BBA - Bioenergetics (v.1837, #11)

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum by Anne-Marie Carey; Kirsty Hacking; Nichola Picken; Suvi Honkanen; Sharon Kelly; Dariusz M. Niedzwiedzki; Robert E. Blankenship; Yuuki Shimizu; Zheng-Yu Wang-Otomo; Richard J. Cogdell (1849-1860).
This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.
Keywords: Purple sulphur bacteria; Light-harvesting complex 2; Bacteriochlorophyll a; Carotenoid; Polypeptide; Spectroscopy;

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila by Dmytro V. Gospodaryov; Oleh V. Lushchak; Bohdana M. Rovenko; Natalia V. Perkhulyn; Mike Gerards; Tea Tuomela; Howard T. Jacobs (1861-1869).
An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20–50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.
Keywords: Alternative respiratory chain enzymes; Tunicate; Gene therapy; Oxidative stress; Ageing;

Chemically modified reaction centers of photosystem II: Exchange of pheophytin a with 7-deformyl-7-hydroxymethyl-pheophytin b by Alexey A. Zabelin; Valentina A. Shkuropatova; Zoya K. Makhneva; Andrey A. Moskalenko; Vladimir A. Shuvalov; Anatoly Ya. Shkuropatov (1870-1881).
The native pheophytin a (Pheo a) in isolated reaction centers of photosystem II (PSII RCs) has been chemically exchanged with extraneous 7-deformyl-7-hydroxymethyl-Pheo b (71-OH-Pheo b) which differs from Pheo a by the C-7 substituent (hydroxymethyl instead of methyl). The two pigments have similar reduction potentials in vitro [M. Meyer, Dissertation, Universität München, 1997], while their absorption spectra show small but distinct differences in the visible region. The resulting 71-OH-Pheo b-modified reaction center preparations were characterized by high-performance liquid chromatography, electronic absorption and light-induced Fourier transform infra red absorption difference spectroscopies, together with photoaccumulation of the reduced pheophytin electron acceptor and NaBH4-treatment. About 70% of the total Pheo a molecules are found to be replaced by 71-OH-Pheo b molecules in modified preparations, indicating that both the photochemically active (PheoD1) and inactive (PheoD2) binding sites were subjected to pigment exchange. The 71-OH-Pheo b molecule located at the PheoD1 site is able to functionally replace the native Pheo a, participating in primary charge separation as an electron acceptor. The Qx absorption band of this modified pheophytin molecule is localized at ~ 546 nm; its Qy band is blue-shifted with respect to the absorption of other reaction center core pigments, being located at ~ 665 nm. The Qy and Qx optical transitions of the 71-OH-Pheo b molecule exchanged into the PheoD2 site are identified at 677 and 543.5 nm, respectively. The photochemically active double-modified PSII RCs additionally containing 7-deformyl-7-hydroxymethyl-131-deoxo-131-hydroxy-Pheo b at the PheoD2 site were obtained by treatment of the 71-OH-Pheo b-modified RCs with NaBH4.Display Omitted
Keywords: Photosystem II; Reaction center; Pheophytin a; 7-Deformyl-7-hydroxymethyl-pheophytin b; Chemical exchange; Charge separation;

We re-determined the near infrared (NIR) spectral signatures (650–980 nm) of the different cytochrome c oxidase redox centres, in the process separating them into their component species. We confirm that the primary contributor to the oxidase NIR spectrum between 700 and 980 nm is cupric CuA, which in the beef heart enzyme has a maximum at 835 nm. The 655 nm band characterises the fully oxidised haem a 3/CuB binuclear centre; it is bleached either when one or more electrons are added to the binuclear centre or when the latter is modified by ligands. The resulting ‘perturbed’ binuclear centre is also characterised by a previously unreported broad 715–920 nm band. The NIR spectra of certain stable liganded species (formate and CO), and the unstable oxygen reaction compounds P and F, are similar, suggesting that the latter may resemble the stable species electronically. Oxidoreduction of haem a makes no contribution either to the 835 nm maximum or the 715 nm band. Our results confirm the ability of NIRS to monitor the CuA centre of cytochrome oxidase activity in vivo, although noting some difficulties in precise quantitative interpretations in the presence of perturbations of the haem a 3/CuB binuclear centre.Display Omitted
Keywords: Near infrared spectroscopy; NIR; Mitochondria; Cytochrome oxidase; Copper; CuA;

High yield of secondary B-side electron transfer in mutant Rhodobacter capsulatus reaction centers by Lucas Kressel; Kaitlyn M. Faries; Marc J. Wander; Charles E. Zogzas; Rachel J. Mejdrich; Deborah K. Hanson; Dewey Holten; Philip D. Laible; Christine Kirmaier (1892-1903).
From the crystal structures of reaction centers (RCs) from purple photosynthetic bacteria, two pathways for electron transfer (ET) are apparent but only one pathway (the A side) operates in the native protein-cofactor complex. Partial activation of the B-side pathway has unveiled the true inefficiencies of ET processes on that side in comparison to analogous reactions on the A side. Of significance are the relative rate constants for forward ET and the competing charge recombination reactions. On the B side, these rate constants are nearly equal for the secondary charge-separation step (ET from bacteriopheophytin to quinone), relegating the yield of this process to < 50%. Herein we report efforts to optimize this step. In surveying all possible residues at position 131 in the M subunit, we discovered that when glutamic acid replaces the native valine the efficiency of the secondary ET is nearly two-fold higher than in the wild-type RC. The positive effect of M131 Glu is likely due to formation of a hydrogen bond with the ring V keto group of the B-side bacteriopheophytin leading to stabilization of the charge-separated state involving this cofactor. This change slows charge recombination by roughly a factor of two and affords the improved yield of the desired forward ET to the B-side quinone terminal acceptor.Display Omitted
Keywords: Photosynthetic reaction center; Charge recombination; High-throughput screening; Ultrafast spectroscopy; Directed evolution; Transmembrane electron transfer;

Evidence of functional trimeric chlorophyll a/c2 -peridinin proteins in the dinoflagellate Symbiodinium by Jing Jiang; Hao Zhang; Gregory S. Orf; Yue Lu; Wenxin Xu; Lucas B. Harrington; Haijun Liu; Cynthia S. Lo; Robert E. Blankenship (1904-1912).
The chlorophyll a-chlorophyll c2 -peridinin-protein (apcPC), a major light harvesting component in peridinin-containing dinoflagellates, is an integral membrane protein complex. We isolated functional acpPC from the dinoflagellate Symbiodinium. Both SDS-PAGE and electrospray ionization mass spectrometry (ESI-MS) analysis quantified the denatured subunit polypeptide molecular weight (MW) as 18 kDa. Size-exclusion chromatography (SEC) and blue native gel electrophoresis (BN-PAGE) were employed to estimate the size of native acpPC complex to be 64–66 kDa. We also performed native ESI-MS, which can volatilize and ionize active biological samples in their native states. Our result demonstrated that the native acpPC complex carried 14 to 16 positive charges, and the MW of acpPC with all the associated pigments was found to be 66.5 kDa. Based on these data and the pigment stoichiometry, we propose that the functional light harvesting state of acpPC is a trimer. Our bioinformatic analysis indicated that Symbiodinium acpPC shares high similarity to diatom fucoxanthin Chl a/c binding protein (FCP), which tends to form a trimer. Additionally, acpPC protein sequence variation was confirmed by de novo protein sequencing. Its sequence heterogeneity is also discussed in the context of Symbiodinium eco-physiological adaptations.
Keywords: Symbiodinium; Chlorophyll a/c 2-peridinin-protein; Light-harvesting complex; Native mass spectrometry;