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R S Czernuszewicz

Publications and source records attributed to R S Czernuszewicz.

10 recordsLinked to original sources
PubMed2001

Resonance Raman spectroscopic study of nitrophorin 1, a nitric oxide-binding heme protein from Rhodnius prolixus, and its nitrosyl and cyano adducts.

The resonance Raman (RR) spectra of nitrophorin 1 (NP1) from the saliva of the blood-sucking insect Rhodnius prolixus, in the absence and presence of nitric oxide (NO) and in the presence of cyanide (CN(-)), have been studied. The NP1 displayed RR spectra characteristic of six-coordinate high-spin (6cHS) ferric heme at room temperature and six-coordinate low-spin heme (6cLS) at low temperature (77 K). NO and CN(-) each bind to Fe(III), both ligands forming 6cLS complexes with NP1. The Fe(III)-NO stretching and bending vibrational frequencies of nitrosyl NP1 were identified at 591 and 578 cm(-1), respectively, on the basis of 15NO isotope shifts. These frequencies are typical of Fe-NO ferric heme proteins, indicating that the NP1 nitrosyl adduct has typical bond strength. Thus, the small NO release rate displayed by NP1 must be due to other protein interactions. Room and cryogenic temperature (77 K) RR spectroscopy and 13C, 15N, and 13C15N isotope substitutions have been used to determine vibrational mode frequencies associated with the Fe(III)-CN(-) bond for the cyano adducts at 454, 443, 397, and 357 cm(-1). The results were analyzed by normal mode calculations to support the assignment of the modes and to assess the NO and CN(-) binding geometries. The observed isotope shifts for the cyano NP1 are smaller than expected and reveal vibrational coupling of Fe(III)-CN(-) modes with heme modes. We also find that the observed frequencies are consistent with the presence of a nearly linear Fe(III)CN(-) linkage (173 degrees ) coexisting with a population with a bent structure (155 degrees ).

Animals↗
PubMed2001

Redox thermodynamics of mutant forms of the rubredoxin from Clostridiumpasteurianum: identification of a stable Fe(III)(S-Cys)3(OH) centre in the C6S mutant.

Redox thermodynamic data provide a detailed insight into control of the reduction potential E degrees' of the [Fe(S-Cys)4] site in rubredoxin. Mutant forms were studied in which specific structural changes were made in both the primary and secondary coordination spheres. Those changes have been probed by resonance Raman spectroscopy. The decrease of approximately 200 mV in E degrees' observed for the [Fe(S-Cys)3(O-Ser)]-/2- couples in the surface ligand mutants C9S and C42S is essentially enthalpic in origin and associated with the substitution of ligand thiolate by ligand olate. However, the pH dependence of the potentials below characteristic pKa(red) approximately equals 7 is an entropic contribution, plausibly associated with increased conformational flexibility induced by a longer Fe(II)-O(H)-Ser bond in the reduced form. The presence of a second surface Ser ligand in the new double mutant protein C9S/C42S affects the enthalpic term primarily for pH>pKa(red) > or = 9.3, but for pH pKa approximately 9: [Fe(III)(S-Cys)3(OH)]- + e- --> [Fe(II)(S-Cys)3(OH)]2-. pH [Fe(II)(S-Cys)3(OH2)]-.

Clostridium↗
PubMed2000

Interaction of oxo-bridged vanadium(III) phenanthroline and bipyridine dimers with DNA.

Cationic mu-oxo V(III) dimers of the type [V2OL4Cl2]2+ (L = 1,10-phenanthroline, 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline; or 2,2'-bipyridine) are shown to interact very strongly with DNA and to lead ultimately to its degradation. Spectroscopic binding studies, electrophoreses, DNA melting temperature experiments, and other tests on the parent 1,10-phenthroline complex all yield results consistent with tight binding. However, the exact nature of the binding--i.e., intercalative, groove binding, electrostatic, or covalent--remains unclear. Resonance Raman spectroscopy is found to be a powerful method for studying the interaction of these mu-oxo V(III) dimers with DNA and shows that in frozen aqueous solution, the parent complex [V2O(phen)4Cl2]2+ undergoes initial aquation, followed by the reaction of the aquated species with the DNA. Once the V(III) dimer is bound to the DNA, redox takes place, leading to the formation of alkaline-sensitive lesions. Hydrogen peroxide is implicated as a partner in this redox event, based on the effects of the enzymes SOD and catalase.

Animals↗
PubMed1996

Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form.

The cDNA encoding the 182 amino acid long precursor stellacyanin from Cucumis sativus was isolated and characterized. The protein precursor consists of four sequence domains: I, a 23 amino acid hydrophobic N-terminal signal peptide with features characteristic of secretory proteins; II, a 109 amino acid copper-binding domain; III, a 26 amino acid hydroxyproline- and serine-rich peptide characteristic of motifs found in the extension family, extracellular structural glycoproteins found in plant cell walls; and IV, a 22 amino acid hydrophobic extension. Maturation of the protein involves posttranslational processing of domains I and IV. The copper-binding domain (domain II), which shares high sequence identity with other stellacyanins, has been expressed without its carbohydrate attachment sites, refolded from the Escherichia coli inclusion bodies, purified, and characterized by electronic absorption, EPR, ESEEM, and RR spectroscopy. Its spectroscopic properties are nearly identical to those of stellacyanin from the Japanese lacquer tree Rhus vernicifera, the most extensively studied and best characterized stellacyanin, indicating that this domain folds correctly, even in the absence of its carbohydrate moiety. The presence of a hydroxyproline- and serine-rich domain III suggests that stellacyanin may have a function other than that of a diffusible electron transfer protein, conceivably participating in redox reactions localized at the plant cell wall, which are known to occur in response to wounding or infection of the plant.

Amino Acid Sequence↗
PubMed1995

The environment of [2Fe-2S] clusters in ferredoxins: the role of residue 45 probed by site-directed mutagenesis.

The biochemical and biophysical properties of the Ala45Ser mutant of the [2Fe-2S] ferredoxin from vegetative cells of the cyanobacterium Anabaena sp. 7120 are described. This novel protein, which incorporates the residue present in many higher plant ferredoxins into the analogous position of a typical cyanobacterial ferredoxin, was prepared to probe the origin of the characteristic spectrochemical and functional differences between the ferredoxins from these two sources. The variant protein was produced by site-directed mutagenesis and was expressed as the holoprotein in Escherichia coli. Although the UV-vis spectrum of the Ala45Ser mutant was indistinguishable from that of the wild-type (WT) protein, the circular dichroism (CD) spectrum of the mutant was distinct and similar in appearance to that of spinach ferredoxin, which possesses a Ser residue at the analogous position. The values of the principal g factors of the EPR spectrum of the dithionite-reduced mutant protein differed from those of the WT spectrum and resembled those of plant ferredoxins containing serine at position 45. Analysis of the mutant EPR spectrum according to the method of Blumberg indicated greater covalent interactions between the localized ferrous site of the cluster and the protein matrix relative to the WT protein. The resonance Raman spectrum of Ala45Ser Anabaena ferredoxin was distinct from the spectrum of the WT protein and showed exceptional similarity to the spectrum of higher plant ferredoxins, such as spinach ferredoxin. The mutant protein spectrum displayed considerably greater deuterium dependent isotope shifts for bands ascribed to terminal Fe-S stretching modes than did the WT spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Anabaena↗
PubMed1994

Resonance Raman spectroscopic evidence for the FeS4 and Fe-O-Fe sites in rubrerythrin from Desulfovibrio vulgaris.

Resonance Raman (RR) spectra of the non-heme iron protein rubrerythrin from Desulfovibrio vulgaris unequivocally demonstrate the presence of both a rubredoxin-type FeS4 site and a (mu-oxo)diiron(III) cluster. The RR spectra of rubrerythrin excited at 496.5 and 568.2 nm are dominated by bands similar to those of rubredoxin and conform to the vibrational pattern expected for a distorted FeS4 tetrahedron of an Fe(S-Cys)4 site. Numerous overtone and combination bands of the Fe-S stretches are also observed, and a band at 650 cm-1 is assigned to a cysteine C-S stretching mode. The 374-, 355-, and 340-cm-1 bands, assigned to the three components of the v3(T2) asymmetric FeS4 stretching mode, are 2-8 cm-1 lower than the corresponding frequencies for the Desulfovibrio gigas rubredoxin FeS4 site. Similar differences in frequencies of bands assigned to SFeS bending modes between rubredoxin and rubrerythrin are also detected. These frequency differences imply either slightly weaker Fe-S bonds or subtle conformational differences among the cysteinyl ligands in the rubrerythrin versus rubredoxin FeS4 sites. The RR spectrum of rubrerythrin excited at 406.7 nm shows dramatically diminished intensities of the FeS4 bands with concomitant enhancement of a band at 514 cm-1. This band shifts 18 cm-1 to lower frequency when the protein is dissolved in H(2)18O. The frequency of this band and the 18O isotope shift are those expected for the symmetric Fe-O-Fe stretch of a bent oxo-bridged diiron(III) cluster and indicate that this cluster has at least one additional bridging ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1987

An investigation of hydrogenase I and hydrogenase II from Clostridium pasteurianum by resonance Raman spectroscopy. Evidence for a [2Fe-2S] cluster in hydrogenase I.

Resonance Raman spectra are reported for hydrogenase I and II from Clostridium pasteurianum. These spectra show overlapping bands with contributions from [4Fe-4S] clusters, known to be present in these enzymes, and from novel FeS centers of hitherto undefined structure. For hydrogenase I there are strong bands at 288 and 394 cm-1, which are seen in [2Fe-2S] proteins and in no other FeS species so far examined. In contrast these bands do not appear for hydrogenase II, whose resonance Raman spectrum is dominated by [4Fe-4S] cluster modes. These results provide the first structural information on the hydrogenase I FeS center involved in H2 activation and demonstrate structural differences between hydrogenase I and hydrogenase II.

Clostridium↗
PubMed1983

Resonance Raman studies of beef heart aconitase and a bacterial hydrogenase.

The resonance Raman (RR) spectra of beef heart aconitase and of an air-stable hydrogenase from Desulfuvibrio desulfuricans, as isolated, are characteristic of 3Fe centers. Activation of aconitase by Fe(II) addition converts the RR spectrum to one characteristic of [4Fe-4S]2+ clusters. Analytical data on aconitase, as isolated, confirms the recent finding (Beinert, H., Emptage, M. H., Dreyer, J.-L., Scott, R. A., Hahn, J. E., Hodgson, K. O., and Thomson, A. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 393-396) of a [3Fe-4S] stoichiometry. The RR spectra of 3Fe centers from aconitase, and the hydrogenase, as well as from several bacterial ferredoxins, conform to the pattern expected for a cube-derived [3Fe-4S] cluster. Perceptible differences are observed among the spectra, which can be explained in terms of differences among the terminal ligands, perhaps limited to their conformations. In the case of aconitase and hydrogenase, frequency shifts suggest additional alterations in the terminal Fe-S bond angles and/or slight differences in core geometry.

Aconitate Hydratase↗