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 of Scenedesmus (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.

Single core polypeptide in the reaction center of the photosynthetic bacterium Heliobacillus mobilis: structural implications and relations to other photosystems.

The gene for a reaction center core polypeptide from the anoxygenic photosynthetic bacterium Heliobacillus mobilis was cloned and sequenced. The deduced amino acid sequence consists of 609 residues with a molecular mass of 68 kDa. An adjacent open reading frame is not transcribed under our experimental conditions. No evidence for a second related reaction center core gene was found. The primary sequence of the reaction center protein (P800 protein) shows a high percentage of sequence identity to photosystem I in a cysteine-containing loop, which is the putative binding site of the iron-sulfur center FX and in the preceding hydrophobic region. Our data imply a homodimeric organization of the reaction center. This is fundamentally different from photosystem I and most other photosynthetic reaction centers, where the reaction center core is composed of two similar but nonidentical subunits.

Role of the carboxy terminus of polypeptide D1 in the assembly of a functional water-oxidizing manganese cluster in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: assembly requires a free carboxyl group at C-terminal position 344.

The D1 polypeptide of the photosystem II (PSII) reaction center is synthesized as a precursor polypeptide which is posttranslationally processed at the carboxy terminus. It has been shown in spinach that such processing removes nine amino acids, leaving Ala344 as the C-terminal residue [Takahashi, M., Shiraishi, T., & Asada, K. (1988) FEBS Lett. 240, 6-8; Takahashi, Y., Nakane, H., Kojima, H., & Satoh, K. (1990) Plant Cell Physiol. 31, 273-280]. We show here that processing on the carboxy side of Ala344 also occurs in the cyanobacterium Synechocystis 6803, resulting in the removal of 16 amino acids. By constructing a deletion strain of Synechocystis 6803 that lacks the three copies of the psbA gene encoding D1, we have developed a system for generating psbA mutants. Using this system, we have constructed mutants of Synechocystis 6803 that are modified in the region of the C-terminus of the D1 polypeptide. Characterization of these mutants has revealed that (1) processing of the D1 polypeptide is blocked when the residue after the cleavage site is changed from serine to proline (mutant Ser345Pro) with the result that the manganese cluster is unable to assemble correctly; (2) the C-terminal extension of 16 amino acid residues can be deleted with little consequence either for insertion of D1 into the thylakoid membrane or for assembly of D1 into a fully active PSII complex; (3) removal of only one more residue (mutant Ala344stop) results in a loss of assembly of the manganese cluster; and (4) the ability of detergent-solubilized PSII core complexes (lacking the manganese cluster) to bind and oxidize exogenous Mn2+ by the secondary donor, Z+, is largely unaffected in the processing mutants (the Ser345Pro mutant of Synechocystis 6803 and the LF-1 mutant of Scenedesmus obliquus) and the truncation mutant Ala344stop. Our results are consistent with a role for processing in regulating the assembly of the photosynthetic manganese cluster and a role for the free carboxy terminus of the mature D1 polypeptide in the ligation of one or more manganese ions of the cluster.

Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I.

Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 Mr. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in highly conserved region that is proposed to bind iron-sulfur center Fx in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%. Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of -414 mV with n = 2 titration behavior. In membranes, the behavior is intermediate between n = 1 and n = 2, and the apparent midpoint potential is -444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A1, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators. These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.

Protein sequences and redox titrations indicate that the electron acceptors in reaction centers from heliobacteria are similar to Photosystem I.

Photosynthetic reaction centers isolated from Heliobacillus mobilis exhibit a single major protein on SDS-PAGE of 47 000 Mr. Attempts to sequence the reaction center polypeptide indicated that the N-terminus is blocked. After enzymatic and chemical cleavage, four peptide fragments were sequenced from the Heliobacillus mobilis apoprotein. Only one of these sequences showed significant specific similarity to any of the protein and deduced protein sequences in the GenBank data base. This fragment is identical with 56% of the residues, including both cysteines, found in the highly conserved region that is proposed to bind iron-sulfur center FX in the Photosystem I reaction center peptide that is the psaB gene product. The similarity to the psaA gene product in this region is 48%.Redox titrations of laser-flash-induced photobleaching with millisecond decay kinetics on isolated reaction centers from Heliobacterium gestii indicate a midpoint potential of -414 mV with n=2 titration behavior. In membranes, the behavior is intermediate between n=1 and n=2, and the apparent midpoint potential is -444 mV. This is compared to the behavior in Photosystem I, where the intermediate electron acceptor A1, thought to be a phylloquinone molecule, has been proposed to undergo a double reduction at low redox potentials in the presence of viologen redox mediators.These results strongly suggest that the acceptor side electron transfer system in reaction centers from heliobacteria is indeed analogous to that found in Photosystem I. The sequence similarities indicate that the divergence of the heliobacteria from the Photosystem I line occurred before the gene duplication and subsequent divergence that lead to the heterodimeric protein core of the Photosystem I reaction center.

Isolation of a photoactive photosynthetic reaction center-core antenna complex from Heliobacillus mobilis.

A photoactive reaction center-core antenna complex was isolated from the photosynthetic bacterium Heliobacillus mobilis by extraction of membranes with Deriphat 160c followed by differential centrifugation and sucrose density gradient ultracentrifugation. The purified complex contained a Mr 47,000 polypeptide(s) that bound both the primary donor (P800) and approximately 24 antenna bacteriochlorophylls g. Time-resolved fluorescence emission spectroscopy indicated that the antenna bacteriochlorophylls g are active in energy transfer to P800, exhibiting a decay time of 25 ps. The complex contained 1.4 menaquinones, 9 Fe, and 3 labile S2- per P800. The complex was photoactive with an exponential decay time of 14 ms for P800+ yet showed no EPR-detectable Fe-S center signal in the g less than or equal to 2.0 region, either by chemical reduction to -600 mV or by illumination of reduced samples. The complex is similar to photosystem I of oxygen-evolving photosynthetic systems in that both the primary donor and a core antenna are bound to the same pigment-protein complex.

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum.

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.

Purification and characterization of a biliverdin-associated protein from the hemolymph of Manduca sexta.

A biliverdin binding protein, insecticyanin, has been isolated from the hemolymph of the fourth instar tobacco hornworm Manduca sexta. The protein has been purified to apparent homogeneity by conventional chromatography with a cumulative yield of 40-50%. The protein (Mw 71 600) is composed of three subunits (Mr 23 000). Each subunit binds one biliverdin molecule. Proton magnetic resonance spectroscopy and absorption spectroscopy demonstrate that the bilin is the biliverdin IX gamma isomer.