An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.     

Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.     

Recognition of joy, anger, and fear by face expression in humans

Behavioral and neurophysiological characteristics of a visual recognition of emotions of joy, anger, and fear were studied in 9 young healthy men and 10 women. It was shown that these emotions were identified by subjects with different rate and accuracy; significant gender differences in recognition of anger and fear were found. Recording of visual evoked potentials (VEP) from the occipital (O1/2), medial temporal (T3/4), inferior temporal (T5/6), and frontal (F3/4) areas revealed differences (related with the type of emotion) in the latencies of P150, N180, P250, and N350 waves and in the amplitude of VEP components with the latencies longer than 250 ms. These differences were maximally expressed in T3/4 derivation. The subjects could be divided in two groups. The first group was characterized by increased VEP latencies and higher amplitudes of VEP components later than 250 ms in response to anger (in comparison with other types of emotions). These phenomena were observed in all the derivations but were most pronounced in T3/4. In the second group, only late P250 and N350 components had shorter latencies during recognition of fear. VEP amplitude variations related with the type of emotions were insignificant and were recorded in the occipital and frontal areas. The two groups of subjects also differed in psychoemotional personality characteristics. It is suggested that primary recognition of facial expression takes place in the temporal cortical areas. A possible correlation of electrophysiological indices of emotion recognition with personality traits is discussed.     

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.     

Lengthy unilateral EEG changes in alert rabbits after a spreading single depressed wave

Effects of a single wave of the cortical spreading depression (SD) on the ECoG of a waking rabbit was studied with chronically implanted intracortical calomel and silverball epidural electrodes. DC potential shifts and integral electrical activity were recorded monopolary in reference to a nasal-bone electrode. ECoG spectral analysis (FFT) showed that an SD wave was accompanied by a suppression of the neocortical activity in a broad frequency range (0.25-80 Hz). However, the SD-related ECoG depression was a rather short phenomenon (5-7 min) as compared to a following rebound effect, i.e., persistent (1.5-2 h) unilateral exaltation of bioelectrical activity. The spectral power in the delta (6-14 fold) and beta bands (2-6-fold) increased, whereas the high-frequency activity (40-80 Hz) remained suppressed. Similar changes in the contralateral neocortex were poorly pronounced or absent; this resulted in a strong interhemispheric asymmetry. It is suggested that (1) exaltation of the delta activity after SD wave is related not only to a dendrite swelling and changes in the extracellular space structure but to increase in synaptic transmission efficiency, probably, by the type of anoxic potentiation, (2) activation of some subcortical structures by the mechanism of their release from the inhibitory neocortical control is an additional factor of the augmentation of the delta and spindle-like beta activity after an SD wave, and (3) the long-term attenuation of the high-frequency gamma activity is a result of its strong suppression during the SD and its reciprocal relations with the exalted delta activity.     

Stabilization of P450 2B4 by its association with P450 1A2 revealed by high-pressure spectroscopy

We studied the effect of intermolecular interactions between cytochromes P450 1A2 (CYP1A2) and 2B4 (CYP2B4) on the barotropic inactivation of the ferrous carbonyl complexes of the hemoproteins. When taken separately, these hemoproteins reveal quite distinct barotropic behavior. While the 2B4(Fe(2+))-CO complex is very sensitive to hydrostatic pressures and undergoes P450 --> P420 transition at rather low pressures (P(1/2) = 297 MPa, DeltaV(0) = -61 ml/mol), the 1A2(Fe(2+))-CO is extremely resistant to barotropic inactivation. Only about 8% of the 1A2 was exposed to pressure-induced P450 --> P420 transition (P(1/2) = 420 MPa, DeltaV(0) = -28 ml/mol). The formation of the mixed oligomers of 2B4 and 1A2 was found to have a dramatic effect on the barotropic behavior of 2B4. In the heterooligomers of 1A2 and 2B4, the 2B4 hemoprotein appears to be largely protected from barotropic inactivation. In 1:1 mixed oligomers no more than 25% of the total P450 content undergoes P450 --> P420 inactivation with the molar reaction volume value (DeltaV(0) = -26 ml/mol) similar to those found for pure 1A2. Moreover, interactions between 1A2 and 2B4 results in a displacement of the Soret band of the ferrous carbonyl complex of CYP2B4 to shorter wavelength (from 451.3 to 448.4 nm) and largely strengthens the dependence of the Soret band wavenumber on hydrostatic pressure below 200 MPa. This effect suggests an important hydration of the CYP2B4 heme moiety in response to the interactions with CYP1A2. We discuss these results in terms of the hypothesis that the heterooligomerization of cytochromes P450 in microsomes plays an important role in the control of the activity and coupling of the microsomal monooxygenase.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

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.