Physiology of sulfide tolerance in a thermophilic Oscillatoria

Oscillatoria amphigranulata is a fast-growing (3 doublings/day) cyanobacterium isolated from sulfide hot springs in New Zealand. Photosynthesis, as measured by incorporation of [¹⁴C]-HCO ₃ ^- , was initially inhibited by 0.3–1.5 mM sulfide at pH 7.9–8.1. However, conversion to sulfide-dependent anoxygenic photosynthesis occurred in about 2 h or less under light intensities of 3–14 klx. Under the stimulation of higher light intensity (8–14 klx) a partial recovery of oxygenic photosynthesis also occurred. It was concluded that oxygenic photosynthesis was responsible for 21–42% of the total incorporation at sulfide concentrations of 1.0–0.3 mM, respectively. This contribution was suppressed at 1.5 mM sulfide and not elicited under lower light intensities (3–7 klx). As judged by the inhibitory effect of 10 g/ml chloramphenicol protein synthesis was required for attainment of both anoxygenic photosynthesis and photosystem II recovery. Sulfide could not be replaced by thiosulfate, elemental sulfur or dithionite as electron donors in photosynthesis, but elemental sulfur could serve as the sole assimilatory source of sulfur. Oxygenic photosynthesis was inhibited by DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] or DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but sulfide relieved the effect of either inhibitor in adapted cells, indicating that electrons derived from sulfide enter the photosynthetic electron transport chain at a point beyond plastoquinone.Uncommon abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DSPD disalicyclidene propanediamine - DNP-INT 2-4-dinitrophenyl ether of 2-iodo-4-nitrothymol - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - PPO 2,5-diphenyloxazole - POPOP 1,4-bis-2-(5-phenyl oxzolyl) benzene     

Control of redox reactivity of flavin and pterin coenzymes by metal ion coordination and hydrogen bonding

The electron-transfer activities of flavin and pterin coenzymes can be fine-tuned by coordination of metal ions, protonation and hydrogen bonding. Formation of hydrogen bonds with a hydrogen-bond receptor in metal–flavin complexes is made possible depending on the type of coordination bond that can leave the hydrogen-bonding sites. The electron-transfer catalytic functions of flavin and pterin coenzymes are described by showing a number of examples of both thermal and photochemical redox reactions, which proceed by controlling the electron-transfer reactivity of coenzymes with metal ion binding, protonation and hydrogen bonding.     

Evidence for two functionally distinct hydrogenases in Anacystis nidulans

The effect of growth conditions on aerobic and anaerobic hydrogenase activities of Anacystis nidulans was studied. It was found that the two hydrogenase activities both of which were confined to the particulate fraction of cell-free extracts correlated in an opposite way with growth temperature: The algae were always grown photoautotrophically in presence of H₂ but after growth at 25° C a significant oxyhydrogen reaction contrasted with negligible photoreduction rates while the opposite was true after growth at 40°C. A similar correlation between incubation temperature and induction of the respective hydrogenase activity was also observed with resting cells.Kinetic analysis of the two different types of hydrogenase — catalysed reactions with Anacystis membranes yielded the following Michaelis-Mentenparameters: K _M=55 M H₂ and v _max=0.12 mol H₂ per min and mg protein for the oxyhydrogen reaction, and K _M=170 M H₂ and v _max=0.3 mol H₂ per min and mg protein for the photoreductions. Also the dependences of oxyhydrogen and of photoreduction activities on pH and on temperature were measured; both pH and temperature profiles were found to be markedly different for each type of H₂-supported reaction.The results are discussed as pointing to the possible occurrence of two functionally distinct hydrogenase enzymes which can be synthesized by Anacystis in response to the conditions of induction.Abbreviations BO p-benzoquinone - CAP chloramphenicol - chl chlorophyll - cytc horse heart cytochrome c - DCMU 3-(34-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenolindophenol - fd ferredoxin - FeCy ferricyanide - MB methylene blue - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethan     

The Class III Cyclobutane Pyrimidine Dimer Photolyase Structure Reveals a New Antenna Chromophore Binding Site and Alternative Photoreduction Pathways

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.     

Femtochemistry in enzyme catalysis: DNA photolyase

Photolyase uses light energy to split UV-induced cyclobutane pyrimidine dimers in damaged DNA. This photoenzyme encompasses a series of elementary dynamical processes during repair function from early photoinitiation by a photoantenna molecule to enhance repair efficiency, to in vitro photoreduction through aromatic residues to reconvert the cofactor to the active form, and to final photorepair to fix damaged DNA. The corresponding series of dynamics include resonance energy transfer, intraprotein electron transfer, and intermolecular electron transfer, bond breaking-making rearrangements and back electron return, respectively. We review here our recent direct studies of these dynamical processes in real time, which showed that all these elementary reactions in the enzyme occur within subnanosecond timescale. Active-site solvation was observed to play a critical role in the continuous modulation of catalytic reactions. As a model system for enzyme catalysis, we isolated the enzyme–substrate complex in the transition-state region and mapped out the entire evolution of unmasked catalytic reactions of DNA repair. These observed synergistic motions in the active site reveal a perfect correlation of structural integrity and dynamical locality to ensure maximum repair efficiency on the ultrafast time scale.     

Rapid X-ray Photoreduction of Dimetal-Oxygen Cofactors in Ribonucleotide Reductase

Prototypic dinuclear metal cofactors with varying metallation constitute a class of O₂-activating catalysts in numerous enzymes such as ribonucleotide reductase. Reliable structures are required to unravel the reaction mechanisms. However, protein crystallography data may be compromised by x-ray photoreduction (XRP). We studied XPR of Fe(III)Fe(III) and Mn(III)Fe(III) sites in the R2 subunit of Chlamydia trachomatis ribonucleotide reductase using x-ray absorption spectroscopy. Rapid and biphasic x-ray photoreduction kinetics at 20 and 80 K for both cofactor types suggested sequential formation of (III,II) and (II,II) species and similar redox potentials of iron and manganese sites. Comparing with typical x-ray doses in crystallography implies that (II,II) states are reached in <1 s in such studies. First-sphere metal coordination and metal-metal distances differed after chemical reduction at room temperature and after XPR at cryogenic temperatures, as corroborated by model structures from density functional theory calculations. The inter-metal distances in the XPR-induced (II,II) states, however, are similar to R2 crystal structures. Therefore, crystal data of initially oxidized R2-type proteins mostly contain photoreduced (II,II) cofactors, which deviate from the native structures functional in O₂ activation, explaining observed variable metal ligation motifs. This situation may be remedied by novel femtosecond free electron-laser protein crystallography techniques.     

Ca2+对叶绿体光还原活性的影响及与钙调素的关系

外加Ｃａ＾２＋能有效地提高杂交水稻汕优６３离体叶绿体的光还原活性,Ｃａ＾２＋专一性螯合剂乙二醇双乙胺醚四乙酸（ＥＧＴＡ）抑制其活性,钙调素（ＣａＭ）抑制剂三氟拉嗪（ＴＦＰ）,Ｃａ＾２＋通道阻断剂Ｃｏ＾２＋,以及Ｚｎ＾２＋抑制离体叶素体的光还原活性,外加Ｃａ＾２＋可以部分地减少ＴＦＰ,Ｃｏ＾２＋和Ｚｎ＾２＋抑制作用,叶绿体的光还原活性与Ｃａ＾２＋和ＣａＭ密切相关。     

Anaerobic hydrogenase activity in Anacystis nidulans H2-dependent photoreduction and related reactions

1. Anaerobic hydrogenase activity in whole cells and cell-free preparations of H₂-induced Anacystis was studied both manometrically and spectrophotometrically in presence of physiological and artificial electron acceptors.2. Up to 90% of the activity measured in crude extracts were recovered in the chlorophyll-containing membrane fraction after centrifugation (144 000 × g, 3 h).3. Reduction of methyl viologen, diquat, ferredoxin, nitrite and NADP by the membranes was light dependent while oxidants of more positive redox potential were reduced also in the dark.4. Evolution of H₂ by the membranes was obtained with dithionite and with reduced methyl viologen; the reaction was stimulated by detergents.5. Both uptake and evolution of H₂ were sensitive to O₂, CO, and thiol-blocking agents. The H₂-dependent reductions were inhibited also by the plastoquinone antagonist dibromothymoquinone, while the ferredoxin inhibitor disalicylidenepropanediamine affected the photoreduction of nitrite and NADP only. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea did not inhibit any one of the H₂-dependent reactions.6. The results present evidence for a membrane-bound ‘photoreduction’ hydrogenase in H₂-induced Anacystis. The enzyme apparently initiates a light-driven electron flow from H₂ to various low-potential acceptors including endogenous ferredoxin.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)