Breaking glass?

Why are women so under-represented at the top echelons of science? Why do so many drop out just as their careers should be blossoming? Are women in science being actively discriminated against by their peers, and, if, so what can be done about this? These were just a few of the puzzles reflected upon at a recent European meeting that examined the role of women in science. Thankfully, there is evidence that, today, there is real opportunity, as well as mere light, emerging through the ceiling that caps so many careers.

Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa.

The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.

Breaking glass?

"The Glass Ceiling for Women in the Life Sciences Meeting", organized by the European Molecular Biology Organization (EMBO), was held on 22-23 June 2001 in Heidelberg, Germany.

Membrane proteins on the move.

The Keystone Symposium on Membrane protein structure/function relationships was held on 5-11 March 2001 in Tahoe City, California, USA.

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

The nitrite reductase from Pseudomonas aeruginosa: essential role of two active-site histidines in the catalytic and structural properties.

Cd(1) nitrite reductase catalyzes the conversion of nitrite to NO in denitrifying bacteria. Reduction of the substrate occurs at the d(1)-heme site, which faces on the distal side some residues thought to be essential for substrate binding and catalysis. We report the results obtained by mutating to Ala the two invariant active site histidines, His-327 and His-369, of the enzyme from Pseudomonas aeruginosa. Both mutants have lost nitrite reductase activity but maintain the ability to reduce O(2) to water. Nitrite reductase activity is impaired because of the accumulation of a catalytically inactive form, possibly because the productive displacement of NO from the ferric d(1)-heme iron is impaired. Moreover, the two distal His play different roles in catalysis; His-369 is absolutely essential for the stability of the Michaelis complex. The structures of both mutants show (i) the new side chain in the active site, (ii) a loss of density of Tyr-10, which slipped away with the N-terminal arm, and (iii) a large topological change in the whole c-heme domain, which is displaced 20 A from the position occupied in the wild-type enzyme. We conclude that the two invariant His play a crucial role in the activity and the structural organization of cd(1) nitrite reductase from P. aeruginosa.

Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition.

The steady-state reaction of trimethylamine dehydrogenase (TMADH) with the artificial electron acceptor ferricenium hexafluorophosphate (Fc(+)) has been studied by stopped-flow spectroscopy, with particular reference to the mechanism of inhibition by trimethylamine (TMA). Previous studies have suggested that the presence of alternate redox cycles is responsible for the inhibition of activity seen in the high-substrate regime. Here, we demonstrate that partitioning between these redox cycles (termed the 0/2 and 1/3 cycles on the basis of the number of reducing equivalents present in the oxidized/reduced enzyme encountered in each cycle) is dependent on both TMA and electron acceptor concentration. The use of Fc(+) as electron acceptor has enabled a study of the major redox forms of TMADH present during steady-state turnover at different concentrations of substrate. Reduction of Fc(+) is found to occur via the 4Fe-4S center of TMADH and not the 6-S-cysteinyl flavin mononucleotide: the direction of electron flow is thus analogous to the route of electron transfer to the physiological electron acceptor, an electron-transferring flavoprotein (ETF). In steady-state reactions with Fc(+) as electron acceptor, partitioning between the 0/2 and 1/3 redox cycles is dependent on the concentration of the electron acceptor. In the high-concentration regime, inhibition is less pronounced, consistent with the predicted effects on the proposed branching kinetic scheme. Photodiode array analysis of the absorption spectrum of TMADH during steady-state turnover at high TMA concentrations reveals that one-electron reduced TMADH-possessing the anionic flavin semiquinone-is the predominant species. Conversely, at low concentrations of TMA, the enzyme is predominantly in the oxidized form during steady-state turnover. The data, together with evidence derived from enzyme-monitored turnover experiments performed at different concentrations of TMA, establish the operation of the branched kinetic scheme in steady-state reactions. With dimethylbutylamine (DMButA) as substrate, the partitioning between the 0/2 and 1/3 redox cycles is poised more toward the 0/2 cycle at all DMButA concentrations studied-an observation that is consistent with the inability of DMButA to act as an effective inhibitor of TMADH.

Internal electron transfer and structural dynamics of cd1 nitrite reductase revealed by laser CO photodissociation.

Laser photolysis techniques have been employed to investigate the internal electron transfer (eT) reaction within Pseudomonas aeruginosa nitrite reductase (Pa-NiR). We have measured the (d1--> c) internal eT rate for the wild-type protein and a site-directed mutant (Pa-NiR H327A) which has a substitution in the d1-heme binding pocket; we found the rate of eT to be fast, keT = 2.5 x 10(4) and 3.5 x 10(4) s-1 for the wild-type and mutant Pa-NiR, respectively. We also investigated the photodissociation of CO from the fully reduced proteins and observed microsecond first-order relaxations; these imply that upon breakage of the Fe2+-CO bond, both Pa-NiR and Pa-NiR H327A populate a nonequilibrium state which decays to the ground state with a complex time course that may be described by two exponential processes (k1 = 3 x 10(4) s-1 and k2 = 0.25 x 10(4) s-1). These relaxations do not have a kinetic difference spectrum characteristic of CO recombination, and therefore we conclude that Pa-NiR undergoes structural rearrangements upon dissociation of CO. The bimolecular rate of CO rebinding is 5 times faster in Pa-NiR H327A than in the wild-type enzyme (1.1 x 10(5) M-1 s-1 compared to 2 x 10(4) M-1 s-1), indicating that this mutation in the active site alters the CO diffusion properties of the protein, probably reducing steric hindrance. CO rebinding to the wild-type mixed valence enzyme (c3+d12+) which is very slow (k = 0.25 s-1) is proposed to be rate-limited by the c --> d1 internal eT event, involving the oxidized d1-heme which has a structure characteristic of the fully oxidized and partially oxidized Pa-NiR.

An exposed tyrosine on the surface of trimethylamine dehydrogenase facilitates electron transfer to electron transferring flavoprotein: kinetics of transfer in wild-type and mutant complexes.

In wild-type trimethylamine dehydrogenase, tyrosine-442 is located at the center of a concave region on the surface of the enzyme that is proposed to form the docking site for the physiological redox acceptor, electron transferring flavoprotein. The intrinsic rate constant for electron transfer in the reoxidation of one-electron dithionite-reduced wild-type trimethylamine dehydrogenase (modified with phenylhydrazine) by electron transferring flavoprotein was investigated by stopped-flow spectroscopy. Analysis of the temperature dependence of the reaction rate by electron transfer theory yielded values for the reorganizational energy of 1.4 eV and the electronic coupling matrix element of 0.82 cm-1. The role played by residue Tyr-442 in facilitating reduction of ETF by TMADH was investigated by isolating three mutant forms of the enzyme in which Tyr-442 was exchanged for a phenylalanine, leucine, or glycine residue. Rates of electron transfer from these mutants of TMADH to ETF were investigated by stopped-flow spectroscopy. At 25 degrees C, modest reductions in rate were observed for the Y442F (1.4-fold) and Y442L (2.2-fold) mutant complexes, but a substantial decrease in rate (30.5-fold) and an elevated dissociation constant for the complex were seen for the Y442G mutant enzyme. Inspection of the crystal structure of wild-type TMADH reveals that Tyr-442 is positioned along one side of a small cavity on the surface of the enzyme: Val 344, located at the bottom of this cavity, is the closest surface residue to the 4Fe-4S center of TMADH and is likely to be positioned on a major electron transfer pathway to ETF. The reduced electron transfer rates in the mutant complexes are probably brought about by decreases in electronic coupling between the electron transfer donor and acceptor within the complex, either directly or indirectly due to unfavorable change in the orientation of the two proteins with respect to one another.

An ultracentrifugal approach to quantitative characterization of the molecular assembly of a physiological electron-transfer complex: the interaction of electron-transferring flavoprotein with trimethylamine dehydrogenase.

The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4 degrees C. Analysis of sedimentation-equilibrium distributions obtained at 15 000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. P. & Winzor, D. J. (1996) Biopolymers 38, 119-130] has yielded an intrinsic dissociation constant of 3-7 microM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules.

Electron tunneling in substrate-reduced trimethylamine dehydrogenase: kinetics of electron transfer and analysis of the tunneling pathway.

The reoxidation of substrate-reduced trimethylamine dehydrogenase by the artificial electron acceptor ferricenium hexafluorophosphate was studied by stopped-flow spectroscopy. The rate constants for the two sequential one-electron transfers from the reduced 4Fe-4S center to ferricenium ions were measured, the first (ka = 49 s-1) being about 7 times greater than the second (kb = 7.3 s-1) at 20 degrees C and neutral pH. The temperature dependence of the second electron transfer was studied over the range 10-40 degrees C, and the rate constant ranged from 5.7 to 19.2 s-1. Analysis of the temperature perturbation of kb by Marcus theory yielded values for the reorganizational energy of 1.95 eV and the electronic coupling matrix element of 0.26 cm-1. An electron tunneling pathway distance of 13 +/- 0.7 A was calculated which correlates with the shortest pathway measured from the 4Fe-4S center to the protein surface using the crystallographic coordinates of trimethylamine dehydrogenase. Tyr-442 is implicated in facilitating electron transfer from the enzyme to ferricenium ions. The data suggest a location for the docking site on the surface of trimethylamine dehydrogenase for the physiological electron acceptor (ETF).

Performance of White Leghorn hens in response to cage density and the introduction of cage mates.

Hens reared at densities of 344 and 516 cm2/bird produced significantly fewer hard-shelled (HS) and shell-less (SL) eggs than hens reared at a density of 1031 cm2/bird. Soft-shelled (SS) egg production was not affected by the density treatment. A decline in ovulation rate would account for the decrease in total hen-day production in response to the higher density treatments. To determine if the introduction of new cage mates would cause hens to lay fewer eggs with poorer shell quality, the performance of hens with visitors was compared with those with nonvisitors. Visitors were introduced monthly for 13 months. Visitors vs. nonvisitors showed no significant difference with regard to HS, SS, and SL egg production, feed efficiency, mortality, egg weight, and specific gravity.

Relationship of plasma corticosterone and adrenal cholesterol and corticosterone to the production of soft-shelled and shell-less eggs.

Two experiments were conducted to determine if plasma corticosterone and adrenal cholesterol and corticosterone levels differed among hens that laid soft-shelled (SS) or shell-less (SL) eggs when compared with hard-shelled (HS) egg layers. For Experiment 1, four groups of White Leghorn hens were bled at 22 to 34 weeks of age in the morning (0600 to 1200 hr) and at 66 to 74 weeks of age in the morning and evening (1500 to 1900 hr). Group 1 consisted of birds that had just laid SS or SL eggs, whereas Group 2 hens had SS or SL eggs in utero. Groups 3 and 4 represented hens with HS eggs in their uteri and hens that had just laid HS eggs, respectively. For Experiment 2, adrenal cholesterol and corticosterone levels were assayed in high (18+%) and low (0%) incidence SS plus SL egg layers at 73 and 78 weeks of age. Both younger and older hens that laid SS or SL eggs in the morning had similar plasma corticosterone concentrations when compared to HS egg layers. Morning plasma corticosterone levels were significantly higher among hens that laid an egg when compared to hens with eggs in utero, regardless of whether the egg was SS, SL, or HS. However, plasma corticosterone levels of hens producing SS or SL eggs in the evening were significantly higher than levels of hens that laid evening HS eggs. Evening HS egg layers demonstrated the oviposition related peak in plasma corticosterone, but concentrations were 1 ng/ml higher among the evening SS or SL egg layers. Adrenal cholesterol and corticosterone concentrations were similar between high and low incidence SS plus SL layers at either age. It is postulated that evening production of SS or SL eggs is a consequence of elevated plasma corticosterone.

Plasma levels of cholesterol and testosterone in White Leghorn hens that laid soft-shelled and shell-less eggs.

Plasma concentrations of cholesterol and testosterone were determined in White Leghorn hens that had just laid soft-shelled (SS) or shell-less (SL) eggs and compared to those that laid hard-shelled (HS) eggs. Hens were bled at two different ages, at 22 to 34 and 66 to 74 weeks of age. Blood samples were collected in the morning hours (0600 to 1200 hr) for both age groups with an additional evening bleed (1500 to 1900 hr) for the 66 to 74-week-old hens. Both plasma constituents were higher in hens that laid SS or SL eggs when compared to those which laid HS eggs, but differences were statistically significant only for the 66 to 74-week-old hens bled in the morning hours. The results of this study indicate that elevated levels of testosterone and its precursor, cholesterol, may be related to the production of eggs with little or no shell calcification.

The effect of caponization and dietary 17 alpha-methyltestosterone on the incidence of leg abnormalities in turkeys.

Six hundred and seventy-two male turkeys were raised according to standard management procedures from 1 day to 3.5 weeks of age. At 3.5 weeks of age, the poults were equally distributed among four treatment groups. One group was subjected to surgical caponization and a second to sham operations. Of the two remaining groups, one served as a control while the other received feed containing 110 ppm of 17 alpha-methyltestosterone. All birds were maintained under identical environmental conditions until 20 weeks of age. Capons demonstrated a significantly higher incidence of leg abnormalities than controls or testosterone fed birds but did not differ significantly from the shams. These data correlated well with the levels of plasma androgen in that capons had significantly lower levels of plasma androgen than controls or testosterone fed turkeys, but concentrations did not differ significantly from shams. Plasma androgen concentrations or percentages of leg abnormalities were not different among shams, controls, or testosterone fed birds. Body weights, feed/gain ratios, mortality, and plasma corticosterone were similar for all four treatment groups.

The effect of varied brooding temperature on the incidence of leg weakness and subsequent performance of turkeys.

Male Nicholas turkeys were used in a study designed to determine the effects of high, moderate, and low brooding temperature on the occurrence of leg abnormalities during a subsequent 20 week grow-out period. Performance data were also obtained. There were no significant differences among the poults brooded under any of the three temperature regimens in the incidence of leg weakness or mortality. Body weights and feed/gain ratios measured at 2, 5, 10, 15, and 20 weeks of age were similar regardless of brooding temperature.