EPR properties of mixed-valent μ-oxo and μ-hydroxo dinuclear iron complexes produced by radiolytic reduction at 77 K

 Radiolytic reduction at 77 K of oxo-/hydroxo-bridged dinuclear iron(III) complexes in frozen solutions forms kinetically stabilized, mixed-valent species in high yields that model the mixed-valent sites of non-heme, diiron proteins. The mixed-valent species trapped at 77 K retain ligation geometry similar to the initial diferric clusters. The shapes of the mixed-valent EPR signals depend strongly on the bridging ligands. Spectra of the Fe(II)OFe(III) species reveal an S=1/2 ground state with small g-anisotropy as characterized by the uniaxial component (g _z –g _av /2<0.03) observable at temperatures as high as ∼100 K. In contrast, hydroxo-bridged mixed-valent species are characterized by large g-anisotropy (g _z –g _av /2>0.03) and are observable only below 30 K. Annealing at higher temperatures causes structural relaxation and changes in the EPR characteristics. EPR spectral properties allow the oxo- and hydroxo-bridged, mixed-valent diiron centers to be distinguished from each other and can help characterize the structure of mixed-valent centers in proteins. Received: 27 June 1998 / Accepted: 25 February 1999     

The lifetime of molecular (Davydov) solitons

The lifetime of Davydov solitons in a one-dimensional system is studied theoretically. The process of thermalization and the properties of solitons at finite temperature are investigated and the processes of soliton creation and disintegration are discussed.     

A Large-Scale Allosteric Transition in Cytochrome P450 3A4 Revealed by Luminescence Resonance Energy Transfer (LRET)

Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys₆₄ and Cys₃₇₇) and the alpha-domain, where Cys₁₂₁ and Cys₄₆₈ are located.     

High-Speed Recording of Vacuum Arc Discharge Images in the Optical Range

Plasma Physics Reports - Switching of a short vacuum gap with an auxiliary discharge over the surface of a dielectric has been studied via high-speed recording of images of discharge plasma...     

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.     

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.     

Resonance Raman and EPR investigations of the D251N oxycytochrome P450cam/putidaredoxin complex

We have performed resonance Raman and electron paramagnetic resonance (EPR) studies on the dioxygen bound state of the D251N mutant of cytochrome P450cam (oxy-P450cam) and its complex with reduced putidaredoxin (Pd). The D251N oxy-P450cam/Pd complex has a perturbed proton delivery mechanism and shows a significantly red-shifted UV-visible spectrum as observed in Benson et al. [Benson, D. E., Suslick, K. S., and Sligar, S. G. (1997) Biochemistry 36, 5104-5107]. The red shift has been interpreted to indicate a major perturbation of the electronic structure of the oxy-heme complex. However, we find no evidence that electron transfer has occurred from Pd to the heme active site of D251N oxy-P450cam. This suggests that both electron and proton transfer are perturbed by the D251N mutation and that these processes may be coupled. Three oxygen isotope sensitive Raman features are identified in the Pd complex, and occur at 1137, 536, and 399 cm(-1). These values are not significantly different from those for WT or D251N oxy-P450cam. However, a careful examination of the oxygen stretching feature near 1137 cm(-1) reveals the presence of three peaks at 1131, 1138, and 1146 cm(-1), which we attribute to the presence of conformational substates in oxy-P450cam. A significant change in the conformational substate population is observed for the D251N oxy-P450cam when the Pd complex is formed. We suggest that the conformational population redistribution of oxy-P450cam, along with the red-shifted electronic spectra, reflects a structural equilibrium of the oxy-heme that is perturbed upon Pd binding. We propose that this structural perturbation is connected to the effector function of Pd and may involve changes in the electron donation properties of the thiolate ligand.     

Resolution of multiple substrate binding sites in cytochrome P450 3A4: the stoichiometry of the enzyme-substrate complexes probed by FRET and Job's titration

To explore the mechanism of homotropic cooperativity in human cytochrome P450 3A4 (CYP3A4) we studied the interactions of the enzyme with 1-pyrenebutanol (1-PB), 1-pyrenemethylamine (PMA), and bromocriptine by FRET from the substrate fluorophore to the heme, and by absorbance spectroscopy. These approaches combined with an innovative setup of titration-by-dilution and continuous variation (Job's titration) experiments allowed us to probe the relationship between substrate binding and the subsequent spin transition caused by 1-PB or bromocriptine or the type-II spectral changes caused by PMA. The 1-PB-induced spin shift in CYP3A4 reveals prominent homotropic cooperativity, which is characterized by a Hill coefficient of 1.8 +/- 0.3 (S50 = 8.0 +/- 1.1 microM). In contrast, the interactions of CYP3A4 with bromocriptine or PMA reveal no cooperativity, exhibiting KD values of 0.31 +/- 0.08 microM and 7.1 +/- 2.3 microM, respectively. The binding of all three substrates monitored by FRET in titration-by-dilution experiments at an enzyme:substrate ratio of 1 reveals a simple bimolecular interaction with KD values of 0.16 +/- 0.09, 4.8 +/- 1.4, and 0.18 +/- 0.09 microM for 1-PB, PMA, and bromocriptine, respectively. Correspondingly, Job's titration experiments showed that the 1-PB-induced spin shift reflects the formation of a complex of the enzyme with two substrate molecules, while bromocriptine and PMA exhibit 1:1 binding stoichiometry. Combining the results of Job's titrations with the value of KD obtained in our FRET experiments, we demonstrate that the interactions of CYP3A4 with 1-PB obey a sequential binding mechanism, where the spin transition is triggered by the binding of 1-PB to the low-affinity site, which becomes possible only upon saturation of the high-affinity site.     

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.