11)(12)(13)(14). In addition, removal of MSP and replacement with calcium and chloride result in kinetic inhibition of the S state transitions (12,(15)(16)(17)(18)(19). MSP can be rebound to PSII (20), and this reconstitution reverses ...

“…Loss of this protein by biochemical removal from isolated PSII (5-7) or genetic deletion (8,9) from algal cells leads to a significant loss of the oxygen-evolving activity and in some conditions loss of manganese atoms from the tetramanganese cluster. The other two proteins, cytochrome c-550 and the 12-kDa protein, however, are present only in algal-type PSII but absent in higher plant PSII.…”

mentioning

confidence: 99%

Analysis of the psbU Gene Encoding the 12-kDa Extrinsic Protein of Photosystem II and Studies on Its Role by Deletion Mutagenesis in Synechocystis sp. PCC 6803

Shen¹,

Ikeuchi²,

Inoue³

1997

The gene encoding the 12-kDa extrinsic protein of photosystem II from Synechocystis sp. PCC 6803 was cloned based on N-terminal sequence of the mature protein. This gene, named psbU, encodes a polypeptide of 131 residues, the first 36 residues of which were absent in the mature protein and thus served as a transit peptide required for its transport into the thylakoid lumen. A psbU gene deletion mutant grew photoautotrophically in normal BG11 medium at almost the same rate as that of the wild type strain. This mutant, however, grew apparently slower than the wild type did upon depletion of Ca 2؉ or Cl ؊ from the growth medium. Photosystem II oxygen evolution decreased to 81% in the mutant as compared with that in the wild type, and the thermoluminescence B-and Q-bands shifted to higher temperatures accompanied by an increase in the Q-band intensity. These results indicate that the 12-kDa protein is not essential for oxygen evolution but may play a role in optimizing the ion (Ca 2؉ and Cl The oxygen-evolving system of cyanobacteria contains three extrinsic proteins, namely, a 33-kDa protein, cytochrome c-550, and a 12-kDa protein. The genes coding for the 33-kDa protein and cytochrome c-550 are psbO and psbV genes, respectively (for reviews see Refs. 1 and 2), whereas the gene for the 12-kDa protein has been tentatively named psbU (3, 4). The 33-kDa protein is commonly found in higher plant and cyanobacterial PSII, 1 and its function has been studied extensively by both in vitro biochemical approaches and in vivo mutagenesis studies.Results from these studies suggested that the 33-kDa protein plays an important role in stabilizing the tetramanganese cluster, which directly catalyzes the water-splitting reaction. Loss of this protein by biochemical removal from isolated PSII (5-7) or genetic deletion (8, 9) from algal cells leads to a significant loss of the oxygen-evolving activity and in some conditions loss of manganese atoms from the tetramanganese cluster. The other two proteins, cytochrome c-550 and the 12-kDa protein, however, are present only in algal-type PSII but absent in higher plant PSII. Our previous in vitro biochemical (10) and in vivo genetic studies (11) have indicated that cytochrome c-550 is required for maintaining both the oxygen evolution and PSII stability in vivo. This cytochrome can bind to PSII essentially independent of the other extrinsic proteins (10). In accordance with this, a double deletion mutant of Synechocystis sp. PCC 6803 lacking both the 33-kDa protein and cytochrome c-550 showed a complete loss of photoautotrophic growth, which is caused by deactivation of oxygen evolution and destabilization of PSII in vivo (12). In contrast, both the single deletion mutant of the 33-kDa protein (9) or cytochrome c-550 (11) were able to grow autotrophically, albeit with reduced rates. These studies suggested that cytochrome c-550 binds to and functions in cyanobacterial PSII independent of the 33-kDa protein in maintaining the oxygen-evolving activity and PSII stability (12). The 12-kDa pro...