Dexter A. Chisholm

Crystal structures of the photosystem II D1 C-terminal processing protease.

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 Å resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains.

Nucleotide sequence of psbC, the gene encoding the CP-43 chlorophyll a-binding protein of Photosystem II, in the cyanobacterium Synechocystis 6803.

The nucleotide sequence for the Photosystem II gene psbC has been determined for the cyanobacterium Synechocystis 6803. The gene overlaps the last 50 bases of the psbD gene, and both genes are transcribed in the same direction, but read in different frames. This arrangement is identical to that found in all chloroplast genomes for which psbC has been sequenced. The Synechocystis nucleotide sequence is 70% homologous to the tobacco gene and the predicted amino acid sequence shows 85% homology. A possible alternative translation start site for psbC has been conserved between seven plant sequences and the cyanobacterial sequence. The hydropathy plot for the cyanobacterial protein is very similar to plots determined for six plant species. Pairs of histidines that may play a role in binding chlorophyll are conserved between the cyanobacterial and plant amino acid sequences.

The D1 C-terminal processing protease of photosystem II from Scenedesmus obliquus. Protein purification and gene characterization in wild type and processing mutants

Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain ofScenedesmus (isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protease can compensate for its loss.

Nucleotide Sequences of Both psbD Genes from the Cyanobacterium Synechocystis 6803

A genetic system has been developed for site-specific mutagenesis of the D2 polypeptide of Photosystem II in the cyanobacterium Synechocystis 6803. In chloroplasts, this polypeptide is encoded by the psbD gene (1). The discovery of significant amino acid homologies between the D2 polypeptide and the M-subunit protein of the bacterial photosynthetic reaction center has led to the proposal that certain amino acid residues in D2 might bind the reaction center chlorophyll, the quinone QA, and the ferrous non-heme iron.

Correlation of the cytochrome c 2 content of cyanobacterial Photosystem II with the EPR properties of the oxygen-evolving complex

The Mn4 cluster of PS II advances through a series of oxidation states (S states) that catalyze the breakdown of water to dioxygen in the oxygen-evolving complex. The present study describes the engineering and purification of highly active PS II complexes from mesophilic His-tagged Synechocystis PCC 6803 and purification of PS II core complexes from thermophilic wild-type Synechococcus lividus with high levels of the extrinsic polypeptide, cytochrome c550. The g = 4.1 S2 state EPR signal, previously not characterized in untreated cyanobacterial PS II, is detected in high yields in these PS II preparations. We present a complete characterization of the g = 4.1 state in cyanobacterial His-tagged Synechocystis PCC 6803 PS II and S. lividus PS II. Also presented are a determination of the stoichiometry of cytochrome c550 bound to His-tagged Synechocystis PCC 6803 PS II and analytical ultracentrifugation results which indicate that cytochrome c550 is a monomer in solution. The temperature-dependent multiline to g = 4.1 EPR signal conversion observed for the S2 state in cyanobacterial PS II with high cytochrome c550 content is very similar to that previously found for spinach PS II. In spinach PS II, the formation of the S2 state g = 4.1 EPR signal has been found to correlate with the binding of the extrinsic 17 and 23 kDa polypeptides. The finding of a similar correlation in cyanobacterial PS II with the binding of cytochrome c550 suggests a functional homology between cytochrome c550 and the 17 and 23 kDa extrinsic proteins of spinach PS II.

Coordination of proton and electron transfer from the redox-active tyrosine, YZ, of Photosystem II and examination of the electrostatic influence of oxidized tyrosine, YD˙(H+)

The redox active tyrosines, YZ and YD, of Photosystem II are oxidized by P680+ to the neutral radical. Such oxidation requires coupling of electron transfer to the transfer of the phenolic proton. Studies of the multiphasic kinetics of YZ oxidation in Mn-depleted PSII core complexes have shown that the relative amplitudes of the kinetic components are pH-dependent with one component showing a pH-dependent t1/2 in the microsecond to tens of microsecond range (pH 4–8). Sjodin and coworkers (M. Sjodin, S. Styring, B. Akemark, L. Sun and L. Hammarstrom, Philos. Trans. R. Soc. London, Ser. B, 2002, 357, 1471–1479) have suggested that the increase in rate of this latter component with pH reflects an increase in the driving force of the reaction by lowering the reduction potential of YZ˙/ YZ, consistent with concerted electron and proton transfer (CEP mechanism). A similar dependence of the rate of YZ oxidation on ΔG° is reported here through modification of the reduction potential of P680+/P680, that is, without modifying either the proton acceptor or the pathway for proton transfer. The results reported here support a CEP mechanism, though formation of the tyrosinate followed by electron transfer cannot be completely ruled out.The presence of oxidized tyrosine YD˙(H+) has been shown to accelerate the photoactivation of the oxygen evolving complex, possibly by an increase in the reduction potential of P680+/P680. The influence of YD˙(H+) on the P680+/P680 reduction potential is examined here by measuring the rate of YZ oxidation in Mn-depleted core complexes from the WT strain and from a YD-less strain of Synechocystis 6803. Also examined is the influence of YD˙(H+) on the P680+–P680 difference spectrum. These comparisons show that the electrostatic contribution of YD˙(H+) to the reduction potential of redox couple P680+/P680 is very small (≤10 mV), implying that the role of YD˙(H+) in photoactivation may have more to do with its providing an oxidizing equivalent during assembly of the manganese cluster.

Site-Directed Mutagenesis in the Photosystem II Gene psbD, Encoding the D2 Protein

Using site-directed mutagenesis, it was shown that two histidine residues (his-197 and his-214) in the Photosystem II protein D2 play a crucial role in the function of the Photosystem II complex in the cyanobacterium Synechocystis 6803. A specific change of either histidine residue into tyrosine and asparagine, respectively, lead to a loss of PS II activity. These histidine residues have been hypothesized to be analogous to the histidine residues of the M-subunit from purple bacteria involved in binding of the reaction center chromophore, and Q and Fe , respectively (see ref. 1 and 2). The data presented here can be interpreted to support this hypothesis. The data obtained are consistent with the hypothesis that the binding determinants for the PS II reaction center are created mainly by D1 and D2.

Engineering and Rapid Purification of Histidine-Tagged Photosystem II from Synechocystis PCC 6803

The cyanobacterium Synechocystis PCC 6803 is especially useful in site-directed mutagenesis studies of photosystem II (PSII). Unfortunately, methods which rely on ion-exchange chromatography for recovery of the mutant PSII (1–4) are lengthy and can even be inadequate for the generation of material in sufficient yield and purity for biophysical study when the level of PSII expression in the mutant is low. Here we present the use of an engineered hexahistidine tag fused to the carboxy-terminus of the CP47 subunit for the rapid purification of PSII core complexes from Synechocystis PCC 6803 by Ni2+-affinity chromatography. A recent paper also reported purification of PSII from Chlamydomonas using a His-tagged D2 subunit (5).