Rapid-Scan Fourier Transform Infrared Monitoring of the Photoactivation Process in Cyanobacterial Photosystem II.

The catalytic site of photosynthetic water oxidation, the Mn4CaO5 cluster, in photosystem II (PSII) is known to be formed by a light-induced process called photoactivation. However, details of its molecular mechanism remain unresolved. In this study, we monitored the photoactivation process in cyanobacterial PSII using rapid-scan, time-resolved Fourier transform infrared (FTIR) spectroscopy. The Mn3+/Mn2+ FTIR difference spectra of PSII, in which D1-D170 was specifically 13C labeled, and PSII from the D1-D170A, D1-E189A, and D1-D342A mutants provide strong evidence that the initial Mn2+ is coordinated by D1-D170 and D1-E189. Protein conformational changes and relocation of photo-oxidized Mn3+ in the dark rearrangement process were detected as slow-phase signals in the amide I and carboxylate regions, whereas similar signals were not observed in D1-E189A PSII. It is thus proposed that relocation of Mn3+ via D1-E189 induces the conformational changes of the proteins to form proper Mn binding sites in the mature protein conformation.

Post-translational amino acid conversion in photosystem II as a possible origin of photosynthetic oxygen evolution.

Photosynthetic oxygen evolution is performed at the Mn cluster in photosystem II (PSII). The advent of this reaction on ancient Earth changed its environment by generating an oxygenic atmosphere. However, how oxygen evolution originated during the PSII evolution remains unknown. Here, we characterize the site-directed mutants at the carboxylate ligands to the Mn cluster in cyanobacterial PSII. A His residue replaced for D1-D170 is found to be post-translationally converted to the original Asp to recover oxygen evolution. Gln/Asn residues in the mutants at D1-E189/D1-D342 are also converted to Glu/Asp, suggesting that amino-acid conversion is a common phenomenon at the ligand sites of the Mn cluster. We hypothesize that post-translational generation of carboxylate ligands in ancestral PSII could have led to the formation of a primitive form of the Mn cluster capable of partial water oxidation, which could have played a crucial role in the evolutionary process of photosynthetic oxygen evolution.

Role of the O4 Channel in Photosynthetic Water Oxidation as Revealed by Fourier Transform Infrared Difference and Time-Resolved Infrared Analysis of the D1-S169A Mutant.

Photosynthetic water oxidation takes place at the Mn4CaO5 cluster in photosystem II. Although the atomic structures of its intermediates called S states have recently been reported, the catalytic mechanism of water oxidation has not been well understood. Here, to investigate the involvement of the O4 site of the Mn4CaO5 cluster and a water channel from O4 in the water oxidation reaction, we examined the effects of D1-S169A mutation, which perturbs the interaction of a water molecule hydrogen-bonded with O4, by thermoluminescence (TL), Fourier transform infrared (FTIR) difference, and time-resolved infrared (TRIR) measurements. The observed upshifts of TL peaks and some changes in FTIR spectra upon S169A mutation revealed the perturbations of the redox potential of the Mn4CaO5 cluster and the interactions of the surrounding hydrogen bond network. In contrast, FTIR oscillation patterns and TRIR traces showed only minor effects of the mutation on the efficiencies and kinetics of individual S-state transitions. It was thus concluded that the O4 site plays a role in retaining the redox potential and the structure of the hydrogen bond network, whereas it is unlikely to be directly involved in the catalytic reaction of substrate water except for proton transfer through the O4 water chain.

Fourier transform infrared and mass spectrometry analyses of a site-directed mutant of D1-Asp170 as a ligand to the water-oxidizing Mn4CaO5 cluster in photosystem II.

The Mn4CaO5 cluster, the catalytic center of water oxidation in photosystem II (PSII), is coordinated by six carboxylate and one imidazole ligands. The roles of these ligands in the water oxidation mechanism remain largely unknown. In this study, we constructed a D1-D170H mutant, in which the Asp ligand bridging Mn and Ca ions was replaced with His, in the cyanobacterium Synechocystis sp. PCC 6803, and analyzed isolated PSII core complexes using Fourier transform infrared (FTIR) difference spectroscopy and mass spectrometry (MS). The S2-minus-S1 FTIR difference spectrum of the PSII complexes of the D1-D170H mutant showed features virtually identical to those of the wild-type PSII. MS analysis further showed that ~70% of D1 proteins from the PSII complexes of D1-D170H possessed the wild-type amino acid sequence, although only the mutated sequence was detected in genomic DNA in the same batch of cells for PSII preparations. In contrast, a D1-S169A mutant as a control showed a modified FTIR spectrum and only a mutated D1 protein. It is thus concluded that the FTIR spectrum of the D1-D170H mutant actually reflects that of wild-type PSII, whereas the Mn4CaO5 cluster is not formed in PSII with D1-D170H mutation. Although the mechanism of production of the wild-type D1 protein in the D1-D170H mutant is unknown at present, a caution is necessary in the analysis of site-directed mutants of crucial residues in the D1 protein, and mutation has to be confirmed not only at the DNA level but also at the amino acid level.

Crystal structure of Cruxrhodopsin-3 from Haloarcula vallismortis.

Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.

Gene orders in the upstream of 16S rRNA genes divide genera of the family Halobacteriaceae into two groups.

In many prokaryotic species, 16S rRNA genes are present in multiple copies, and their sequences in general do not differ significantly owing to concerted evolution. At the time of writing, the genus Haloarcula of the family Halobacteriaceae comprises nine species with validly published names, all of which possess two to four highly heterogeneous 16S rRNA genes. Existence of multiple heterogeneous 16S rRNA genes makes it difficult to reconstruct a biological phylogenetic tree using their sequence data. If the orthologous gene is able to be discriminated from paralogous genes, a tree reconstructed from orthologous genes will reflect a simple biological phylogenetic relationship. At present, however, we have no means to distinguish the orthologous rRNA operon from paralogous ones in the members of the family Halobacteriaceae. In this study, we found that the dihydroorotate oxidase gene, pyrD, was present in the immediate upstream of one 16S rRNA gene in each of ten strains of the family Halobacteriaceae whose genome sequences have been determined, and the direction of the pyrD gene was opposite to that of the 16S rRNA genes. In two other strains whose genome sequences have been determined, the pyrD gene was present in far separated positions. We designed PCR primer sets to amplify DNA fragments encompassing a region from the conserved region of the pyrD gene to a conserved region of the tRNA-Ala gene or the 23S rRNA gene to determine the 16S rRNA gene sequences preceded by the pyrD gene, and to see if the pyrD gene is conserved in the immediate upstream of rRNA operon(s) in the type strains of the type species of 28 genera of the family Halobacteriaceae. Seventeen type strains, including the ten strains mentioned above, gave amplified DNA fragments of approximately 4000 bp, while eleven type strains, including the two strains mentioned above, did not give any PCR products. These eleven strains are members of the Clade I haloarchaea, originally defined by Walsh et al. (2004) and expanded by Minegishi et al. (2010). Analysis of contig sequences of three strains belonging to the Clade I haloarchaea also revealed the absence of the pyrD gene in the immediate upstream of any 16S rRNA genes. It may be scientifically sound to hypothesize that during the evolution of members of the family Halobacteriaceae, a pyrD gene transposition event happened in one group and this was followed by subsequent speciation processes in each group, yielding species/genera of the Clade I group and 'the rest' of the present family Halobacteriaceae.

Functionally important structural elements of the cyanobacterial clock-related protein Pex.

Pex, a clock-related protein involved in the input pathway of the cyanobacterial circadian clock system, suppresses the expression of clock gene kaiA and lengthens the circadian period. Here, we determined the crystal structure of Anabaena Pex (AnaPex; Anabaena sp. strain PCC 7120) and Synechococcus Pex (SynPex; Synechococcus sp. strain PCC 7942). Pex is a homodimer that forms a winged-helix structure. Using the DNase I protection and electrophoresis mobility shift assays on a Synechococcus kaiA upstream region, we identified a minimal 25-bp sequence that contained an imperfectly inverted repeat sequence as the Pex-binding sequence. Based on crystal structure, we predicted the amino acid residues essential for Pex's DNA-binding activity and examined the effects of various Ala-substitutions in the alpha3 helix and wing region of Pex on in vitro DNA-binding activity and in vivo rhythm functions. Mutant AnaPex proteins carrying a substitution in the wing region displayed no specific DNA-binding activity, whereas those carrying a substitution in the alpha3 helix did display specific binding activity. But the latter were less thermostable than wild-type AnaPex and their in vitro functions were defective. We concluded that Pex binds a kaiA upstream DNA sequence via its wing region and that its alpha3 helix is probably important to its stability.

Salinibacter sensory rhodopsin: sensory rhodopsin I-like protein from a eubacterium.

Halobacterium salinarum sensory rhodopsin I (HsSRI), a dual receptor regulating both negative and positive phototaxis in haloarchaea, transmits light signals through changes in protein-protein interactions with its transducer, halobacterial transducer protein I (HtrI). Haloarchaea also have another sensor pigment, sensory rhodopsin II (SRII), which functions as a receptor regulating negative phototaxis. Compared with HsSRI, the signal relay mechanism of SRII is well characterized because SRII from Natronomonus pharaonis (NpSRII) is much more stable than HsSRI and HsSRII, especially in dilute salt solutions and is much more resistant to detergents. Two genes encoding SRI homologs were identified from the genome sequence of the eubacterium Salinibacter ruber. Those sequences are distantly related to HsSRI ( approximately 40% identity) and contain most of the amino acid residues identified as necessary for its function. To determine whether those genes encode functional protein(s), we cloned and expressed them in Escherichia coli. One of them (SrSRI) was expressed well as a recombinant protein having all-trans retinal as a chromophore. UV-Vis, low-temperature UV-Vis, pH-titration, and flash photolysis experiments revealed that the photochemical properties of SrSRI are similar to those of HsSRI. In addition to the expression system, the high stability of SrSRI makes it possible to prepare large amounts of protein and enables studies of mutant proteins that will allow new approaches to investigate the photosignaling process of SRI-HtrI.

A photochromic photoreceptor from a eubacterium.

Sensory rhodopsin I (SRI) is one of the most interesting photo- sensory receptors because of its function in using the photochromic reaction to mediate opposing signals which depend on the color of light. It was initially thought that SRI exists only in the archaea, but we recently reported for the first time a newly functional SRI from a eubacterium, Salinibacter ruber (SrSRI). The amino acid sequence of SrSRI shows 43% identity with the well-known SRI (HsSRI) and contains most of the amino acid residues identified as necessary for SRI function. The photochemical properties of SrSRI are similar to those of HsSRI. In addition, SrSRI is a highly stable protein, even in dilute salt conditions. Thus, SrSRI could be a key protein for characterizing its association with the SrSRI transducer protein, SrHtrI, and for elucidating structural changes of SRI and HtrI that occur during their function. Recently, new approaches to manipulate cellular functions with rhodopsins have been established. SRI can activate and deactivate a kinase, CheA, by the photochromic reaction. Kinases are key molecules for signal transduction in various organisms, and SRI could potentially manipulate their cellular functions.