Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Spatio-temporal variability in the photosynthetic characteristics of Zostera tasmanica measured by PAM

Rapid light curves (RLCs), based on pulse amplitude modulated (PAM) fluorometry, were used to investigate the spatio-temporal variability in photosynthesis versus irradiance parameters (α, I_k and P_max) and the F_v/F_m ratio of the seagrass Zostera tasmanica (formerly Heterozostera tasmanica). Spatial variation was examined across scales ranging from within a leaf (cms) to across the bed (ms), using a nested analysis of covariance sampling design. Overall, significant variation was identified at all scales examined, excluding the largest scale (area). Patterns of variability differed among individual parameters; however a high percentage of the variation was consistently assigned to the covariates, age (within and between leaves) for all parameters, except P_max.     

Water deficit in field-grown Gossypium hirsutum primarily limits net photosynthesis by decreasing stomatal conductance,increasing photorespiration,and increasing the ratio of dark respiration to gross photosynthesis

Much effort has been expended to improve irrigation efficiency and drought tolerance of agronomic crops; however, a clear understanding of the physiological mechanisms that interact to decrease source strength and drive yield loss has not been attained. To elucidate the underlying mechanisms contributing to inhibition of net carbon assimilation under drought stress, three cultivars of Gossypium hirsutum were grown in the field under contrasting irrigation regimes during the 2012 and 2013 growing season near Camilla, Georgia, USA. Physiological measurements were conducted on three sample dates during each growing season (providing a broad range of plant water status) and included, predawn and midday leaf water potential (Ψ_PD and Ψ_MD), gross and net photosynthesis, dark respiration, photorespiration, and chlorophyll a fluorescence. End-of-season lint yield was also determined. Ψ_PD ranged from −0.31 to −0.95 MPa, and Ψ_MD ranged from −1.02 to −2.67 MPa, depending upon irrigation regime and sample date. G. hirsutum responded to water deficit by decreasing stomatal conductance, increasing photorespiration, and increasing the ratio of dark respiration to gross photosynthesis, thereby limiting P_N and decreasing lint yield (lint yield declines observed during the 2012 growing season only). Conversely, even extreme water deficit, causing a 54% decline in P_N, did not negatively affect actual quantum yield, maximum quantum yield, or photosynthetic electron transport. It is concluded that P_N is primarily limited in drought-stressed G. hirsutum by decreased stomatal conductance, along with increases in respiratory and photorespiratory carbon losses, not inhibition or down-regulation of electron transport through photosystem II. It is further concluded that Ψ_PD is a reliable indicator of drought stress and the need for irrigation in field-grown cotton.     

Decoupling of differentiation between traits and their underlying genes in response to divergent selection

We dissected the relationship between genetic differentiation (Q(ST)) for a trait and its underlying genes (G(STq), differentiation for a quantitative locus) in an evolutionary context, with the aim of identifying the conditions in which these two measurements are decoupled. We used two parameters (θ(B) and θ(W)) scaling the contributions of inter- and intrapopulation allelic covariation between genes controlling the trait of interest. We monitored the changes in θ(B) and θ(W), Q(ST) and G(STq) over successive generations of divergent and stabilizing selection, in simulations for an outcrossing species with extensive gene flow. The dynamics of these parameters are characterized by two phases. Initially, during the earliest generations, differentiation of the trait increases very rapidly and the principal and immediate driver of Q(ST) is θ(B). During subsequent generations, G(STq) increases steadily and makes an equal contribution to Q(ST). These results show that selection first captures beneficial allelic associations at different loci at different populations, and then targets changes in allelic frequencies. The same patterns are observed when environmental change modifies divergent selection, as shown by the very rapid response of θ(B) to the changes of selection regimes. We compare our results with previous experimental findings and consider their relevance to the detection of molecular signatures of natural selection.     

Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c1 and its physiological redox partners

The transient electron transfer (ET) interactions between cytochrome c₁ of the bc₁-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c₅₅₂ and cytochrome c₅₅₀ have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c₁ were generated and used together with a soluble cytochrome c₅₅₂ module as a model system for interprotein ET reactions. Both c₁ fragments lack the membrane anchor; the c₁ core fragment (c_1CF) consists of only the hydrophilic heme-carrying domain, whereas the c₁ acidic fragment (c_1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c₁ reveals fast bimolecular rate constants (10⁷ to 10⁸ M^− 1 s^− 1) for the ET reaction with its physiological substrates c₅₅₂ and c₅₅₀, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c₁-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c₅₅₀ over c₅₅₂ reacting with cyochrome c₁ was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.     

Determination of catalase activity using chromogenic probe involving iso-nicotinicacidhydrazide and pyrocatechol

A biocatalatic pathway involving chromogenic probe has been proposed for the determination of catalase activity by means of iso-nicotinicacidhydrazide (INH) and pyrocatechol (PC). The assay is based on the enzymatic consumption of hydrogen peroxide using INH-PC system. The response of the catalase activity was ascertained by the rate of the reaction involving 14.10 mM H₂O₂. On addition of H₂O₂, INH-PC indicator system formed a chromogenic product with absorbance maxima at 490 nm. Hence the activity of catalase was directly measured by the chromogenic response in the formation of the coupled product. The catalase assay was elaborated by the kinetic response of the INH-PC system. The linearity of the catalase activity and H₂O₂ was in the range 0.2-7.0 units and 1.76-7.0 mM, respectively in 3 ml solution. The catalytic efficiency and catalytic power were calculated. The Michaelis-Menten constant of INH, PC and H₂O₂ were found to be 0.344, 0.176 and 8.82 mM, respectively. The indicator reaction was applied in the determination of catalase activity in mycelia mats and culture media.     

Interconversions of different forms of vitamin B6 in tobacco plants

There are six different vitamin B₆ (VB₆) forms, pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxal 5′-phosphate (PLP), pyridoxamine 5′-phosphate (PMP), and pyridoxine 5′-phosphate (PNP), of which PLP is the active form. Although plants are a major source of VB₆ in the human diet, and VB₆ plays an important role in plants, the mechanisms underlying the interconversions of different VB₆ forms are not well understood. In this study, in vitro tobacco plants were grown on Murashige and Skoog (MS) basal media supplemented with 100 mg/L of PM, PL or PN and the abundance of the different B₆ vitamers in leaf tissue was quantified by high performance liquid chromatography (HPLC). The total amount of VB₆ was about 3.9 μg/g fresh weight of which PL, PM, PN, PLP and PMP accounted for 23%, 14%, 37%, 20% and 6%, respectively. Tobacco plants contained a trace amount of PNP. Supplementation of the culture medium with any of the non-phosphorylated vitamers resulted in an increase in total VB₆ by about 10-fold, but had very little impact on the concentrations of the endogenous phosphorylated vitamers. Administration of either PM or PN increased their endogenous levels more than the levels of any other endogenous B₆ vitamers. PL supplementation increased the levels of plant PN and PM significantly, but not that of PL, suggesting that efficient conversion pathways from PL to PN and PM are present in tobacco. Additionally, maintenance of a stable level of PLP in the plant is not well-correlated to changes in levels of non-phosphorylated forms.     

Absolute correlation between lag time and growth rate in the spontaneous formation of several amyloid-like aggregates and fibrils

The formation of polypeptide aggregates, including amyloid fibrils and prions, is a biochemical process of considerable interest in the context of its association with ageing and neurodegeneration. Aggregation occurs typically with a lag phase and a growth phase that reflect an underlying nucleation-polymerisation mechanism. While the propensity of nucleation can be estimated from the lag time t(l), the efficiency of growth is represented by the growth rate k(g). Here, I have analysed the absolute k(g) and t(l) values from a total of 298 samples prepared from insulin, glucagon and different sequence variants of the Alzheimer's Abeta(1-40) peptide. Although these samples differ in the conditions of aggregation, systematic comparison reveals an overall similarity in the plot of k(g)versus t(l). The plot fits readily with the simple equation k(g)=alpha/t(l) and by using a proportionality factor alpha of 4.5. In contrast to the individual values of k(g) and t(l) that depend substantially on sequential and environmental parameters, alpha seems much less affected by such factors. These data suggest mechanistic similarities in the nucleation behaviour of different amyloid-like fibrils and aggregates.     

Cholesterol-induced protein sorting: an analysis of energetic feasibility

The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.     

The high-resolution NMR structure of the early folding intermediate of the Thermus thermophilus ribonuclease H

Elucidation of the high-resolution structures of folding intermediates is a necessary but difficult step toward the ultimate understanding of the mechanism of protein folding. Here, using hydrogen-exchange-directed protein engineering, we populated the folding intermediate of the Thermus thermophilus ribonuclease H, which forms before the rate-limiting transition state, by removing the unfolded regions of the intermediate, including an α-helix and two β-strands (51 folded residues). Using multidimensional NMR, we solved the structure of this intermediate mimic to an atomic resolution (backbone rmsd, 0.51 Å). It has a native-like backbone topology and shows some local deviations from the native structure, revealing that the structure of the folded region of an early folding intermediate can be as well defined as the native structure. The topological parameters calculated from the structures of the intermediate mimic and the native state predict that the intermediate should fold on a millisecond time scale or less and form much faster than the native state. Other factors that may lead to the slow folding of the native state and the accumulation of the intermediate before the rate-limiting transition state are also discussed.     

The reaction of neuroglobin with potential redox protein partners cytochrome b5 and cytochrome c

Previously identified, potentially neuroprotective reactions of neuroglobin require the existence of yet unknown redox partners. We show here that the reduction of ferric neuroglobin by cytochrome b(5) is relatively slow (k=6 x 10(2)M(-1)s(-1) at pH 7.0) and thus is unlikely to be of physiological significance. In contrast, the reaction between ferrous neuroglobin and ferric cytochrome c is very rapid (k=2 x 10(7)M(-1)s(-1)) with an apparent overall equilibrium constant of 1 microM. Based on this data we propose that ferrous neuroglobin may well play a role in preventing apoptosis.     

Cotton bracts are adapted to a microenvironment of concentrated CO2 produced by rapid fruit respiration

Background and Aims

Elucidation of the mechanisms by which plants adapt to elevated CO₂ is needed; however, most studies of the mechanisms investigated the response of plants adapted to current atmospheric CO₂. The rapid respiration rate of cotton (Gossypium hirsutum) fruits (bolls) produces a concentrated CO₂ microenvironment around the bolls and bracts. It has been observed that the intercellular CO₂ concentration of a whole fruit (bract and boll) ranges from 500 to 1300 µmol mol⁻¹ depending on the irradiance, even in ambient air. Arguably, this CO₂ microenvironment has existed for at least 1·1 million years since the appearance of tetraploid cotton. Therefore, it was hypothesized that the mechanisms by which cotton bracts have adapted to elevated CO₂ will indicate how plants will adapt to future increased atmospheric CO₂ concentration. Specifically, it is hypothesized that with elevated CO₂ the capacity to regenerate ribulose-1,5-bisphosphate (RuBP) will increase relative to RuBP carboxylation.

Methods

To test this hypothesis, the morphological and physiological traits of bracts and leaves of cotton were measured, including stomatal density, gas exchange and protein contents.

Key results

Compared with leaves, bracts showed significantly lower stomatal conductance which resulted in a significantly higher water use efficiency. Both gas exchange and protein content showed a significantly greater RuBP regeneration/RuBP carboxylation capacity ratio (J_max/V_cmax) in bracts than in leaves.

Conclusions

These results agree with the theoretical prediction that adaptation of photosynthesis to elevated CO₂ requires increased RuBP regeneration. Cotton bracts are readily available material for studying adaption to elevated CO₂.     

Thermoregulation in male temperate bats depends on habitat characteristics

Several energy-saving strategies have evolved in animals, one example being the short-term reduction of metabolism and body temperature (torpor) in endotherms. For bats, pronounced torpor behaviour has been described. The aim of this study was to assess individual variation in torpor expression of male Myotis daubentonii, and to analyse whether this variation is related to habitat characteristics. For that we measured skin temperatures of bats from different habitats using radio transmitters and also recorded ambient temperature. Skin temperature was corrected for ambient temperature and individual body mass. Cluster analysis of residuals revealed two different thermoregulatory strategies. Males in cluster 1 were more often encountered torpid and reached lower minimum skin temperatures than males in cluster 2. The differences in behaviour were related to environmental variables (water surface area near the roost, roost altitude, precipitation, ambient temperature in the warmest quarter of the year). Males from cluster 1 occupied less favourable habitats (less water surface, higher altitudes, wetter and colder climate) than males from cluster 2. Our data suggest a linkage between torpor behaviour and habitat characteristics. These characteristics could be used to identify favourable and marginal habitats for M. daubentonii.     

Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc₁ complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b_H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b_H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

The use of small subcutaneous transponders for quantifying thermal biology and torpor in small mammals

Remote measurements of body temperature (T_b) in animals require implantation of relatively large temperature-sensitive radio-transmitters or data loggers, whereas rectal temperature (T_rec) measurements require handling and therefore may bias the results. We investigated whether ∼0.1 g temperature-sensitive subcutaneously implanted transponders can be reliably used to quantify thermal biology and torpor use in small mammals. We examined (i) the precision of transponder readings as a function of temperature and (ii) whether subcutaneous transponders can be used to remotely record subcutaneous temperature (T_sub). Five adult male dunnarts (Sminthopsis macroura, body mass 24 g) were implanted with subcutaneous transponders to determine T_sub as a function of time and ambient temperature (T_a), and in comparison to thermocouple readings of T_rec. Transponder temperature was highly correlated with water bath temperature (r²=0.96–0.99) over a range of approximately 10.0–40.0 °C. Transponders provided reliable data (±0.6 °C) over the T_sub of 21.4–36.9 °C and could be read from a distance of up to 5 cm. Below 21.4 °C, accuracy was reduced to ±2.8 °C, but individual transponder accuracy varied. Consequently, small subcutaneous transponders are useful to remotely quantify thermal physiology and torpor patterns without having to disturb the animal and disrupt torpor. Even at T_sub<21.4 °C where the accuracy of the temperature readings was reduced, transponders do provide reliable data on whether and when torpor is used.     

Genetic consequences of a century of protection: serial founder events and survival of the little spotted kiwi (Apteryx owenii)

We present the outcome of a century of post-bottleneck isolation of a long-lived species, the little spotted kiwi (Apteryx owenii, LSK) and demonstrate that profound genetic consequences can result from protecting few individuals in isolation. LSK were saved from extinction by translocation of five birds from South Island, New Zealand to Kapiti Island 100 years ago. The Kapiti population now numbers some 1200 birds and provides founders for new populations. We used 15 microsatellite loci to compare genetic variation among Kapiti LSK and the populations of Red Mercury, Tiritiri Matangi and Long Islands that were founded with birds from Kapiti. Two LSK native to D''Urville Island were also placed on Long Island. We found extremely low genetic variation and signatures of acute and recent genetic bottleneck effects in all four populations, indicating that LSK have survived multiple genetic bottlenecks. The Long Island population appears to have arisen from a single mating pair from Kapiti, suggesting there is no genetic contribution from D''Urville birds among extant LSK. The N_e/N_C ratio of Kapiti Island LSK (0.03) is exceptionally low for terrestrial vertebrates and suggests that genetic diversity might still be eroding in this population, despite its large census size.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

Generation, characterization and crystallization of a highly active and stable cytochrome bc1 complex mutant from Rhodobacter sphaeroides

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure-function relation of the cytochrome bc₁ complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc₁ complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 °C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Å resolution.     

Effects of changes in the shape of the intracellular action potential on the peak-to-peak ratio of single muscle fibre potentials

In situ recording of the intracellular action potential (IAP) of human muscle fibres is not yet feasible, and consequently, knowledge about certain IAP characteristics of these IAPs is still limited. The ratio between the amplitudes of the second and first phases (the so-called peak-to-peak ratio, PPR) of a single fibre action potential (SFAP) is known to be closely related to the IAP profile. The PPR of experimentally recorded SFAPs has been found to be largely independent of changes in the fibre-to-electrode (radial) distance. The main goal of this paper is to analyze the effect of changes in different aspects of the IAP spike on the relationship between PPR and radial distance. Based on this analysis, we hypothesize about the characteristics of IAPs obtained experimentally. It was found that the sensitivity of the SFAP PPR to changes in radial distance is essentially governed by the duration of the IAP spike. Assuming that, for mammals, the duration of the IAP rising phase lies within the range 0.2-0.4 ms, we tentatively suggest that the duration of the IAP spike should be over approximately 0.75 ms, with the shape of the spike strongly asymmetric. These IAP characteristics broadly coincide with those observed in mammal IAPs.