Distribution of the extrinsic proteins as a potential marker for the evolution of photosynthetic oxygen-evolving photosystem II

Distribution of photosystem II (PSII) extrinsic proteins was examined using antibodies raised against various extrinsic proteins from different sources. The results showed that a glaucophyte (Cyanophora paradoxa) having the most primitive plastids contained the cyanobacterial-type extrinsic proteins (PsbO, PsbV, PsbU), and the primitive red algae (Cyanidium caldarium) contained the red algal-type extrinsic proteins (PsO, PsbQ', PsbV, PsbU), whereas a prasinophyte (Pyraminonas parkeae), which is one of the most primitive green algae, contained the green algal-type ones (PsbO, PsbP, PsbQ). These suggest that the extrinsic proteins had been diverged into cyanobacterial-, red algal- and green algal-types during early phases of evolution after a primary endosymbiosis. This study also showed that a haptophyte, diatoms and brown algae, which resulted from red algal secondary endosymbiosis, contained the red algal-type, whereas Euglena gracilis resulted from green algal secondary endosymbiosis contained the green algal-type extrinsic proteins, suggesting that the red algal- and green algal-type extrinsic proteins have been retained unchanged in the different lines of organisms following the secondary endosymbiosis. Based on these immunological analyses, together with the current genome data, the evolution of photosynthetic oxygen-evolving PSII was discussed from a view of distribution of the extrinsic proteins, and a new model for the evolution of the PSII extrinsic proteins was proposed.     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.     

Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.     

In vitro growth and development of bovine oocyte-granulosa cell complexes on the flat substratum: effects of high polyvinylpyrrolidone concentration in culture medium

The aim of this study was to establish a culture system to support the growth of bovine oocytes as enclosed in granulosa cell complexes that extend on a flat substratum. Such systems have been established for mouse oocytes but are not applicable to larger animals because it is difficult to maintain an appropriate association between the oocyte and companion somatic cells. Growing bovine oocytes with a mean diameter of 95 microm were isolated from early antral follicles: the growing stage corresponds to that of oocytes in preantral follicles of 12-day-old mice. Oocyte-granulosa cell complexes were cultured for 14 days in modified TCM199 medium supplemented with 5% fetal bovine serum, 4 mM hypoxanthine, and 0.1 microg/ml estradiol. The novel modification made for this medium was a high concentration, 4% (w/v), of polyvinylpyrrolidone (PVP; molecular weight of 360000). The flat substratum used was either an insert membrane fit in the culture plate or the bottom surface of the wells of 96-well culture plates. PVP influenced the organization of complexes, resulting in a firm association between the oocyte and the innermost layer of surrounding cells. More oocytes enclosed by a complete cell layer were recovered from the medium supplemented with 4% PVP than from the control medium. Similarly, of the oocytes initially introduced into the growth culture, a significantly larger proportion developed to the blastocyst stage from medium containing 4% PVP than from medium without PVP. When PVP medium was used, the overall yield of blastocysts was similar between the system with the insert membranes (12%) and that with the 96-well culture plates (9%). A calf was produced from one of four embryos derived from oocytes grown in 96-well culture plates, matured, and fertilized in vitro and then transferred to a recipient cow.     

Identification of crucial histidines involved in carbon-nitrogen triple bond synthesis by aldoxime dehydratase

Aldoxime dehydratase (OxdA), which is a novel heme protein, catalyzes the dehydration of an aldoxime to a nitrile even in the presence of water in the reaction mixture. The combination of site-directed mutagenesis of OxdA (mutation of all conserved histidines in the aldoxime dehydratase superfamily), estimation of the heme contents and specific activities of the mutants, and CD and resonance Raman spectroscopic analyses led to the identification of the proximal and distal histidines in this unique enzyme. The heme contents and CD spectra in the far-UV region of all mutants except for the H299A one were almost identical to those of the wild-type OxdA, whereas the H299A mutant lost the ability of binding heme, demonstrating that His(299) is the proximal histidine. On the other hand, substitution of alanine for His(320) did not affect the overall structure of OxdA but caused loss of its ability of carbon-nitrogen triple bond synthesis and a lower shift of the Fe-C stretching band in the resonance Raman spectrum for the CO-bound form. Furthermore, the pH dependence of the wild-type OxdA closely followed the His protonation curves observed for other proteins. These findings suggest that His(320) is located in the distal heme pocket of OxdA and would donate a proton to the substrate in the aldoxime dehydration mechanism.     

Contribution of comparative fish studies to general endocrinology: structure and function of some osmoregulatory hormones

Fish endocrinologists are commonly motivated to pursue their research driven by their own interests in these aquatic animals. However, the data obtained in fish studies not only satisfy their own interests but often contribute more generally to the studies of other vertebrates, including mammals. The life of fishes is characterized by the aquatic habitat, which demands many physiological adjustments distinct from the terrestrial life. Among them, body fluid regulation is of particular importance as the body fluids are exposed to media of varying salinities only across the thin respiratory epithelia of the gills. Endocrine systems play pivotal roles in the homeostatic control of body fluid balance. Judging from the habitat-dependent control mechanisms, some osmoregulatory hormones of fish should have undergone functional and molecular evolution during the ecological transition to the terrestrial life. In fact, water-regulating hormones such as vasopressin are essential for survival on the land, whereas ion-regulating hormones such as natriuretic peptides, guanylins and adrenomedullins are diversified and exhibit more critical functions in aquatic species. In this short review, we introduce some examples illustrating how comparative fish studies contribute to general endocrinology by taking advantage of such differences between fishes and tetrapods. In a functional context, fish studies often afford a deeper understanding of the essential actions of a hormone across vertebrate taxa. Using the natriuretic peptide family as an example, we suggest that more functional studies on fishes will bring similar rewards of understanding. At the molecular level, recent establishment of genome databases in fishes and mammals brings clues to the evolutionary history of hormone molecules via a comparative genomic approach. Because of the functional and molecular diversification of ion-regulating hormones in fishes, this approach sometimes leads to the discovery of new hormones in tetrapods as exemplified by adrenomedullin 2.     

Binding and Functional Properties of Five Extrinsic Proteins in Oxygen-evolving Photosystem II from a Marine Centric Diatom,Chaetoceros gracilis

Oxygen-evolving photosystem II (PSII) isolated from a marine centric diatom, Chaetoceros gracilis, contains a novel extrinsic protein (Psb31) in addition to four red algal type extrinsic proteins of PsbO, PsbQ′, PsbV, and PsbU. In this study, the five extrinsic proteins were purified from alkaline Tris extracts of the diatom PSII by anion and cation exchange chromatographic columns at different pH values. Reconstitution experiments in various combinations with the purified extrinsic proteins showed that PsbO, PsbQ′, and Psb31 rebound directly to PSII in the absence of other extrinsic proteins, indicating that these extrinsic proteins have their own binding sites in PSII intrinsic proteins. On the other hand, PsbV and PsbU scarcely rebound to PSII alone, and their effective bindings required the presence of all of the other extrinsic proteins. Interestingly, PSII reconstituted with Psb31 alone considerably restored the oxygen evolving activity in the absence of PsbO, indicating that Psb31 serves as a substitute in part for PsbO in supporting oxygen evolution. A significant difference found between PSIIs reconstituted with Psb31 and with PsbO is that the oxygen evolving activity of the former is scarcely stimulated by Cl⁻ and Ca²⁺ ions but that of the latter is largely stimulated by these ions, although rebinding of PsbV and PsbU activated oxygen evolution in the absence of Cl⁻ and Ca²⁺ ions in both the former and latter PSIIs. Based on these results, we proposed a model for the association of the five extrinsic proteins with intrinsic proteins in diatom PSII and compared it with those in PSIIs from the other organisms.     

Mammalian Mitochondrial DNA Replication Intermediates Are Essentially Duplex but Contain Extensive Tracts of RNA/DNA Hybrid

We demonstrate, using transmission electron microscopy and immunopurification with an antibody specific for RNA/DNA hybrid, that intact mitochondrial DNA replication intermediates are essentially duplex throughout their length but contain extensive RNA tracts on one strand. However, the extent of preservation of RNA in such molecules is highly dependent on the preparative method used. These findings strongly support the strand-coupled model of mitochondrial DNA replication involving RNA incorporation throughout the lagging strand.