Scott D. Betts

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O2 evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).

Functional reconstitution of photosystem II with recombinant manganese-stabilizing proteins containing mutations that remove the disulfide bridge.

The 33-kDa extrinsic subunit of PSII stabilizes the O2-evolving tetranuclear Mn cluster and accelerates O2 evolution. We have used site-directed mutagenesis to replace one or both Cys residues in spinach MSP with Ala. Previous experiments using native and reduced MSP led to the conclusion that a disulfide bridge between these two cysteines is essential both for its binding and its functional properties. We report here that the disulfide bridge, though essential for MSP stability, is otherwise dispensible. The mutation C51A by itself had a delayed effect on MSP function: [C51A]MSP restored normal rates of O2 evolution to PSII but was defective in stabilizing this activity during extended illumination. In contrast, the Cys-free double mutant, [C28A,C51A]MSP, was functionally identical to the wild-type protein. Based on results presented here, we propose a light-dependent interaction between MSP and PSII that occurs only during the redox cycling of the Mn cluster and which is destabilized by the single mutation, C51A.

Spectroscopic characterization of CP26, a chlorophyll ab binding protein of the higher plant Photosystem II complex

Abstract A spectroscopic study is presented of the minor chlorophyll a b binding protein CP26 isolated from spinach by means of a dodecylmaltoside/betaine washing procedure. The preparations are characterized by a chlorophyll a chlorophyll b ratio of 3.3 ± 0.1, and most likely contain 2 chlorophyll b (Chl b) and 6–7 chlorophyll a (Chl a) molecules per monomeric protein. Some of the spectroscopic properties of CP26 show strong similarities to those of the major chlorophyll a b light-harvesting protein LHC-II, which is in line with the sequence homologies between the two proteins. Spectroscopic differences in the Chl b absorption region are caused by the variation in Chl b content in both proteins. A strongly blue-shifted Chl b band at 637 nm in CP26 has similar linear and circular dichroism properties as the spectral component at 640 nm of LHC-II. It is suggested that these spectral features arise from a conserved Chl b molecule, and that the blue shifts are caused by charged amino acids in the vicinity of these Chl b molecules. The other Chl b band in CP26 is observed at 650 nm. Differences in the Chl a absorption region mainly concern the reduced absorption at 670 nm for CP26, whereas a strong band near 675 nm is very similar to the band at 676 nm for LHC II. Tentative assignments of several absorption bands of CP26 and LHC II to specific pigments are made on the basis of the recently reported three-dimensional structure of LHC II and of the primary amino acid sequences of both proteins. In the carotenoid region LHC II and CP26 show slightly different linear and circular dichroism signals. However, the differences are not large enough to exclude a similar arrangement of the two lutein molecules in LHC II and CP26.

Nucleotide sequence of cDNA encoding the precursor of the 23 kDa photosystem II protein of tomato

Photosystem II (PSII) in higher plants is a multiprotein transmembrane complex residing in the thylakoids of chloroplasts. Oxygen evolution activity of inside-out thylakoid vesicles is inhibited by treatment with 250 mM NaC1, which releases water-soluble polypeptides of 17 and 23 kDa from PSII [1]. Rebinding of the purified 23 kDa protein restores activity; this observation first implicated the 23 kDa protein as a component of the oxygen-evolving complex. The cDNA encoding the 23 kDa PSII protein from tomato (Lycopersicon esculentum) was isolated from a cDNA library probed with the corresponding cDNA from mustard (Sinapis alba) (Fig. 1). The amino acid sequence of the mature protein is 86~o identical to that for tobacco (Nicotiana tabacum) [2], also a member of the Solanaceae family. The mustard and spinach (Spinacia oIeracea) proteins share, respectively, 85 To and 81 ~o sequence identity with the tomato protein [ 3, 4]. Acknowledgements

Two-Dimensional Crystals of the CP47-D1-D2-Cytochrome b559 Complex of Photosystem II

Substantial progress has been made in understanding the structure and function of Photosystem II, both in terms of the catalytic activity and in terms of the polypeptides. Several groups have succeeded in reducing the oxygen-evolving reaction to a complex comprised of seven polypeptides, which, when supplemented with chloride and calcium, evolve oxygen at substantial rates. The complex includes six intrinsic membrane proteins, which serve the most essential photochemical and structural functions, and an extrinsic 33 kDa protein necessary for the stabilization of oxygen evolution. Recently, a smaller protein complex consisting of the D1, D2, 47 kDa and cytochrome b559 subunits was isolated in the non-ionic detergent Dodecylmaltoside (1,2).

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.

Bacterial Overexpression and Site-directed Mutagenesis of the PS2 Manganese Stabilizing Protein: Progress in Elucidation of Structure and Function

Manganese Stabilizing Protein (MSP) was discovered and purified by Kuwabara and Murata (1), and has since proven to be an essential extrinsic component of PS2 (reviewed in 2,3). Extraction of MSP modifies the tetranuclear Mn cluster of the O2-evolving complex; 2 Mn are released as Mn2+, the S2 and S3 states are abnormally stable, and the S3 → S4 → S0 step is slowed by a factor of 3–5. The isolated spinach protein is comprised of 247 amino acids with a molecular mass of 26,535 and a pi of 5.2. Analyses of secondary structure by CD shows that the protein in solution is comprised of α-helix (10%), β-sheet (33–38%) and about 50% turns and random coil (4,5). The technique of site-directed mutagenesis is a useful tool for probing protein structure and function. For MSP, two approaches have been applied. In the first, mutagenesis in vivo has been utilized in cyanobacteria (6). In the second, a method first used by Seidler and Michel (7) for overexpression and processing of precursor eukaryotic proteins in E. coli has provided a means for mutagenesis of MSP that is facilitated by the ease with which the overexpressed protein can be rebound to MSP-depleted PS2 samples. We have modified this method so that MSP inclusion bodies can be harvested from E. coil purified, and used to reconstitute high levels of activity (8). Here, we report on the use of recombinant wildtype and mutant forms of MSP in experiments designed to characterize the structure and function of this important PS2 protein.