Iron-sulfur cluster dynamics in biotin synthase: A new [2Fe-2S] cluster

Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S]²⁺ cluster per monomer. However, active BioB contains in addition a [4Fe-4S]²⁺ cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S]¹⁺ cluster on reduction with dithionite and report the observation of a new [2Fe-2S]¹⁺ cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.     

Elevated Proton Leak of the Intermediate OH in Cytochrome c Oxidase

The kinetics of the formation and relaxation of transmembrane electric potential (Δψ) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa₃-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Δψ kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O_H) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Δψ (τ^app ≤ 0.5-0.8 s). By contrast, when the enzyme in the O_H state is rapidly re-reduced by sequential electron delivery, Δψ dissipates much slower (τ^app > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Δψ dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O_H state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.     

Diffusion and Partitioning of Fluorescent Lipid Probes in Phospholipid Monolayers

The pressure-dependent diffusion and partitioning of single lipid fluorophores in DMPC and DPPC monolayers were investigated with the use of a custom-made monolayer trough mounted on a combined fluorescence correlation spectroscopy (FCS) and wide-field microscopy setup. It is shown that lipid diffusion, which is essential for the function of biological membranes, is heavily influenced by the lateral pressure and phase of the lipid structure. Both of these may change dynamically during, e.g., protein adsorption and desorption processes. Using FCS, we measured lipid diffusion coefficients over a wide range of lateral pressures in DMPC monolayers and fitted them to a free-area model as well as the direct experimental observable mean molecular area. FCS measurements on DPPC monolayers were also performed below the onset of the phase transition (Π < 5 mN/m). At higher pressures, FCS was not applicable for measuring diffusion coefficients in DPPC monolayers. Single-molecule fluorescence microscopy and differential scanning calorimetry clearly showed that this was due to heterogeneous partitioning of the lipid fluorophores in condensed phases. The results were compared with dye partitioning in giant lipid vesicles. These findings are significant in relation to the application of lipid fluorophores to study diffusion in both model systems and biological systems.     

The effects of mowing and agricultural landscape management on population movements of the common quail

Aim Information on the movements of bird populations in extensive landscapes is needed to assess environmental effects and conservation strategies over appropriate temporal and spatial scales. The common quail (Coturnix coturnix) is a game bird that breeds mainly in dense cereal crops. These crops cover huge areas of the Iberian Peninsula. The aim of this work is to relate cereal mowing, which causes rapid and massive habitat loss for the common quail, to population movements of the species during its breeding season. Location Spain, southern Europe. Methods We used ring recoveries to analyse, using circular statistics, the orientation of movements by male common quails in the northern half of Spain. Forward stepwise multiple regressions were applied to correlate (1) the Julian day of cereal harvesting at 770 locations with the respective latitude, longitude and elevation, and (2) the number of hunted birds with hunting pressure, breeding densities and mean mowing date by province. Finally, data concerning the number of quails hunted at the end of the breeding season were compared by province and year using two‐way ANOVA. Results Our results show that during the breeding season in the northern half of Spain, male quails orient their movements towards higher areas, and mainly to the northern Castilian Plateau. These are the areas of Spain where cereals are harvested latest. Moreover, records show that the number of quails hunted is significantly higher in these areas than in other areas of the country, independently of hunting pressure and breeding densities, and it is extremely high in the northern Castilian Plateau. Main conclusions Cereal mowing combined with some Spanish geographical characteristics acts as a funnel, forcing quail populations to concentrate in certain areas of Spain at the end of the breeding season. This implies that the number of quails hunted in these areas may be high, and that they therefore constitute priority conservation areas.     

Rewarming rates of two large hibernators: Comparison of a monotreme and a eutherian

We measured body temperatures in two large hibernating mammals, the eutherian alpine marmot (Marmota marmota) and the egg-laying echidna (Tachyglossus aculeatus) from unrestrained animals in their natural environment. In both species hibernation is broken every 13 days on average by rewarming to euthermic temperatures. We found that the time course of a rewarming could be closely fitted with a sigmoid curve, allowing calculation of peak rewarming rate and corresponding body temperature. Maximum rewarming rates were twice as high in marmots as in echidnas (12.1±1.3 °C h⁻¹, n=10 cf. 6.2±1.2 °C h⁻¹, n=10). Peak rewarming rates were positively correlated with body temperature in echidnas, but negatively correlated in marmots.     

Annual growth of Cassiope tetragona as a proxy for Arctic climate: developing correlative and experimental transfer functions to reconstruct past summer temperature on a millennial time scale

Annual growth of the polar evergreen shrub Cassiope tetragona on Svalbard was evaluated as a proxy for Arctic summer temperatures. Transfer functions were derived from temperature‐growth correlations of shoots and from a temperature‐growth response, obtained from experimental warming using open top chambers (OTC) in high Arctic tundra vegetation at Isdammen approximately 1.5 km southeast of Longyearbyen, Svalbard (78°N, 15 E) and in Longyeardalen, 3 km west of Isdammen from 2004 to 2006. Air temperatures, monitored throughout the summer months, were 1.3 °C higher inside the OTCs than in the control plots. Annual stem growth was measured by tagging stems and leaves, and in the lab with shoots harvested from OTCs and control plots. Annual growth parameters assessed were leaf production, sum of length and weight of individual leaves, and stem length increment derived from leaf scar distances and the distances between wintermarksepta in the stem. Wintermarksepta are formed at the end of the summer growth period when the pith is narrowing and consist of dense and dark tissue ( Fig. 1b ). The variation of annual growth in a 34‐year site chronology (based on Cassiope shoots from the surroundings of the OTCs and control plots) correlated strongly with the mean summer temperature on Svalbard. The number of leaf pairs, leaf length and stem length also increased in the OTC warmed plots in the second and third year of warming. Transfer functions were derived from the temperature‐annual growth correlations from a single shoot from Longyeardalen, from the cross‐dated Isdammen site chronology and from the growth response to experimental warming. Based on leaf scar distances and distances between wintermarksepta of well‐preserved subfossil shoots in arctic tundra soil, annual stem length increase was assessed for the layers of a soil core collected at the Isdammen site. Based on the derived transfer functions summer temperature of the period relating to the 15 cm deep tundra soil core layer, radiocarbon dated at 4230±40 bp , may have been 3.0 °C lower than the present‐day 6.2 °C value. These results indicate that the transfer functions can be used to reconstruct past temperatures, beyond the time range of instrumental temperature and ice core records of Svalbard.

Figure 1 Open in figure viewer PowerPoint (a) Morphology of shoot of Cassiope tetragona collected May 26, 2007, Longyeardalen. Removal of the front row of leaves shows four leaf pairs of the 2006 summer, the leaf primordium for the 2007 growing season and the stem length increase summer 2006. (b) Wintermarksepta, darker colored than the pith tissue, indicating the winterperiod in a longitudinal section of an air‐dried shoot collected August 26, 2006, Longyeardalen.     

Phosphatidyl alcohols: Effect of head group size on domain forming properties and interactions with sterols

In this study, we have examined the membrane properties and sterol interactions of phosphatidyl alcohols varying in the size of the alcohol head group coupled to the sn-3-linked phosphate. Phosphatidyl alcohols of interest were dipalmitoyl derivatives with methanol (DPPMe), ethanol (DPPEt), propanol (DPPPr), or butanol (DPPBu) head groups. The Phosphatidyl alcohols are biologically relevant, because they can be formed in membranes by the phospholipase D reaction in the presence of alcohol. The melting behavior of pure phosphatidyl alcohols and mixtures with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or cholesterol was assessed using high sensitivity differential scanning calorimetry (DSC). DPPMe had the highest melting temperature (∼ 49 °C), whereas the other phosphatidyl alcohols had similar melting temperatures as DPPC (∼ 40-41 °C). All phosphatidyl alcohols, except DPPMe, also showed good miscibility with DPPC. The effects of cholesterol on the melting behavior and membrane order in multilamellar bilayer vesicles were assessed using steady-state anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DSC. The ordering effect of cholesterol in the fluid phase was lower for all phosphatidyl alcohols as compared to DPPC and decreased with increasing head group size. The formation of ordered domains containing the phosphatidyl alcohols in complex bilayer membranes was determined using fluorescence quenching of DPH or the sterol analogue cholesta-5,7,(11)-trien-3-beta-ol (CTL). The phosphatidyl alcohols did not appear to form sterol-enriched ordered domains, whereas DPPMe, DPPEt appeared to form ordered domains in the temperature window examined (10-50 °C). The partitioning of CTL into bilayer membranes containing phosphatidyl alcohols was to a small extent increased for DPPMe and DPPEt, but in general, sterol interactions were weak or unfavorable for the phosphatidyl alcohols. Our results show that the biophysical and sterol interacting properties of phosphatidyl alcohols, having identical acyl chain structures, are markedly dependent on the size of the head group.     

The conformation of end-groups is one determinant of carotenoid topology suitable for high fidelity molecular recognition: A study of β- and ε-end-groups

Conformation affects a carotenoid’s ability to bind selectively to proteins. We calculated adiabatic energy profiles for rotating the ring end-groups around the C6C7 bond and for flexing of the ring with respect to the polyene chain. The choice of computational methods is important. A low, 4.2 kcal/mol barrier to rotation exists for a β-ring. An 8.3 kcal/mol barrier exists for rotation of an ε-ring. Rotation of the ε-ring is sensitive to substitution at C3. In the absence of external forces neither β- nor ε-rings are rotationally constrained. The nearly parallel alignment of the β-ring to the C6C7 bond axis contrasts to the more perpendicular orientation of the ε-ring. Flexion of a β-ring to the minimized ε-ring conformation requires ∼23 kcal/mol; extension of the ε-ring to the minimized β-ring conformation requires ∼8 kcal/mol. Selectivity associated with β- versus ε-rings is dominated by the inability of the β-ring to flex to minimize protein/ring steric interactions and maximize van der Waal’s attractions with the binding site.     

Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H₂O₂ generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-, and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.