Cytochrome P450 from Photobacterium profundum SS9, a piezophilic bacterium, exhibits a tightened control of water access to the active site

We report cloning, expression in Escherichia coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further toward the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoylglycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoylglycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around -100 to -200 mL/mol and P(1/2) of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium toward the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.     

EPR and ENDOR characterization of intermediates in the cryoreduced oxy-nitric oxide synthase heme domain with bound L-arginine or N(G)-hydroxyarginine

Reconstitution of the endothelial nitric oxide synthase heme domain (NOS) with the catalytically noncompetent 4-aminotetrahydrobiopterin has allowed us to prepare at -40 degrees C the oxyferrous-NOS-substrate complexes of both L-arginine (Arg) and N(G)-hydroxyarginine (NOHA). We have radiolytically cryoreduced these complexes at 77 K and used EPR and ENDOR spectroscopies to characterize the initial products of reduction, as well as intermediates that arise during stepwise annealing to higher temperatures. Peroxo-ferri-NOS is the primary product of 77 K cryoreduction when either Arg or NOHA is the substrate. Proton ENDOR spectra of this state suggest that the peroxo group is H-bonded to a [guanidinium-water] network that forms because the binding of O2 to the ferroheme of NOS recruits H2O. At no stage of reaction/annealing does one observe an EPR signal from a hydroperoxo-ferri state with either substrate. Instead, peroxo-ferri-NOS-substrate complexes convert to a product-state intermediate at the extremely low temperature of 165-170 K. EPR and proton ENDOR spectra of the intermediate formed with Arg as substrate support the suggestion that the reaction involves the formation and attack of Compound I. Within the time/temperature resolution of the present experiments, samples with Arg and NOHA as substrate behave the same in the initial steps of cryoreduction/annealing, despite the different acid/base characteristics of the two substrates. This leads us to discuss the possibility that ambient-temperature catalytic conversion of both substrates is initiated by reduction of the oxy-ferroheme to the hydroperoxo-ferriheme through a coupled proton-electron transfer from a heme-pocket reductant, and that Arg may provide the stoichiometrically second proton of catalysis.     

NADP pool state and activity of NADP-dependent dehydrogenases in the heart tissue of adult and old rats during immobilization stress

To reveal the reasons of age-specific change of heart sensitivity to stress, the content of oxidized and reduced NADP and activity of NADP-dependent dehydrogenase in myocardium of adult and old immobilized rats have been studied. It has been established that in the process of aging the amount of reduced NADP decreases in the old rat heart. At the same time the activity of NADP-dependent dehydrogenase decreases in postmitochondrial fraction of old rat myocardium as compared to adult rats. There is no change in the amount of reduced NADP in the heart of both rat age groups after 30-minute immobilization. The activity of NADP-dependent dehydrogenase in postmitochondrial fraction of myocardium in adult rats remains on the initial level while activation of NADP-dependent malate dehydrogenase and glucose 6-phosphate dehydrogenase appears in old rats.     

Suppression of gamma EEG activity as an index of a spreading depression wave in the neocortex of a waking rabbit

A spreading depression (SD) can spontaneously develop in seizures, attacks of migraine, vascular disorders and other pathological states of the brain. However, problems in technique of recording the DC-potential in the neocortex of humans and waking animals substantially restrict the possibilities of studying functional consequences of the SD. In this article, the EEG pattern was studied in detail at the moment of the SD development. Specific features were revealed, which make it possible to detect the SD without recording shifts of the DC-potential. At the moment of the SD arrival, the interhemispheric balance drastically disturbs because of a strong decrease in the high-frequency activity. By the time indices, the course of the suppression of the gammal and gamma2 EEG frequencies is the most reliable symptom of the SD wave development. The EEG spectral power in the delta band increases with a certain delay in reference to the deep depression of the high-frequency activity and is, in essence, an SD aftereffect. The found EEG signs of an SD wave can substantially simplify the identification of this phenomenon both in experiment and clinical conditions in certain pathological states of the brain.     

Gender differences in regulating emotions in response to text with violent content

Gender difference in intensity of emotional response depending on the cognitive disclosure of the emotions were analyzed in detail. Texts with emotionally negative content were presented to the subjects, and the parameters of their cardiovascular activity were recorded either before or after filling a special checklist to assess the impression caused by the texts and to reveal changes in the physiological activation of the subjects in response to these texts. Unlike women, men intensified their affective involvement into processing the content of the texts after filling the checklist. The results are interpreted in the context of current research on physiological emotional processes during their cognitive disclosure; the role of gender differences in the regulation of these processes was also studied.     

Interhemispheric EEG interrelations in recognition of masked visual images accompanied by music

Changes in the EEG depend on the style of accompanying music. Classical music produces more patterns with increased diagonal coherences in the γ frequency band, where two foci of integration are formed at points T ₄ and T ₆. During listening to rock music, a larger proportion of all changes occurred in the θ-and α₁ frequency bands; an integration focus was formed at point T ₆ in the α₁ and γ frequency bands. An increase in diagonal coherences that was observed during the recognition was especially pronounced in the Δ-, θ-and α₁ frequency bands. In addition, there was an increase in the coherence between the potentials recorded at points O ₁-P ₄ and O ₂-F ₇, and it was observed in all the frequency bands. During the recognition of masked visual images accompanied by classical or rock music, there were more patterns with an increased diagonal coherence of the currently dominant activity. Before erroneous recognition, a widely generalized increase in coherence over the brain cortex was observed in the Δ, θ, and α₁ bands, which was an informative indicator of the subsequent erroneous recognition. In tests with recognition unaccompanied by music, as well as with music, the main change in symmetrical coherences was either a decrease in or an absence of changes in this indicator versus the background value.     

Conversion of methane in a coaxial microwave torch

A microwave coaxial plasmatron (microwave torch) is used as a plasmachemical converter of methane into hydrogen and hydrocarbons. The measured energy cost of methane decomposition is close to its minimum theoretical value. Such a low energy cost is unsurpassed for reactors operating at atmospheric pressure. A model of the plasmachemical converter is constructed. The results of calculations in the frame-work of this model agree well with experimental data.