Conformational structure and binding mode of glyceraldehyde-3-phosphate dehydrogenase to tRNA studied by Raman and CD spectroscopy.
(17/2113)
Recently it has been suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) play a role in nuclear tRNA export. As the structural basis of binding of GAPDH to tRNA is as yet unknown, we have employed Raman and CD spectroscopy as probes of the solution structures of GAPDH from rabbit and tRNA(Phe) from brewers yeast. Additionally, we have obtained the Raman and CD spectra of GAPDH when bound to tRNA(Phe). In the complex we find the following results: (a) The most part of the tRNA(Phe) structure is conserved, but with a slight perturbation toward a B-like form. (b) No significant changes in the secondary structure of the protein upon binding are observed. (c) The surface enhanced Raman spectra are consistent with a GAPDH-tRNA(Phe) complex molecular model that involves the insertion of TRNA(Phe) into the GAPDH tetramer groove containing the R and P axes. (d) The specific interactions that occur between GAPDH and the tRNA(Phe) involve, mainly, stacking between nucleobases and aromatic amino-acid residues, and ionic interactions of basic amino-acid residues with phosphate groups of the ribose-phosphate backbone. The above stacking interactions are also supported by the significant relatedness that we have found between an amino-acid sequence (residues 303-308) of GAPDH and RNP2 binding motifs of some RNA binding proteins. (+info)
A pure S = 3/2 [Fe4S4]+ cluster in the A33Y variant of Pyrococcus furiosus ferredoxin.
(18/2113)
The properties of the [4Fe-4S]2+/+ cluster in wild-type and the A33Y variant of Pyrococcus furiosus ferredoxin have been investigated by the combination of EPR, variable-temperature magnetic circular dichroism (VTMCD) and resonance Raman (RR) spectroscopies. The A33Y variant involves the replacement of an alanine whose alpha-C is less than 4 A from one of the cluster iron atoms by a tyrosine residue. Although the spectroscopic results give no indication of tyrosyl cluster ligation, the presence of a tyrosine residue in close proximity to the cluster results in a 38-mV decrease in the midpoint potential of the [4Fe-4S]2+/+ couple and has a marked effect on the ground state properties of the reduced cluster. The mixed spin [4Fe-4S]+ cluster in the wild-type protein, 80% S = 3/2 (E/D = 0.22, D = +3.3 cm(-1)) and 20% S = 1/2 (g = 2.10, 1.87, 1.80), is converted into a homogeneous S = 3/2 (E/D = 0.30, D = -0.7 cm(-1)) form in the A33Y variant. As the first example of a pure S = 3/2 [4Fe-4S]+ cluster in a ferredoxin, this variant affords the opportunity for detailed characterization of the excited electronic properties via VTMCD studies and demonstrates that the protein environment can play a crucial role in determining the ground state properties of [4Fe-4S]+ clusters. (+info)
Pentacoordinate hemin derivatives in sodium dodecyl sulfate micelles: model systems for the assignment of the fifth ligand in ferric heme proteins.
(19/2113)
Ferric iron protoporhyrin IX derivatives in SDS micelles have been investigated by means of visible absorption, resonance Raman, and XANES spectroscopies to establish specific correlations between the marker bands of the pentacoordinate derivatives obtained from the three different techniques. Hydroxyl and 1,2-dimethyl imidazole coordinated hemins display the typical spectroscopic marker bands of a pentacoordinate high-spin ferric iron derivative in both Raman and XANES spectra. In turn, the optical absorption spectra of these two derivatives are very different. This difference is in line with the assignment of hydroxyl as the fifth coordination ligand to free hemin in SDS micelles, as demonstrated by the isotopic shift of the frequency of Fe-OH bond with H(2)(18)O. The present assignments are relevant to the identification of the coordination state and the nature of the fifth ligand in ferric heme proteins. (+info)
Iron coordination structures of oxygen sensor FixL characterized by Fe K-edge extended x-ray absorption fine structure and resonance raman spectroscopy.
(20/2113)
FixL is a heme-based O(2) sensor protein involved in a two-component system of a symbiotic bacterium. In the present study, the iron coordination structure in the heme domain of Rhizobium meliloti FixLT (RmFixLT, a soluble truncated FixL) was examined using Fe K-edge extended x-ray absorption fine structure (EXAFS) and resonance Raman spectroscopic techniques. In the EXAFS analyses, the interatomic distances and angles of the Fe-ligand bond and the iron displacement from the heme plane were obtained for RmFixLT in the Fe(2+), Fe(2+)O(2), Fe(2+)CO, Fe(3+), Fe(3+)F(-), and Fe(3+)CN(-) states. An apparent correlation was found between the heme-nitrogen (proximal His-194) distance in the heme domain and the phosphorylation activity of the histidine kinase domain. Comparison of the Fe-CO coordination geometry between RmFixLT and RmFixLH (heme domain of RmFixL), based on the EXAFS and Raman results, has suggested that the kinase domain directly or indirectly influences steric interaction between the iron-bound ligand and the heme pocket. Referring to the crystal structure of the heme domain of Bradyrhizobium japonicum FixL (Gong, W., Hao, B., Mansy, S. S., Gonzalez, G., Gilles-Gonzalez, M. A., and Chan, M. K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 15177-15182), we discussed details of the iron coordination structure of RmFixLT and RmFixLH in relation to an intramolecular signal transduction mechanism in its O(2) sensing. (+info)
Hydration of human nails investigated by NIR-FT-Raman spectroscopy.
(21/2113)
The human nail, although it is usually stable against outer influences, becomes soft and flexible after soaking in water. Frequent washing increases brittleness of nails. Hydration of nails is thought to be the most important factor influencing the physical properties of nails and possibly acts through changes in keratin structure. Here NIR-FT-Raman has been used to examine molecular structural changes of intact moisten nails. Raman spectra were obtained both in vitro from nail samples and in vivo before and after soaking in water. The water uptake of normal nail samples during the first 15 min was reflected in the increasing intensity ratio of the nu(OH)/nu(CH(2)) bands. A saturating effect appeared soon after 10 min which is explained by a defined water holding capacity. R(nu) representation of the low frequency range of the Raman spectra showed that mainly bound water is found both in dry and in wet nails. This implies water-protein interaction. Protein backbone vibration at 932 cm(-1) indicating alpha-helical proteins increased in intensity in the wet nails. The nu(S-S) which is sensitive to changes in conformation of proteins showed a 4% higher intensity. Additional protein-water interactions could lead to a slight change of the dihedral angle of the C-S-S-C bonds and to geometric changes in coiling behavior of the alpha-helical protein. Suggesting a separation between matrix proteins and fiber proteins giving them a greater freedom of flexibility. The in vivo spectra detected from the distal part of the nail resembled spectra in vitro. Raman spectra of the proximal part of the nail showed that it was fully saturated with water. The proximal part of the nail did not show changes in water content and protein structure during nail moisturizing in the Raman spectra. Our results suggest that the softening of the nail following hydration may be due to changed matrix protein molecular structure induced by water. (+info)
Characterization of the Asp94 and Glu242 mutants in myeloperoxidase, the residues linking the heme group via ester bonds.
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The heme group of all mammalian peroxidases is covalently linked to the protein matrix via two esterbonds, as we have recently shown by Fourier transform infrared (FTIR) difference spectroscopy [Kooter, I. M., Pierik, A.J., Merkx, M., Averill, B.A., Moguilevsky, N., Bollen, A. & Wever, R. (1997) J. Am. Chem. Soc. 119, 11542-11543]. We have examined the effects of mutation of Asp94 and Glu242, responsible for those ester bonds in myeloperoxidase, on the spectroscopic properties and catalytic activity of this enzyme. Mutation of Asp94 in myeloperoxidase results in two species. The first species has spectroscopic characteristics similar to that of wild-type myeloperoxidase. The second species has spectroscopic characteristics similar to that of Met243-->Gln mutant, and it is therefore concluded that, besides loss of the ester bond involving Asp94, this species also has lost the sulfonium ion linkage that is also characteristic of myeloperoxidase. The Asp94-->Asn mutant still has about 30% residual peroxidase activity while for the Asp94-->Val mutant only a few percentage activity is left. When Glu242 is mutated the sulfonium ion linkage is not affected, but this residue together with its neighbouring residue Met243, according to resonance Raman spectra, is responsible for the low symmetry of the heme group. Mutation of either of these residues results in loss of the bowed distortion from the planar conformation, and in a heme group with higher symmetry. For the Glu242-->Gln mutant 8% residual peroxidase activity is found. (+info)
Redox-linked transient deprotonation at the binuclear site in the aa(3)-type quinol oxidase from Acidianus ambivalens: implications for proton translocation.
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The hyperthermophilic archaeon Acidianus ambivalens expresses a membrane-bound aa(3)-type quinol oxidase, when grown aerobically, that we have studied by resonance Raman spectroscopy. The purified aa(3) oxidase, which does not contain bound quinol, undergoes a reversible slow conformational change at heme a(3) upon reduction, as indicated by a change in the frequency of its heme formyl stretching mode, from 1,660 cm(-1) to 1,667 cm(-1). In contrast, upon reduction of the integral membrane enzyme or the purified enzyme preincubated with decylubiquinol, this mode appears at 1,667 cm(-1) much more rapidly, suggesting a role of the bound quinol in controlling the redox-linked conformational changes. The shift of the formyl mode to higher frequency is attributed to a loss of hydrogen bonding that is associated with a group having a pKa of approximately 3.8. Based on these observations, a crucial element for proton translocation involving a redox-linked conformational change near the heme a(3) formyl group is postulated. (+info)
The heme complex of Hmu O, a bacterial heme degradation enzyme from Corynebacterium diphtheriae. Structure of the catalytic site.
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Hmu O, a heme degradation enzyme in Corynebacterium diphtheriae, forms a stoichiometric complex with iron protoporphyrin IX and catalyzes the oxygen-dependent conversion of hemin to biliverdin, carbon monoxide, and free iron. Using a multitude of spectroscopic techniques, we have determined the axial ligand coordination of the heme-Hmu O complex. The ferric complex shows a pH-dependent reversible transition between a water-bound hexacoordinate high spin neutral pH form and an alkaline form, having high spin and low spin states, with a pK(a) of 9. (1)H NMR, EPR, and resonance Raman of the heme-Hmu O complex establish that a neutral imidazole of a histidine residue is the proximal ligand of the complex, similar to mammalian heme oxygenase. EPR of the deoxy cobalt porphyrin IX-Hmu O complex confirms this proximal histidine coordination. Oxy cobalt-Hmu O EPR reveals a hydrogen-bonding interaction between the O(2) and an exchangeable proton in the Hmu O distal pocket and two distinct orientations for the bound O(2). Mammalian heme oxygenase has only one O(2) orientation. This difference and the mixed spin states at alkaline pH indicate structural differences in the distal environment between Hmu O and its mammalian counterpart. (+info)