Neil R. Bowlby

Isolation and characterization of the 47 kDa protein and the D1-D2-cytochrome b-559 complex

The 47 kDa polypeptide and a protein complex consisting of the D1 (32 kDa), D2 (34 kDa) and cytochrome b-559 (9 kDa) species were isolated from a Tris-washed Photosystem II core complex solubilized with dodecylmaltoside in the presence of LiClO4. Although the 43 kDa chlorophyll-binding protein is readily dissociated from the Photosystem II complex under our conditions, two cycles of exposure to high concentrations of detergent and LiClO4 were required for complete removal of the 47 kDa chlorophyll-binding protein from the D1-D2-cytochrome b-559 complex. Spectroscopic characterization of these two species revealed that the 47 kDa protein binds chlorophyll a, whereas the D1-D2-cytochrome b-559 complex shows an enrichment in Pheo a and heme on a chlorophyll basis. A spin-polarized EPR triplet can be observed at liquid helium temperatures in the D1-D2-cytochrome b-559 complex, but no such triplet is observed in the purified 47 kDa species. The zero-field splitting parameters of the P-680+ triplet indicate that the triplet spin is localized onto one chlorophyll molecule. Resonance Raman spectroscopy showed that: (i) beta-carotene is bound to the reaction center in its all-trans conformation; (ii) all chlorophyll a molecules are five-coordinate; and (iii) the C-9 keto group of one of the chlorine pigments is hydrogen-bonded. Our results support the proposal that the D1-D2 complex binds the P-680+ and Pheo a species that are involved in the primary charge separation.

Chlorophyll and cytochrome b‐559 content of the photochemical reaction center of photosystem II

Photosystem II reaction centers comprised of the D1, D2 and cytochrome b‐559 polypeptides were isolated from well‐defined oxygen‐evolving reaction center preparations using either a combination of LiClO4 and dodecylmaltoside, or Triton X‐100 alone. Yields of chlorophyll and cytochrome b‐559 for both preparative methods were compared, and pigment contents were compared based on 2.0 b‐559 per reaction center, a standard derived from photosystem II components found in the starting material. Results obtained with dodecylmaltoside suggest that the reaction center of photosystem II binds 10–12 Chl a along with 2 Cyt b‐559 molecules. Reaction centers prepared with Triton X‐100 bind about 8 Chl a per 2 Cyt b‐559 molecules, a finding which indicates that Triton X‐100 extracts pigments from the reaction center polypeptides. Our results contradict the widely held notion that the pigment binding properties of the photosystem II reaction center are similar to those of the bacterial reaction center.

Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem II

An intrinsic 22 kDa polypeptide is found associated with the oxygen‐evolving photesytem II (PSII) core complex in all green plants and cyanobacteria so far examined, although it does not appear to be required for oxygen evolution. Amino acid sequence information obtained from the purified 22 kDa protein was used to construct a probe that was employed to isolate a full‐length cDNA clone encoding the 274‐residue precursor of the 22 kDa protein. Hydropathy plot analysis predicts the existence of four membrane‐spanning helices in the mature protein. The two halves of the approximattely 200‐residue mature protein show high sequence similarity to each other, suggesting that the psbS gene arose from an internal gene duplication. The 22 kDa protein has some sequence similarity to chlorophyll a/b‐binding, proteins.

Comparative structural and catalytic properties of oxygen-evolving photosystem II preparations

Biochemical techniques now exist to produce the oxygen-evolving complex of photosystem II (PSII) and its associated photochemical redox reactions in various states of purity. These preparations permit one to assess the structural roles of polypeptides in promoting activity by using selective extraction techniques which remove certain polypeptides, to carry out reconstitution studies which re-establish activity, and, in the case of more recently developed, highly purified preparations discussed in this overview, to identify the minimal polypeptide complement necessary for photosynthetic oxygen evolution activity. These comparative investigations also suggest a tentative structure for an oxygen-evolving PSII core complex whose primary constituents are a hydrophobic complex of polypeptide, manganese, calcium and chloride, and the 33 kDa extrinsic polypeptide.

Genetic Manipulation of the Cyanobacterium Synechocystis sp. PCC 6803 (Development of Strains Lacking Photosystem I for the Analysis of Mutations in Photosystem II)

We have taken a genetic approach to eliminating the presence of photosystem I (PSI) in site-directed mutants of photosystem II (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. By selecting under light-activated heterotrophic conditions, we have inactivated the psaA-psaB operon encoding the PSI reaction center proteins in cells containing deletions of the three psbA genes. We have also introduced deletions into both copies of psbD in a strain containing a mutation that inactivates psaA (ADK9). These strains, designated D1-/PSI- and D2-/PSI-, may serve as recipient strains for the incorporation of site-directed mutations in either psbA2 or psbD1. The characterization of these cells, which lack both PSI and PSII, is described.

Effects of cholate on Photosystem II: Selective extraction of a 22 kDa polypeptide and modification of QB-site activity

Abstract A quinone-mediated two-electron gate is shared by Photosystem II (PS II) and the photosystem of purple bacteria. In the bacterial reaction center, electron transfer from the reduced primary quinone acceptor, Q−A, to the secondary quinone, QB, as well as the sensitivity of this electron transfer step to inhibition by terbutryn, are regulated by the H subunit of the reaction center. Sequential removal of three polypeptides (10 and 22, followed by 28 kDa) from O2 evolving PS II reaction center complex preparations impairs QB activity. Removal of the 22 kDa protein does not abolish the herbicide sensitivity of electron transfer mediated by an added p-benzoquinone, but another of these proteins, a species of 28 kDa that binds chlorophyll, appears to regulate the ability of the herbicide DCMU to interfere with PS II electron transfer (Bowlby, N.R. et al. (1990) Curr. Res. Photosynth. I, 539–542). In this communication, we show that exposure of PS II to Na-cholate yields a preparation in which substantial depletion of a 22 kDa intrinsic protein has occurred. In the depleted preparations, a hydrophilic oxidant (Fe(CN)3−6), rather than a lipophilic p-benzoquinone acceptor, supports the highest rates of O2 evolution in a reaction that shows little sensitivity to the QB inhibitor DCMU. Although this observation might indicate a disruption of QB function that is associated with removal of the 22 kDa protein, this is not the case. It is shown here that under some conditions, Na-cholate will modify the response of PS II to Fe(CN)3−6 without release of the 22 kDa protein.

Chlorophyll-Protein Interactions in Photosystem II. Resonance Raman Spectroscopy of the D1-D2-cytochrome b559 Complex and the 47 kDa Protein

Recent advances in the biochemistry of plant photosystem II (PSII) have led to the proposal that the complex composed of the D1 (32 kDa), D2 (34 kDa), and cytochrome b559(4 and 9 kDa) polypeptides constitutes the reaction center core (1). This complex, though similar in many respects to the reaction center complex of purple bacteria, has unique characteristics. Chief among these is its pigment stoichiometry. Unlike the bacterial reaction center complex, which binds six chlorin molecules, the D1-D2-cytochrome b559 complex has as many as 13 chlorin molecules (2).

A technique for the determination of protein concentration by neutron activation analysis of silver binding

A method for the quantitative determination of small amounts of protein samples was developed employing neutron activation analysis. Current methods of protein concentration determination are severely limited as a result of differences in the specific characteristics of each protein. Silver binding has been used as a sensitive colorimetric method to indicate the presence of protein. However, silver-protein complexes can have a variety of absorbance spectra unique to each protein, which complicate the analysis. Various amounts of specific proteins were equilibrated in an excess of silver nitrate prior to the reduction of the silver by the addition of NaBH4, HCHO, and NaOH. The protein-silver complex was rapidly separated from the unbound silver by centrifugation chromatography and the amount of bound silver was determined by INAA. The amount of silver was proportional to the amount of protein present in each sample. When the silver was not reduced prior to removal of the unbound silver by chromatography, only negligible amounts of silver remained bound to the protein. The stoichiometry of bound silver to protein on a molar basis showed relatively small differences for the proteins that were examined. This ratio was found to depend on the conditions of the binding and reduction of the silver. The results suggest that the binding of silver is not specific to any charged or polar groups on these proteins and may, therefore, provide a means of determination of the concentration of protein that has general application for all proteins.

X-Ray Absorption Spectroscopy of the Photosynthetic Oxygen Evolving Complex

X-ray absorption spectroscopy (XAS) allows one to probe directly and specifically the coordination environment of a metal ion in a metalloprotein. XAS has been utilized extensively by Klein, Sauer, and coworkers[1], and more recently by Goerge et al.[2] to investigate the average environment of the Mn atoms in the photosynthetic oxygen evolving complex (OEC). Previous studies have utilized either BBY membrane preparations or PSII preparations from photosynthetic bacteria. We have recently measured XAS data for the Mn in highly purified reaction center complexes[3]. The higher Mn concentrations of these samples, coupled with the use of a high resolution solid state detector arry, has allowed us to obtain a significant improvement in the signal-to-noise ratio for our XAS data. Our EXAFS data for the S1 state of the OEC (an average of 25 scans) are shown in Figure 1. In the following, we describe these data and their structural interpretation, and compare our interpretations with those previously proposed.

Alteration of QB Activity in Photosystem II

The PSII RC complex preparation developed by Ghanotakis and Yocum (1) has 9 subunits and retains native QB activity. The 6 subunit “core” preparations described by Ikeuchi et al. (2) and Ghanotakis et al. (3) have impaired QB activity; highest rates of O2 evolution are seen with Fe(CN)6 3− as a DCMU insensitive electron acceptor. Removal of the 28, 22, and 10 kDa intrinsic PSII proteins inhibits electron transfer through the iron-quinone acceptor complex. The inhibition of QA − → QB electron transfer caused by chromatographic removal of the 28 kDa protein is accompanied by the appearance of an EPR signal at g=6.1 ascribed to the non-heme iron of the QA-Fe-QB acceptor complex. Our results suggest that the 28 kDa chl-a binding protein plays a role in the regulation of QB-site activity in the 9 subunit PSII RC complex, perhaps by directly providing a ligand to the non-heme iron.