Mechanism of Nitrogenase Inhibition in Soybean Nodules : Pulse-Modulated Spectroscopy Indicates that Nitrogenase Activity Is Limited by O(2)

A novel, pulse-modulated spectroscopic system for measuring fractional leghemoglobin oxygenation and infected cell O₂ concentration (O_i) in intact attached nodules of soybean (Glycine max) is described. The system is noninvasive and uses a pulsed (1000 Hertz) light-emitting diode coupled to an optical fiber to illuminate the nodule with light at 660 nanometer. A second optical fiber receives a portion of the light reflected from the nodule and directs this to a photodiode. A lock-in amplifier measures only the signal from the photodiode which is in phase with the pulsed light from the light-emitting diode, and the voltage output from the amplifier, proportional to reflectance, is used to calculate fractional leghemoglobin oxygenation and the nanomolar concentration of free O₂ in the infected cells of the nodule (O_i). The system was used to show that inhibition of nitrogenase activity in soybean nodules by NO₃⁻ treatment, stem-girdling, continuous darkness, or nodule disturbance is caused by a reduction in O_i and limitation of respiration in support of nitrogenase activity. A plot of nitrogenase activity (measured as peak H₂ evolution in Ar:O₂) versus O_i for the various treatments was consistent with the concept that O_i limits in vivo nitrogenase activity in legume nodules under adverse conditions. The potential for using O_i to estimate nitrogenase activity in laboratory and field-grown legumes is discussed.     

Proline Accumulation, Nitrogenase (C2H2 reducing) Activity and Activities of Enzymes related to Proline Metabolism in Drought-Stressed Soybean Nodules

We examined the effect of drought stress on proline accumulation,nitrogenase activity and activities of enzymes related to prolinemetabolism in soybean (Glycine max [L.] Merr.) nodules. Nitrogenase(C₂H₂ reducing) activity was inhibited 90% or more as a resultof drought stress. This inhibition was substantially reversedafter a 4 h recovery period. Pyrroline-5-carboxylate reductaseactivity in extracts of drought-stressed nodules from 25-d-oldplants was 55% higher than in unstressed nodules, but the sameactivity in preparations from 55-d-old plants was similar tothat of control plants. Extracts of recovering nodules on plantsof both ages had activities near those of controls. Droughtstress increased the activity of the pentose phosphate pathwayby about 65% in extracts of nodules from 55-d-old plants, butthere was no effect in extracts of nodules from younger plants(25-d-old). Proline dehydrogenase activity was 3.7 and 1.6 timeshigher in bacteroids isolated from nodules taken from 25- and55-d-old stressed plants, respectively, than in comparable controlbacteroids. This activity remained high in bacteroids from bothsets of recovering nodules. The amount of proline in extractsfrom stressed nodules was 3- to 4-fold higher than in unstressednodules, despite increased proline dehydrogenase activity andremained high in nodules collected 4 h after rewatering. Thisincrease was observed in both cytoplasmic and bacteroid fractions.The possible physiological significance of these results isdiscussed. Key words: Proline metabolism, pentose phosphate pathway, drought stress, soybean nodules     

Hydrogen Effect on Nitrogenase (C2H2 Reduction) Response to Oxygen in Bradyrhizobium japonicum-Phaseoleae Symbioses

The influence of hydrogenase in Bradyrizobum-Phaseoleae symbioseswas studied ex-planta and in-planra in soybean (Glycine max)and cowpea (Vigna unguiculata). The hydrogenase was activatedby the addition of hydrogen in the incubation gas phase whichmodified the response of nitrogenase activity of Hup⁺ (hydrogenuptake positive) symbiosis to the external oxygen partial pressure.For bacteroids the hydrogenase expression increased nitrogenaseactivity at supraoptimal pO₂, acting possibly as a respiratoryprotection of nitrogenase. However, at suboptimal pO₂, nitrogenaseactivity of Hup⁺ bacteroids decreased with hydrogen, a phenomenonattributed to the lower efficiency of ATP synthesis from hydrogenthan from carbon substrates oxidation. For undisturbed nodules,the hydrogenase expression in soybean increased the optimalpO₂ for ARA (COP), from 35.3 to 40.3 kPa O₂, and the ARA atsupraoptimal pO₂; at suboptimal PO₂ there was a negative effectof hydrogenase on ARA, although this inhibition was less thanon bacteroids and was not detected if plants were grown at 15°C rather than 20 °C root temperature. No H₂ effectwas detected on cowpea nodules. The results on soybean nodulesare consistent with the concept that symbiotic nitrogen fixationis oxygen-limited and that hydrogenase activity has no beneficialeffect on nitrogen fixation in O₂ limitation. Key words: Glycine max, hydrogenase, nitrogenase, nitrogen fixation, nodules, Vigna unguiculata     

Calcium in the Control of Nitrogenase Activity in Vicia faba L. Root Nodules: Calcium Release from Symbiosomes and the Accompanying Decrease in Their Nitrogenase Activity Is Blocked by Verapamil

Treatment of root nodules or symbiosomes isolated from them with calcium chelator EGTA alone or together with calcium ionophore A23187 for 3 h under microaerophilic conditions considerably decreased their nitrogenase activity (NA). Under these experimental conditions, cytochemical electron-microscopic analysis revealed considerable calcium depletion of symbiosomes in the infected nodule cells treated with EGTA and A23187. Ca²⁺ channel blockers, verapamil and ruthenium red, inhibited EGTA-induced Ca²⁺ release from symbiosomes. In this case, NA insignificantly increased in the whole nodules and reached its initial level in symbiosomes. The experiments on isolated symbiosomes with arsenazo III, a Ca²⁺ indicator, demonstrated that verapamil inhibited Ca²⁺ release from them induced by valinomycin in the presence of K⁺ ions. These data suggest the presence on the peribacteroid membrane of a verapamil-sensitive transporter responsible for Ca²⁺ release from symbiosomes. A possible role of this transporter in the interaction between symbiotic partners in the infected cells of root nodules is discussed.     

Nitrogenase Activity and Nodule Gas Permeability Response to Rhizospheric NH(3) in Soybean

This study was conducted on soybean (Glycine max L. Merr.) nodules to determine if exogenous NH₃ exerts a controlling influence over nitrogenase activity through changes in nodule gas permeability (P), and if decreasing carbohydrate availability, as a result of low-light treatment, increases the sensitivity of root nodules to NH₃. Nodulated root systems of intact plants were exposed to one of several NH₃ concentrations ranging from 0 to 821 microliters per liter for an 8-hour period. Treatments were conducted under high-light (2300 micromoles per square meter per second) or low-light (800 micromoles per square meter per second) conditions. Increasing the NH₃ concentration and length of exposure of NH₃ caused a progressive decline in acetylene reduction activity (ARA). There was generally a greater reduction in ARA under the low-light treatment compared to the high-light treatment at a particular NH₃ concentration. The NH₃ concentration necessary to decrease P was greater than that needed to decrease ARA, and there was no evidence of a causal relationship between P and ARA in response to NH₃.     

A New Chloroplast Protein Is Induced by Growth on Low CO(2) in Chlamydomonas reinhardtii

The biosynthesis of a 36 kilodalton polypeptide of Chlamydomonas reinhardtii was induced by photoautotrophic growth on low CO₂. Fractionation studies using the cell-wall-deficient strain of C. reinhardtii, CC-400, showed that this polypeptide was different from the low CO₂-induced periplasmic carbonic anhydrase. In addition, the 36 kilodalton polypeptide was found to be localized in intact chloroplasts isolated from low CO₂-adapting cultures. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO₂-grown C. reinhardtii cells.     

Reduction in Turgor Pressure as a Result of Extremely Brief Exposure to CO(2)

CO₂ depresses water influx into sunflower hypocotyl segments of low water potential; by contrast, it stimulates flux into segments of high water potential. When segments of high potential were placed in a series of mannitol concentrations and allowed to achieve steady rates of water uptake, influx into CO₂-treated tissue in a solution of 3 atm equalled that into control tissue in water. Reasons are given for deducing that a change in internal osmotic concentration (π_i) of the order of 40% would be necessary to account for this result on the basis of π_i. Direct measurements (by cryoscopy and by the minimum volume method) detected no difference in the steady state value for π_i as between CO₂-treated and control tissue. It was therefore concluded that CO₂ had caused some reduction in turgor pressure.

Water uptake into tissue treated with CO₂ for only the first 2 minutes of a 30-minute period was equal to that into tissue treated continuously with CO₂, i.e. 3 times the control value. Ten seconds' CO₂ treatment produced a significant stimulation. When the cycles of treatment were repeated the samples receiving flashes of CO₂ maintained a rate of water uptake superior to that of the control, whereas influx into continuously treated tissue fell below the control value after 1 hour.

CO₂ treatment applied in a moist air chamber stimulated subsequent water influx when the tissue was transferred to water. Fifteen seconds' treatment was sufficient to produce a marked effect. Even when a transition period of 30 minutes in the moist chamber was interposed between CO₂ treatment (5 minutes) and transfer to water, a stimulation was observed. The CO₂ effect could be achieved at zero degrees; 5 minutes' treatment in the moist chamber at zero degrees, followed by a 15-minute transition period at the same temperature, substantially increased subsequent water uptake at 25°.

     

Resistance Mutation R292K Is Induced in Influenza A(H6N2) Virus by Exposure of Infected Mallards to Low Levels of Oseltamivir

Resistance to neuraminidase inhibitors (NAIs) is problematic as these drugs constitute the major treatment option for severe influenza. Extensive use of the NAI oseltamivir (Tamiflu®) results in up to 865 ng/L of its active metabolite oseltamivir carboxylate (OC) in river water. There one of the natural reservoirs of influenza A, dabbling ducks, can be exposed. We previously demonstrated that an influenza A(H1N1) virus in mallards (Anas platyrhynchos) exposed to 1 µg/L of OC developed oseltamivir resistance through the mutation H274Y (N2-numbering). In this study, we assessed the resistance development in an A(H6N2) virus, which belongs to the phylogenetic N2 group of neuraminidases with distinct functional and resistance characteristics. Mallards were infected with A(H6N2) while exposed to 120 ng/L, 1.2 µg/L or 12 µg/L of OC in their sole water source. After 4 days with 12 µg/L of OC exposure, the resistance mutation R292K emerged and then persisted. Drug sensitivity was decreased ≈13,000-fold for OC and ≈7.8-fold for zanamivir. Viral shedding was similar when comparing R292K and wild-type virus indicating sustained replication and transmission. Reduced neuraminidase activity and decrease in recovered virus after propagation in embryonated hen eggs was observed in R292K viruses. The initial, but not the later R292K isolates reverted to wild-type during egg-propagation, suggesting a stabilization of the mutation, possibly through additional mutations in the neuraminidase (D113N or D141N) or hemagglutinin (E216K). Our results indicate a risk for OC resistance development also in a N2 group influenza virus and that exposure to one NAI can result in a decreased sensitivity to other NAIs as well. If established in influenza viruses circulating among wild birds, the resistance could spread to humans via re-assortment or direct transmission. This could potentially cause an oseltamivir-resistant pandemic; a serious health concern as preparedness plans rely heavily on oseltamivir before vaccines can be mass-produced.     

Nitrogenase Activity (C2H2) of Isolated Peanut and Cowpea Bacteroids under Nitrogen, Argon, and Helium Atmospheres

Peanut nodules have been reported to have several times highernitrogenase activity (C₂H₂) than cowpea and siratro nodulesinduced by the same rhizobial strains. The unique morphologicalmodification of the peanut bacteroids has been considered tobe the cause for such enhanced activity. To investigate thispossibility, nitrogenase activities of isolated peanut and cowpeabacteroids were compared. Peanut bacteroids showed low initialrates of C₂H₂ reduction which increased with time, but for cowpeabacteroids higher initial rates decreased with time. Moreover,the gases used as diluent for O₂ (N₂, Ar, or He) were foundto influence O₂ tolerance and C₂H₂-reduction rates of bacteroids.     

Early Exposure of Bay Scallops (Argopecten irradians) to High CO2 Causes a Decrease in Larval Shell Growth

Ocean acidification, characterized by elevated pCO₂ and the associated decreases in seawater pH and calcium carbonate saturation state (Ω), has a variable impact on the growth and survival of marine invertebrates. Larval stages are thought to be particularly vulnerable to environmental stressors, and negative impacts of ocean acidification have been seen on fertilization as well as on embryonic, larval, and juvenile development and growth of bivalve molluscs. We investigated the effects of high CO₂ exposure (resulting in pH = 7.39, Ω_ar = 0.74) on the larvae of the bay scallop Argopecten irradians from 12 h to 7 d old, including a switch from high CO₂ to ambient CO₂ conditions (pH = 7.93, Ω_ar = 2.26) after 3 d, to assess the possibility of persistent effects of early exposure. The survival of larvae in the high CO₂ treatment was consistently lower than the survival of larvae in ambient conditions, and was already significantly lower at 1 d. Likewise, the shell length of larvae in the high CO₂ treatment was significantly smaller than larvae in the ambient conditions throughout the experiment and by 7 d, was reduced by 11.5%. This study also demonstrates that the size effects of short-term exposure to high CO₂ are still detectable after 7 d of larval development; the shells of larvae exposed to high CO₂ for the first 3 d of development and subsequently exposed to ambient CO₂ were not significantly different in size at 3 and 7 d than the shells of larvae exposed to high CO₂ throughout the experiment.     

Osmotic Response of Sugar Beet Source Leaves at CO(2) Compensation Point

As sugar beet source leaves lowered the CO₂ concentration to compensation point in a closed atmosphere, leaf thickness and relative water content decreased. Leaf water potential declined rapidly from −0.5 to −1.4 megapascals. At 340 microliters CO₂ per liter, water potential and sucrose, glucose, and fructose contents were steady in photosynthesizing source leaves. Within 90 minutes after leaves were exposed to a CO₂ concentration at the compensation point, leaf sucrose content declined to 60% of the preteatment level, rapidly in the first 30 minutes and then more slowly. During the subsequent 200 minutes, sucrose content increased to 180% of pretreatment level. Glucose and fructose remained unchanged during the treatment. Degradation of starch was sufficient to account for the additional sucrose that accumulated. Labeled carbon lost from starch appeared in sucrose and several other compounds that likely contributed to the recovery in leaf water content.     

Synthesis and characterisation of the carboxylate linked osmium clusters [{Os3H(CO)10}2(CO2CH2CO2)], [{Os3H(CO)10}2(CO2C2H4CO2)], [{Os3H(CO)10}2(C4O4)] and [{Os3H(CO)10}2(C4O4){Co2(CO)6}]

Reactions between the activated cluster [Os₃(CO)₁₀(NCMe)₂] and malonic acid, succinic acid and dicarboxylic acetylene, respectively, lead to the formation of the linked cluster complexes [{Os₃H(CO)₁₀}₂(CO₂CH₂CO₂)] (1), [{Os₃H(CO)₁₀}₂(CO₂C₂H₄CO₂)] (2), and [{Os₃H(CO)₁₀}₂(C₄O₄)] (3) in good yield. Cluster 3 was subsequently treated with [Co₂(CO)₈] and this results in the addition of a “Co₂(CO)₆” group giving [{Os₃H(CO)₁₀}₂(C₂O₄){Co₂(CO)₆}] (4). The X-ray crystal structures are reported for 2–4. In each structure the two triangular triosmium units are linked by the carboxylate groups and within each complex the carboxylate groups are chelating and bridge two osmium atoms.     

C(3)-C(4) Intermediate Species in Alternanthera (Amaranthaceae) : Leaf Anatomy, CO(2) Compensation Point, Net CO(2) Exchange and Activities of Photosynthetic Enzymes

Two naturally occurring species of the genus Alternanthera, namely A. ficoides and A. tenella, were identified as C₃-C₄ intermediates based on leaf anatomy, photosynthetic CO₂ compensation point (Γ), O₂ response of г, light intensity response of г, and the activities of key enzymes of photosynthesis. A. ficoides and A. tenella exhibited a less distinct Kranz-like leaf anatomy with substantial accumulation of starch both in mesophyll and bundle sheath cells. Photosynthetic CO₂ compensation points of these two intermediate species at 29°C were much lower than in C₃ plants and ranged from 18 to 22 microliters per liter. Although A. ficoides and A. tenella exhibited similar intermediacy in г, the apparent photorespiratory component of O₂ inhibition in A. ficoides is lower than in A. tenella. The г progressively decreases from 35 microliters per liter at lowest light intensity to 18 microliters per liter at highest light intensity in A. tenella. It was, however, constant in A. ficoides at 20 to 25 microliters per liter between light intensities measured. The rates of net photosynthesis at 21% O₂ and 29°C by A. ficoides and A. tenella were 25 to 28 milligrams CO₂ per square decimeter per hour which are intermediate between values obtained for Tridax procumbens and A. pungens, C₃ and C₄ species, respectively. The activities of key enzymes of C₄ photosynthesis, phosphoenolpyruvate carboxylase, pyruvate Pi dikinase, NAD malic enzyme, NADP malic enzyme and phosphoenolpyruvate carboxykinase in the two intermediates, A. ficoides and A. tenella are very low or insignificant. Results indicated that the relatively low apparent photorespiratory component in these two species is presumably the basis for the C₃-C₄ intermediate photosynthesis.     

Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO₂ atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO₂ levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO₂ assimilation rate per unit area measured in the growth room at high CO₂ was not higher in the selected plants. The CO₂ assimilation rate versus intercellular CO₂ curve and the CO₂ compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO₂ compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO₂ survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.     

Mechanism of Plant Growth Stimulation by Naphthenic Acid: II. Enzymes of CO(2) Fixation, CO(2) Compensation Point, Bean Embryo Respiration

Potassium naphthenate, 20 mm, was applied to the foliage of 14-day-old plants of bush bean, Phaseolus vulgaris L, cv Top Crop, maize, Zea mays L, cv Golden Bantam, spring wheat, Triticum vulgare Vill., cv Neepawa, and a 2 mm solution to 21-day-old plants of sugar beet, Beta vulgaris L, cv CS-43. Seven days after application, the activities of ribulose diphosphate carboxylase and phosphoenolpyruvic carboxylase in leaves of naphthenate-treated bean and maize were greater than in the leaves of untreated plants. The increase in activity of the carboxylases in treated spring wheat lacked statistical significance. At the same time after treatment, the CO(2) compensation point of bean was smaller than that of control plants, as was the average CO(2) compensation point of sugar beet measured at intervals up to 21 days after spraying. Respiratory rates of embryos of bean seeds soaked for 12, 24, and 48 hours in 43.5 mum K naphthenate were greater than those of seeds soaked in water. Ascorbate oxidase activity in bean leaves, determined 7, 14, and 21 days after K naphthenate application, was also stimulated. Foliar application of 10 mm cyclohexanecarboxylic acid to bean was followed in 7 and 14 days by a greater activity of catalase than in control plants. Higher activity of the enzyme, measured 6, 7, 12, and 14 days after spraying, also resulted from K naphthenate application. The results indicate that the higher rates of photosynthesis in naphthenate-treated plants may be due in part to increased rates of CO(2) fixation, and that greater photosynthetic efficiency, together with a more plentiful supply of ATP arising from increased electron flow in respiration, is involved in the greater growth of plants to which naphthenate has been applied.     

Effect of Oxygen on the Contribution of Respiration to the CO(2) Compensation Point in Wheat and Bean Leaves

The CO₂ compensation point at 21% O₂ (Γ₂₁) and at 2% O₂ (Γ₂), and the rate of dark CO₂ efflux at 21% O₂ (R_n) were measured in adult wheat (Triticum aestivum L, cv Gabo) leaves at the end of the night and after a period of photosynthesis of 5 h at 800 μbar CO₂. The values of Γ₂₁ and R_n significantly increased after the light period, due to the stimulation of respiration by carbohydrates. In contrast, Γ₂ did not increase after the same period of photosynthesis, suggesting that the respiratory component of Γ₂ was not stimulated by carbohydrates. In a different experiment, Γ₂₁, Γ₂, and R_n were studied during the growth period of bean (Phaseolus vulgaris L, cv Hawkesbury Wonder) leaves. The values of Γ₂₁ and R_n were high in young leaves, and decreased rapidly in parallel during maturation. However, Γ₂ presented relatively low values in growing bean leaves, and a model predicted that the observed values of Γ₂ should have been considerably higher if their respiratory component was considered to be as large as that of Γ₂₁. The results suggest that the rate of respiration in the light contributing to the CO₂ compensation point in wheat and bean leaves is smaller at low O₂ levels than at ambient levels.     

Relation of CO(2) Compensation Concentration to Apparent Photosynthesis in Maize

Significant differences in CO₂ compensation concentration measured in the field among varieties of the species Zea mays L. are reported for the first time. CO₂ compensation concentrations were significantly (P≤ 0.01) and negatively correlated with apparent photosynthesis at 300 μl CO₂/liter air. The Michaelis constant (as defined) for a leaf was significantly (P≤ 0.01) and positively correlated with apparent photosynthesis among varieties. While the first correlation is similar to behavior of CO₂ compensation among species of different photosynthetic efficiency, the latter correlation is the converse of the behavior of Km among species.     

Localized Tetrazolium Reduction in Relation to N(2) Fixation, CO(2) Fixation, and H(2) Uptake in Aquatic Filamentous Cyanobacteria

The aquatic filamentous cyanobacteria Anabaena oscillarioides and Trichodesmium sp. reveal specific cellular regions of tetrazolium salt reduction. The effects of localized reduction of five tetrazolium salts on N(2) fixation (acetylene reduction), CO(2) fixation, and H(2) utilization were examined. During short-term (within 30 min) exposures in A. oscillarioides, salt reduction in heterocysts occurred simultaneously with inhibition of acetylene reduction. Conversely, when salts failed to either penetrate or be reduced in heterocysts, no inhibition of acetylene reduction occurred. When salts were rapidly reduced in vegetative cells, CO(2) fixation and H(2) utilization rates decreased, whereas salts exclusively reduced in heterocysts were not linked to blockage of these processes. In the nonheterocystous genus Trichodesmium, the deposition of reduced 2,3,5-triphenyl-2-tetrazolium chloride (TTC) in the internal cores of trichomes occurs simultaneously with a lowering of acetylene reduction rates. Since TTC deposition in heterocysts of A. oscillarioides occurs contemporaneously with inhibition of acetylene reduction, we conclude that the cellular reduction of this salt is of use in locating potential N(2)-fixing sites in cyanobacteria. The possible applications and problems associated with interpreting localized reduction of tetrazolium salts in cyanobacteria are presented.     

Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO₂, little O₂ inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO₂-concentrating system. The CO₂-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO₂ concentrations have inorganic carbon transport activity, but cells grown at 5% CO₂ do not. Four membrane-associated polypeptides (M_r 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO₂ concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO₂-concentrating system activity in response to CO₂ limitation.     

Synthesis and characterization of two isomers of Os3(CO)10(C5H4N-2-C(H)NR) and the conversion to ortho-metallated HOs3(C5H3N-2-C(H)NR)(CO)9. Part III. X-ray Structure of HOs3(C5H3N-2-C(H)Ni-Pr)(CO)9

Os₃(CO)₁₀(MeCN)₂ reacts at room temperature in MeCN or toluene with R-Pyca2 to yield two isomers of Os₃(CO)₁₀(R-Pyca) that differ in the bonding of the R-Pyca ligand to the Os₃(CO)₁₀ unit. In all cases Os₃(CO)₁₀(R-Pyca(4e)) (isomer A; 4a: R = c-Pr, 4b: R = i-Pr, 4c: R = neo-Pent, 4d: R = t-Bu), containing a chelating 4e donating R-Pyca ligand and three OsS bonds, could be isolated. In the case of R = c-Pr and R = i-Pr Os₃(CO)₁₀(R-Pyca(6e)) (isomer B; 5a: R = c-Pr, 5b: R = i-Pr), in which only two OsS bonds are present and the R-Pyca ligand is bonded as a 6e donating ligand bridging two non-bonded Os atoms, could be isolated as a minor product.At 70 °C Os₃(CO)₁₀(R-Pyca(4e)) (4b and 4d) loses one carbonyl and the pyridine moiety of the R-Pyca ligand is ortho-metallated to form HOs₃(C₅H₃N-2-C(H)NR)(CO)₉ (6b: R = i-Pr and 6d: R = t-Bu). Under the same conditions Os₃(CO)₁₀(i-Pr-Pyca(6e)) (5b) reacts to Os₂(CO)₆(6e)) (7b) containing a bridging 6e donating ligands. The latter two reactions were followed with FT-IR spectroscopy in a high temperature IR cell.The structure of the complexes in solution have been studied by ¹H and ¹C NMR and IR spectroscopy. The stoichiometries of 4a and 5a were determined by FAB-mass spectrometry while an exact mass determination was carried out for 4a.The crystal structure of 6b has been determined. Crystal of 6b are monoclinic, space group P2₁/n, with a = 7.808(2),b = 17.613(3),c = 16.400(8)Å, β = 94.09(3)° and Z = 4. The structure was refined to R = 0.039. The molecule contains a triangular array of osmium atoms [Os(1)Os(2) = 2.898(2)Å, Os(1)Os(3) = 2.886(2)Åand Os(2)O(3) = 2.911(2)Å] and nine terminally bonded carbonyl ligands. The C₅H₃N-2-C(H)N-i-Pr ligand is chelate bonded to Os(2) with the pyridine and imine nitrogens atoms axially and equatorially coordinated respectively [Os(2)N(1) = 2.00(2)Åand Os(2)N(2) = 2.11(2)Å]. The i-Pr-Pyca ligand is ortho-metallated at C(1) and forms a four membered ring containing Os(2), Os(3), C(1) and N(1), the Os(3)C(1) distance being 2.12(2)Å. The hydride, which could not be located unequivocally from a difference Fourier map is proposed to bridge the Os(2)(3) bond on the basis of stereochemical considerations.