A resonance Raman study of Ambystoma tigrinum hemoglobins: evidence for intraspecies hemepocket variations

Using resonance Raman difference spectroscopy, the Raman-active vibrational modes of hemoglobins from adult, neotenic, and larval forms of the salamander, Ambystoma tigrinum have been compared to each other and to human hemoglobin. The local heme environment of the adult and neotenic proteins were identical and differed from that of the larval protein. Differences were observed in modes sensitive to porphyrin pi electron density and axial ligation. Systematic differences were also observed between human and adult salamander hemoglobins particularly in modes sensitive to the heme vinyl environment. The relationship between these environmental differences, oxygen binding affinity, and the effects of allosteric modulators are discussed.     

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and spectroscopic characterization of exogenous ligand binding to isolated factor F430 and its configurational isomers

Binding of axial ligands to the nickel(II) of isolated factor F430 from the methyl reductase enzyme of Methanobacterium thermoautotrophicum is demonstrated. Evidence of bis-ligand coordination is obtained from the x-ray absorption, optical, and resonance Raman spectral characterization of F430 and its 12,13-diepimeric isomer in the presence of a large excess of cyanide, pyridine, or 1-methylimidazole. Significant broadening and 5-10-nm red shifts of the main 430-nm optical absorption band and shifts of up to 30 cm-1 for the high-frequency Raman lines are observed upon coordination of these axial ligands. The Raman spectra of native F430 and the diepimer with a particular axial ligand are nearly identical. Nickel x-ray absorption edge spectra of the diepimer in the absence and presence of these exogenous ligands are indicative of conversion from a square-planar to a tetragonally distorted octahedral geometry. Analyses of the nickel extended x-ray absorption fine structure data for the ligated diepimer complexes yield detailed structural information for these complexes. Implications of these data with respect to the enzymatic mechanism and the structure of the enzyme-bound factor are discussed.     

Ligand-induced heme ruffling and bent no geometry in ultra-high-resolution structures of nitrophorin 4

The nitrophorins are a family of proteins that use ferric heme to transport nitric oxide (NO) from the salivary glands of blood-sucking insects to their victims, resulting in vasodilation and reduced blood coagulation. We have refined atomic resolution structures of nitrophorin 4 (NP4) from Rhodnius prolixus complexed with NO (1.08 A) and NH(3) (1.15 A), yielding a highly detailed picture of the iron coordination sphere. In NP4-NO, the NO nitrogen is coordinated to iron (Fe-N distance = 1.66 A) and is somewhat bent (Fe-N-O angle = 156 degrees ), with bending occurring in the same plane as the proximal histidine ring. The Fe(NO)(heme)(His) coordination geometry is unusual but consistent with an Fe(III) oxidation state that is stabilized by a highly ruffled heme. Heme ruffling occurs in both structures, apparently due to close contacts between the heme and leucines 123 and 133, but increases on binding NO even though the steric contacts have not changed. We also report the structure of NP4 in complexes with histamine (1.50 A) and imidazole (1.27 A). Unexpectedly, two mobile loops that rearrange to pack against the bound NO in NP4-NO, also rearrange in the NP4-imidazole complex. This conformational change is apparently driven by the nonpolar nature of the NO and imidazole (as bound) ligands. Taken together, the desolvation of the NO binding pocket through a change in protein conformation, and the bending of the NO moiety, possibly through protein-assisted heme ruffling, may lead to a nitrosyl-heme complex that is unusually resistant to autoreduction.     

Resonance Raman investigation of nickel microperoxidase-11

Resonance Raman and UV-visible absorption spectra show that nickel(II) microperoxidase-11 (NiMP-11) is four-coordinate in aqueous solution in the pH range from 1.0 to 13.0. In aqueous solutions of NiMP-11 in the absence of cetyltrimethylammonium bromide (CTAB), NiMP-11 is aggregated. In CTAB micellar solutions, where aggregation of NiMP-11 does not occur, the Raman spectra of NiMP-11 are similar to that of nickel(II) cytochrome c (NiCyt-c). The presence of the peptide segment shifts the equilibrium heavily in favor of the nonplanar form, just as does the entire protein component in the case of NiCyt-c. This further elucidates the structural mechanism by which the protein segment ruffles the heme, most likely modulating the redox potential as indicated for the cytochromes c3 [Ma, J.-G., et al. (1998) Biochemistry 37, 12431-12442]. Furthermore, the hydrophobic environment that is provided by the CTAB micelle is found to be crucial to the native folding of the pentapeptide and formation of two hydrogen bonds in the peptide backbone. These two H-bonds act to contract the peptide segment exerting the force on the macrocycle that causes the ruffling and makes the redox potential more negative than if the heme were to remain planar. The structure of the heme and pentapeptide may also be associated with redox-linked triggering of the formation and release of cytochrome-protein complexes.     

Calcium-dependent heme structure in the reduced forms of the bacterial cytochrome c peroxidase from Paracoccus pantotrophus

This work reports for the first time a resonance Raman study of the mixed-valence and fully reduced forms of Paracoccus pantotrophus bacterial cytochrome c peroxidase. The spectra of the active mixed-valence enzyme show changes in the structure of the ferric peroxidatic heme compared to the fully oxidized enzyme; these differences are observed upon reduction of the electron-transferring heme and upon full occupancy of the calcium site. For the mixed-valence form in the absence of Ca(2+), the peroxidatic heme is six-coordinate and low-spin on the basis of the frequencies of the structure-sensitive Raman lines: the enzyme is inactive. With added Ca(2+), the peroxidatic heme is five-coordinate high-spin and active. The calcium-dependent spectral differences indicate little change in the conformation of the ferrous electron-transferring heme, but substantial changes in the conformation of the ferric peroxidatic heme. Structural changes associated with Ca(2+) binding are indicated by spectral differences in the structure-sensitive marker lines, the out-of-plane low-frequency macrocyclic modes, and the vibrations associated with the heme substituents of that heme. The Ca(2+)-dependent appearance of a strong gamma 15 saddling-symmetry mode for the mixed-valence form is consistent with a strong saddling deformation in the active peroxidatic heme, a feature seen in the Raman spectra of other peroxidases. For the fully reduced form in the presence of Ca(2+), the resonance Raman spectra show that the peroxidatic heme remains high-spin.     

Conserved nonplanar heme distortions in cytochromesc

A nonplanar distortion of the heme ofc-type cytochromes is conserved in the proteins isolated from diverse species based upon a comprehensive analysis of available highresolution X-ray crystal structures. This distortion is induced through the cysteine thioether linkages between the porphyrin pyrrole groups and the polypeptide and results in an asymmetric pyrrole distortion. This asymmetry in the heme distortion is also conserved. For other heme proteins which lack these covalent bonds, nearly planar porphyrins are observed. Resonance Raman evidence indicates that nonplanar distortion of porphyrins containing metals, like iron, with large core sizes (?2.00 Å) is energetically unfavorable and can occur only in the presence of significant environmental perturbations. Further, energy minimization and dynamics calculations on the ferric form of yeast iso-1-cytochromec, starting from the crystallographic coordinates and using a molecular mechanics force field which accurately reproduces nonplanar distortions in metalloporphyrins, suggest that this distortion is indeed maintained by the protein tertiary structure. It is proposed that this protein-linked heme distortion modulates electron transfer function through modification of redox potentials of the porphyrin ring and the protein binding properties ofc-type cytochromes.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Experimental Feeding of Hydrilla verticillata Colonized by Stigonematales Cyanobacteria Induces Vacuolar Myelinopathy in Painted Turtles (Chrysemys picta)

Vacuolar myelinopathy (VM) is a neurologic disease primarily found in birds that occurs when wildlife ingest submerged aquatic vegetation colonized by an uncharacterized toxin-producing cyanobacterium (hereafter “UCB” for “uncharacterized cyanobacterium”). Turtles are among the closest extant relatives of birds and many species directly and/or indirectly consume aquatic vegetation. However, it is unknown whether turtles can develop VM. We conducted a feeding trial to determine whether painted turtles (Chrysemys picta) would develop VM after feeding on Hydrilla (Hydrilla verticillata), colonized by the UCB (Hydrilla is the most common “host” of UCB). We hypothesized turtles fed Hydrilla colonized by the UCB would exhibit neurologic impairment and vacuolation of nervous tissues, whereas turtles fed Hydrilla free of the UCB would not. The ability of Hydrilla colonized by the UCB to cause VM (hereafter, “toxicity”) was verified by feeding it to domestic chickens (Gallus gallus domesticus) or necropsy of field collected American coots (Fulica americana) captured at the site of Hydrilla collections. We randomly assigned ten wild-caught turtles into toxic or non-toxic Hydrilla feeding groups and delivered the diets for up to 97 days. Between days 82 and 89, all turtles fed toxic Hydrilla displayed physical and/or neurologic impairment. Histologic examination of the brain and spinal cord revealed vacuolations in all treatment turtles. None of the control turtles exhibited neurologic impairment or had detectable brain or spinal cord vacuolations. This is the first evidence that freshwater turtles can become neurologically impaired and develop vacuolations after consuming toxic Hydrilla colonized with the UCB. The southeastern United States, where outbreaks of VM occur regularly and where vegetation colonized by the UCB is common, is also a global hotspot of freshwater turtle diversity. Our results suggest that further investigations into the effect of the putative UCB toxin on wild turtles in situ are warranted.     

The approximate entropy of the electromyographic signals of tremor correlates with the osmotic fragility of human erythrocytes

Background  

The main problem of tremor is the damage caused to the quality of the life of patients, especially those at more advanced ages. There is not a consensus yet about the origins of this disorder, but it can be examined in the correlations between the biological signs of aging and the tremor characteristics.