ESR spectroscopy demonstrates that cytochrome b 559 remains low potential in Ca 2+ -reactivated, salt-washed PSII particles

Cytochrome b 559 in various Photosystem II preparations was studled by using low temperature ESR spectroscopy. This technique was used because it is able to distinguish high from low potential forms of the cytochrome owing to the g-value differences between these species. Moreover, by using low temperature irradiation to oxidize cyt b 559 we have avoided the use of redox mediators. Previous work (Ghanotakis DF., Topper J.N. and Yocum, C.F. (1984) Biochim. Biophys. Acta 767 , 524–531) demonstrated that reduction and extraction of manganese of the oxygen evolving complex, which might be expected to alter the redox properties of cyt b 559 , occurs when certain PSII preparations are exposed to reductants. The ESR data presented here show that a mixture of high potential and lower potential cyt b 559 species is observed in the oxygen evolving Photosystem II complex. Treatment of PSII membranes with 0.8 M Tris converts the high potential form(s) to those of lower potential. Exposure of the membranes to 2M NaCl shifts a significant amount of high potential cyt b 559 to lower potential form(s); addition of CaCl 2 reconstituted oxygen evolution activity but did not restore cyt b 559 to its high potential form(s).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43546/1/11120_2004_Article_BF00029738.pd

Characterization of a photosystem II reaction center complex isolated by exposure of PSII membranes to a non-ionic detergent and high concentrations of NaCl

A highly resolved PSII reaction center complex has been prepared by exposure of PSII membranes to the detergent octylglucopyranoside at elevated ionic strengths; oxygen evolution activity is about 1,000 μmoles O 2 /hr/mg Chl in the presence of CaCl 2 . A Mn quantitation and a kinetic study of Z, the donor to P 680 , reveals that on a Chl basis this new preparation shows an almost four-fold enrichment in Mn and the electron transport components of PSII.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43532/1/11120_2004_Article_BF00118314.pd

One-step isolation and biochemical characterization of a highlyactive plant PSII monomeric core

We describe a one-step detergent solubilization protocol for isolating a highly active form of Photosystem II (PSII) from Pisum sativum L. Detailed characterization of the preparation showed that the complex was a monomer having no light harvesting proteins attached. This core reaction centre complex had, however, a range of low molecular mass intrinsic proteins as well as the chlorophyll binding proteins CP43 and CP47 and the reaction centre proteins D1 and D2. Of particular note was the presence of a stoichiometric level of PsbW, a low molecular weight protein not present in PSII of cyanobacteria. Despite the high oxygen evolution rate, the core complex did not retain the PsbQ extrinsic protein although there was close to a full complement of PsbO and PsbR and partial level of PsbP. However, reconstitution of PsbP and PsbPQ was possible. The presence of PsbP in absence of LHCII and other chlorophyll a/b binding proteins confirms that LHCII proteins are not a strict requirement for the assembly of this extrinsic polypeptide to the PSII core in contrast with the conclusion of Caffarri et al. (2009)

Copper effect on the protein composition of photosystem II

The definitive version is available at: http://www.blackwell-synergy.com/doi/full/10.1111/j.1399-3054.2000.1100419.xWe provide data from in vitro experiments on the polypeptide composition, photosynthetic electron transport and oxygen evolution activity of intact photosystem II (PSII) preparations under Cu(II) toxicity conditions. Low Cu(II) concentrations (Cu(II) per PSII reaction centre unit≤230) that caused around 50% inhibition of variable chlorophyll a fluorescence and oxygen evolution activity did not affect the polypeptide composition of PSII. However, the extrinsic proteins of 33, 24 and 17 kDa of the oxygen-evolving complex of PSII were removed when samples were treated with 300 μM CuCl2 (Cu(II) per PSII reaction centre unit=1 400). The LHCII antenna complex and D1 protein of the reaction centre of PSII were not affected even at these Cu(II) concentrations. The results indicated that the initial inhibition of the PSII electron transport and oxygen-evolving activity induced by the presence of toxic Cu(II) concentrations occurred before the damage of the oxygen-evolving complex. Indeed, more than 50% inhibition could be achieved in conditions where its protein composition and integrity was apparently preserved.This work was supported by the Dirección General de Investigación Científica y Técnica (Grant PB98-1632).Peer reviewe

Characterization of inhibitory effects of NH 2 OH and its N-methyl derivatives on the O 2 -evolving complex of Photosystem II

Inorganic cofactors (Mn, Ca 2+ and Cl - ) are essential for oxidation of H 2 O to O 2 by Photosystem II. The Mn reductants NH 2 OH and its N-methyl derivatives have been employed as probes to further examine the interactions between these species and Mn at the active site of H 2 O oxidation. Results of these studies show that the size of a hydroxylamine derivative regulates its ability to inactivate O 2 evolution activity, and that this size-dependent inhibition behavior arises from the protein structure of Photosystem II. A set of anions (Cl - , F - and SO 4 2- ) is able to slow NH 2 OH and CH 3 NHOH inactivation of intact Photosystem II membranes by exerting a stabilizing influence on the extrinsic 23 and 17 kDa polypeptides. In contrast to this non-specific anion effect, only Cl - is capable of attenuating CH 3 NHOH and (CH 3 ) 2 NOH inhibition in salt-washed preparations lacking the 23 and 17 kDa polypeptides. However, Cl - fails to protect against NH 2 OH inhibition in salt-washed membranes. These results indicate that the attack by NH 2 OH and its N-methyl derivatives on Mn occurs at different sites in the O 2 -evolving complex. The small reductant NH 2 OH acts at a Cl - -insensitive site whereas the inhibitions by CH 3 NHOH and (CH 3 ) 2 NOH involve a site that is Cl - sensitive. These findings are consistent with earlier studies showing that the size of primary amines controls the Cl - sensitivity of their binding to Mn in the O 2 -evolving complex.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43537/1/11120_2004_Article_BF00046773.pd

Chlorophyll a/b binding (CAB) polypeptides of CP29, the internal chlorophyll a/b complex of PSII: characterization of the tomato gene encoding the 26 kDa (type 1) polypeptide, and evidence for a second CP29 polypeptide

CP29, the core chlorophyll a/b (CAB) antenna complex of Photosystem II (PSII), has two nuclearencoded polypeptides of approximately 26 and 28 kDa in tomato ( Lycopersicon esculentum ). Cab9, the gene for the Type 1 (26 kDa) CP29 polypeptide was cloned by immunoscreening a tomato leaf cDNA library. Its identity was confirmed by sequencing tryptic peptides from the mature protein. Cab9 is a single-copy gene with five introns, the highest number found in a CAB protein. In vitro transcription-translation gave a 31 kDa precursor which was cleaved to about 26 kDa after import into isolated tomato chloroplasts. The Cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides, which define the members of this extended gene family. Outside of the conserved regions, it is only slightly more closely related to other PSII CABs than to PSI CABs. Sequence analysis of tryptic peptides from the Type II (28 kDa) CP29 polypeptide showed that it is also a member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach. All members of the CAB family have absolutely conserved His, Gln and Asn residues which could ligate the Mg atoms of the chlorophylls, and a number of conserved Asp, Glu, Lys and Arg residues which could form H-bonds to the polar groups on the porphyrin rings. The two conserved regions comprise the first and third predicted trans-membrane helices and the stroma-exposed segments preceding them.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47577/1/438_2004_Article_BF00259681.pd

Excitation energy transfer in native and unstacked thylakoid membranes studied by low temperature and ultrafast fluorescence spectroscopy

In this work, the transfer of excitation energy was studied in native and cation-depletion induced, unstacked thylakoid membranes of spinach by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission spectra at 5 K show an increase in photosystem I (PSI) emission upon unstacking, which suggests an increase of its antenna size. Fluorescence excitation measurements at 77 K indicate that the increase of PSI emission upon unstacking is caused both by a direct spillover from the photosystem II (PSII) core antenna and by a functional association of light-harvesting complex II (LHCII) to PSI, which is most likely caused by the formation of LHCII-LHCI-PSI supercomplexes. Time-resolved fluorescence measurements, both at room temperature and at 77 K, reveal differences in the fluorescence decay kinetics of stacked and unstacked membranes. Energy transfer between LHCII and PSI is observed to take place within 25 ps at room temperature and within 38 ps at 77 K, consistent with the formation of LHCII-LHCI-PSI supercomplexes. At the 150-160 ps timescale, both energy transfer from LHCII to PSI as well as spillover from the core antenna of PSII to PSI is shown to occur at 77 K. At room temperature the spillover and energy transfer to PSI is less clear at the 150 ps timescale, because these processes compete with charge separation in the PSII reaction center, which also takes place at a timescale of about 150 ps. © 2007 Springer Science+Business Media B.V

Mn 2+ reduces Y z + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn 2+ . Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y z + , the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y z + reduction by benzidine was a linear function of benzidine concentration. The rate of Y z + reduction by Mn 2+ at pH 6 increased linearly at low Mn 2+ concentrations and reached a maximum at the Mn 2+ concentrations equal to several times the reaction center concentration. The rate was inhibited by K + , Ca 2+ and Mg 2+ . These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y z + reduction at pH 7.5 was biphasic with a fast 400 μs phase that suggests binding of Mn 2+ near Y z + at a site that may be one of the native manganese binding sites.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43534/1/11120_2004_Article_BF00048306.pd