HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Inter-beat intervals of cardiac-cell aggregates during exposure to 2.45 GHz CW,pulsed, and square-wave-modulated microwaves

Inter-beat intervals of aggregated cardiac cells from chicken embryos were studied during 190 s exposures to 2.45 GHz microwaves in an open-ended coaxial device. Averaged specific-absorption rates (SARs) and modulation conditions were 1.2–86.9 W/kg continuous-wave (CW). 1.2–12.2 W/kg pulse modulation (PW, duty cycle ∽ 11%). and 12.0–43.5 W/kg square-wave modulation (duty cycle = 50%). The inter-beat interval decreased during microwave exposures at 42.0 W/kg and higher when CW or square-wave modulation was used, which is consistent with established effects of elevated temperatures. However, increases in the inter-beat interval during CW exposures at 1.2–12.2 W/kg, and decreases in the inter-beat interval after PW exposures at 8.4–12.2 W/kg. are not consistent with simple thermal effects. Analysis of variance indicated that SAR. modulation, and the modulation-SAR interaction were all significant factors in altering the interbeat interval. The latter two factors indicated that the cardiac cells were affected by athermal as well as thermal effects of microwave exposure. © 1993 Wiley-Liss. Inc.     

Characterization of nonsymbiotic tomato hemoglobin

The nonsymbiotic tomato hemoglobin SOLly GLB1 (Solanum lycopersicon) is shown to form a homodimer of approximately 36 kDa with a high affinity for oxygen. Furthermore, our combined ultraviolet/visible, resonance Raman, and continuous wave electron paramagnetic resonance (EPR) measurements reveal that a mixture of penta- and hexacoordination of the heme iron is found in the deoxy ferrous form, whereas the ferric form shows predominantly a bis-histidine ligation (F8His-Fe(2+/3+)-E7His). This differs from the known forms of vertebrate hemoglobins and myoglobins. We have successfully applied our recently designed pulsed-EPR strategy to study the low-spin ferric form of tomato hemoglobin. These experiments reveal that, in ferric SOLly GLB1, one of the histidine planes is rotated 20 degrees (+/-10 degrees ) away from a N(heme)-Fe-N(heme) axis. Additionally, the observed g-values indicate a quasicoplanarity of the histidine ligands. From the HYSCORE (hyperfine sublevel correlation) measurements, the hyperfine and nuclear quadrupole couplings of the heme and histidine nitrogens are identified and compared with known EPR/ENDOR data of vertebrate Hbs and cytochromes. Finally, the ligand binding kinetics, which also indicate that the ferrous tomato Hb is only partially hexacoordinated, will be discussed in relation with the heme-pocket structure. The similarities and differences with other known nonsymbiotic plant hemoglobins will be highlighted.     

Correlation between Hemichrome Stability and the Root Effect in Tetrameric Hemoglobins

Oxidation of Hbs leads to the formation of different forms of Fe(III) that are relevant to a range of biochemical and physiological functions. Here we report a combined EPR/x-ray crystallography study performed at acidic pH on six ferric tetrameric Hbs. Five of the Hbs were isolated from the high-Antarctic notothenioid fishes Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, and one was isolated from the sub-Antarctic notothenioid Cottoperca gobio. Our EPR analysis reveals that 1), in all of these Hbs, at acidic pH the aquomet form and two hemichromes coexist; and 2), only in the three Hbs that exhibit the Root effect is a significant amount of the pentacoordinate (5C) high-spin Fe(III) form found. The crystal structure at acidic pH of the ferric form of the Root-effect Hb from T. bernacchii is also reported at 1.7 Å resolution. This structure reveals a 5C state of the heme iron for both the α- and β-chains within a T quaternary structure. Altogether, the spectroscopic and crystallographic results indicate that the Root effect and hemichrome stability at acidic pH are correlated in tetrameric Hbs. Furthermore, Antarctic fish Hbs exhibit higher peroxidase activity than mammalian and temperate fish Hbs, suggesting that a partial hemichrome state in tetrameric Hbs, unlike in monomeric Hbs, does not remove the need for protection from peroxide attack, in contrast to previous results from monomeric Hbs.     

Strange kinetic phase in the extremely early folding process of beta-lactoglobulin

A continuous-wave probed laser-induced temperature jump system was constructed and applied to monitor the changes in tryptophan fluorescence of the beta-lactoglobulin during its folding; the kinetic phases were traced from 300 ns to 10 ms after a temperature jump. Notably, an early phase with typical squeezed-exponential characteristics, [exp[-(kt)(beta)], beta>1.0], was observed around several tens of microseconds after the temperature jump, which is actually the earliest phase ever observed for beta-lactoglobulin. This process can be explained by conformational shift occurring within the unfolded ensemble (U-->U'), which is followed by the non-native intermediate (I) formation of this protein.     

EPR properties of a g=2 broad signal trapped in an S1 state in Ca-depleted Photosystem II

We investigated a new EPR signal that gives a broad line shape around g=2 in Ca²⁺-depleted Photosystem (PS) II. The signal was trapped by illumination at 243 K in parallel with the formation of Y_Z. The ratio of the intensities between the g=2 broad signal and the Y_Z signal was 1:3, assuming a Gaussian line shape for the former. The g=2 broad signal and the Y_Z signal decayed together in parallel with the appearance of the S₂ state multiline at 243 K. The g=2 broad signal was assigned to be an intermediate S₁X state in the transition from the S₁ to the S₂ state, where X represents an amino acid radical nearby manganese cluster, such as D1-His337. The signal is in thermal equilibrium with Y_Z. Possible reactions in the S state transitions in Ca²⁺-depleted PS II were discussed.     

Comparative effects of pulsed and continuous-wave 2.8-GHz microwaves on temporally defined behavior

The effects of pulsed-(PW) and continuous-wave (CW) 2.8-GHz microwaves were compared on the performance of rodents maintained by a temporally defined schedule of positive reinforcement. The schedule involved food-pellet reinforcement of behavior according to a differential-reinforcement-of-low-rate (DRL) contingency. The rats were independently exposed to PW and to CW fields at power densities ranging from 1 to 15 mW/cm². Alterations of normal performance were more pronounced after a 30-minute exposure to the PW field than to the CW field. The rate of emission of appropriately timed responses declined after exposure to PW at 10 and 15 mW/cm², whereas exposure at the same power levels to the CW field did not consistently affect the rate of responding. Change in performance associated with microwave exposure was not necessarily related to a general decline in responding: in some instances, increases in overall rates of responding were observed.     

Low-power compact continuous-wave stimulated emission depletion microscopy

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.     

Optimization for continuous-wave terahertz reflection imaging for biological tissues

Continuous-wave terahertz reflection imaging is a potential tool for biological tissues. Based on our home-made continuous-wave terahertz reflection imaging system, the effect of both polarization mode and reflection window on the imaging performance is studied theoretically and experimentally, showing good agreement. By taking obtaining sample information and image contrast into consideration, p-polarized terahertz waves are recommended. Moreover, considering the sample boundary identification and the image contrast, selection criteria for reflection window are proposed. This work will help to improve the performance of continuous-wave terahertz reflection imaging and accelerate the THz imaging in biological application.