Ronald S. Hutchison

[23] Measurement of equilibrium midpoint potentials of thiol/disulfide regulatory groups on thioredoxinactivated chloroplast enzymes

Publisher Summary This chapter presents an experimental strategy and protocol for measuring the reversible exchange of a protein intramolecular disulfide between thioredoxin f and chloroplast target enzymes. These data can in turn be used to determine the equilibrium redox midpoint potentials that control the change in catalytic activity of these enzymes. The chapter uses the titration of fructose-l,6-bisphosphatase (FBPase), a nuclear-encoded protein located in the chloroplast stroma, to illustrate the procedures and considerations involved with determination of equilibrium redox midpoint potentials of the thiol/disulfide conversions controlling chloroplast enzyme activation. Chloroplast FBPase is a tetramer composed of four identical 45-kDa subunits forming a holoenzyme of 180 kDa. The thioredoxin f-dependent reduction of the regulatory disulfide on FBPase can be measured using the thiol-labeling reagent monobromobimane (mBBr) that reacts rapidly and exclusively with accessible thiol groups, producing a fluorescent, chemically modified protein. The thermodynamics of the thiol/disulfide exchange between thioredoxin and the regulatory sulfhydryl groups of the various light-regulated chloroplast enzymes is of increasing interest with the emergence of evidence that differences in the redox properties among the enzymes may play an important role in the regulation of photosynthesis.

Alterations in Carboxylate Ligation at the Active Site of Photosystem II

Photosystem II (PSII) is the photosynthetic enzyme catalyzing the oxidation of water and reduction of plastoquinone (Q). This reaction occurs at a catalytic site containing four manganese atoms and cycling among five oxidation states, the S n states, where n refers to the number of oxidizing equivalents stored. Biochemical and spectroscopic techniques have been used previously to conclude that aspartate 170 in the D1 subunit influences the structure and function of the PSII active site (Boerner, R. J., Nguyen, A. P., Barry, B. A., and Debus, R. J. (1992)Biochemistry 31, 6660–6672). Substitution of glutamate for aspartate 170 resulted in an assembled manganese cluster, which was capable of enzymatic turnover, but at lower steady-state oxygen evolution rates. Here, we obtained the difference (light-minus-dark) Fourier transform IR spectrum associated with the S2Q−-minus-S1Q transition by illumination of oxygen-evolving wild-type and DE170D1 PSII preparations at 200 K. These spectra are known to be dominated by contributions from carboxylic acid and carboxylate residues that are close to or ligating the manganese cluster. Substitution of glutamate for aspartate 170 results in alterations in the S2Q−-minus-S1Q spectrum; the alterations are consistent with a change in carboxylate coordination to manganese or calcium. In particular, the spectra are consistent with a shift from bridging/bidentate carboxylates in wild-type PSII to unidentate carboxylate ligation in DE170D1 PSII.

Mutagenesis of the Symmetry Related H117 Residue in the Photosystem II D2 Protein of Chlamydomonas : Implications for Energy Transfer from Accessory Chlorophylls

Similar to the quinone-type, bacterial photosynthetic reaction center (BRC), the photosystem II (PSII) reaction center complex contains four-five polypeptides. These polypeptides include: the D1, D2, cytochrome b559 polypeptides, plus a small polypeptide, psbI known from its gene product [1–5]. Two of the polypeptides (D1 and D2) have substantial sequence and topological similarity to the L and M subunits of the BRC [2, 4, 5]. Regions of the L and M and D1 and D2 proteins which are most highly conserved are the non-heme Fe and the chlorophyll special pair (ChlSP)ligands and adjacent residues. Similar to the BRC the PII complex has two quinones (QA and QB) per reaction center complex, but in contrast to the BRC has 6 chlorophylls per 2 pheophytins [6] (Figure 1). Two of the chlorophylls make up the chlorophyll special pair, two chlorophyll monomers participate in electron transfer between the Chlsp and pheophytin, and an additional pair of accessory chlorophylls is thought to be located at the margins of the reaction center complex [6–8]. It has been proposed that the accessory chlorophylls function as antennae or are involved in an electron transfer cycle around PSII (including cytochrome b559) which protects PSU from photoinhibitory protein degradation [7–9].

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.