Induction of photochemical auto-reduction of cytochrome-c oxidase by an organic peroxide.
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Cytochrome-c oxidase aa3 (CcO) from Paracoccus denitrificans interacts with tertiary butyl hydroperoxide (t-Bu-O-O-H, TBHP) by forming an adduct as indicated by an absorption shift at 408/432 nm and the induction of photochemical autoreduction. The adduct was stable at room temperature for several days even under aerobic conditions. Upon irradiation (413 nm) of the adduct, a photoproduct, similar to the oxygenated mixed valence species (607 nm form), was formed, as indicated by the 418/442 and 607 nm signals in the absorption-difference spectrum. It is concluded that the adduct formation changes the photochemical properties of heme a3. A molecular model for the binding mechanism of TBHP to CcO and for the photochemistry of heme a3-TBHP adduct is proposed. (+info)
Replacement of the distal glycine 139 transforms human heme oxygenase-1 into a peroxidase.
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The human heme oxygenase-1 crystal structure suggests that Gly-139 and Gly-143 interact directly with iron-bound ligands. We have mutated Gly-139 to an alanine, leucine, phenylalanine, tryptophan, histidine, or aspartate, and Gly-143 to a leucine, lysine, histidine, or aspartate. All of these mutants bind heme, but absorption and resonance Raman spectroscopy indicate that the water coordinated to the iron atom is lost in several of the Gly-139 mutants, giving rise to mixtures of hexacoordinate and pentacoordinate ligation states. The active site perturbation is greatest when large amino acid side chains are introduced. Of the Gly-139 mutants investigated, only G139A catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, but most of them exhibit a new H(2)O(2)-dependent guaiacol peroxidation activity. The Gly-143 mutants, all of which have lost the water ligand, have no heme oxygenase or peroxidase activity. The results establish the importance of Gly-139 and Gly-143 in maintaining the appropriate environment for the heme oxygenase reaction and show that Gly-139 mutations disrupt this environment, probably by displacing the distal helix, converting heme oxygenase into a peroxidase. The principal role of the heme oxygenase active site may be to suppress the ferryl species formation responsible for peroxidase activity. (+info)
Non-invasive raman spectroscopic detection of carotenoids in human skin.
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Carotenoids are thought to play a significant part in the skin's anti-oxidant defense system, and may help prevent malignancy. Inability to measure skin carotenoid content readily has, however, made it difficult to establish the relationship between carotenoid concentration and the occurrence of cutaneous malignancy. We have measured in vivo carotenoid concentration using a noninvasive optical method, Raman spectroscopy. To validate our instrumentation, abdominoplasty skin was evaluated by both Raman spectroscopy and high-performance liquid chromatography determination for carotenoid content. Evaluation of the Raman signal in specific carotenoid solutions was also performed. Precision of Raman measurements within skin sites, within subjects, and between subjects was measured. Sensitivity of the method was evaluated as a function of anatomical region and the distribution of carotenoids within the stratum corneum. Lastly, we evaluated the Raman signal in actinic keratosis and basal cell carcinoma lesions and perilesional skin and compared this with region-matched sites in healthy subjects. Our results indicate that the Raman scattering method reflects the presence of carotenoids in human skin and is highly reproducible. Evaluation of five anatomical regions demonstrated significant differences in carotenoid concentration by body region with the highest carotenoid concentration noted in the palm. Comparison of carotenoid concentrations in basal cell carcinomas, actinic keratosis, and their perilesional skin demonstrate a significantly lower carotenoid concentration than in region-matched skin of healthy subjects. These results represent the first evidence that carotenoid concentration in the skin correlate with the presence or absence of skin cancer and precancerous lesions. (+info)
Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy.
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Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine ([H(3)N(CH(2))(3)NH(2)(CH(2))(4)NH(3)](3+)) and spermine ([(CH(2))(4)(NH(2)(CH(2))(3)NH(3))(2)](4+)). In solutions containing 60 mM DNA phosphate (approximately 20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H(3)) are less affected than phosphates by polyamine-DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei. (+info)
Primordial carbonylated iron-sulfur compounds and the synthesis of pyruvate.
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Experiments exploring the potential catalytic role of iron sulfide at 250 degrees C and elevated pressures (50, 100, and 200 megapascals) revealed a facile, pressure-enhanced synthesis of organometallic phases formed through the reaction of alkyl thiols and carbon monoxide with iron sulfide. A suite of organometallic compounds were characterized with ultraviolet-visible and Raman spectroscopy. The natural synthesis of such compounds is anticipated in present-day and ancient environments wherever reduced hydrothermal fluids pass through iron sulfide-containing crust. Here, pyruvic acid was synthesized in the presence of such organometallic phases. These compounds could have provided the prebiotic Earth with critical biochemical functionality. (+info)
Chromophore reorientation during the photocycle of bacteriorhodopsin: experimental methods and functional significance.
(70/2113)
Light-induced isomerization leads to orientational changes of the retinylidene chromophore of bacteriorhodopsin in its binding pocket. The chromophore reorientation has been characterized by the following methods: polarized absorption spectroscopy in the visible, UV and IR; polarized resonance Raman scattering; solid-state deuterium nuclear magnetic resonance; neutron and X-ray diffraction. Most of these experiments were performed at low temperatures with bacteriorhodopsin trapped in one or a mixture of intermediates. Time-resolved measurements at room temperature with bacteriorhodopsin in aqueous suspension can currently only be carried out with transient polarized absorption spectroscopy in the visible. The results obtained to date for the initial state and the K, L and M intermediates are presented and discussed. The most extensive data are available for the M intermediate, which plays an essential role in the function of bacteriorhodopsin. For this intermediate the various methods lead to a consistent picture: the curved all-trans polyene chain in the initial state straightens out in the M intermediate (13-cis) and the chain segment between C(5) and C(13) tilts upwards in the direction of the cytoplasmic surface. The kink at C(13) allows the positions of beta-ionone ring and Schiff base nitrogen to remain approximately fixed. (+info)
Structural changes in cytochrome c oxidase induced by cytochrome c binding. A resonance raman study.
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Electrostatically stabilized complexes of fully oxidized cytochrome c oxidase from Paracoccus denitrificans and horse heart cytochrome c were studied by resonance Raman spectroscopy. The experiments were carried out with the wild-type oxidase and a variant in which a negatively charged amino acid in the binding domain (D257) is replaced by an asparagine. It is shown that cytochrome c induces structural changes at heme a and heme a(3) which are reminiscent to those found in mammalian cytochrome c oxidase-cytochrome c complex. The spectral changes are attributed to subtle changes in the heme-protein interactions implying that there is a structural communication from the binding domain even to the remote catalytic center. Only for the heme a modes minor spectral differences were found in the response of the wild-type and the D257N variant oxidase upon cytochrome c binding indicating that electrostatic interactions of aspartate 257 are not crucial for the perturbation of the catalytic site structure in the complex. On the other hand, in none of the complexes, structural changes were detected in the bound cytochrome c. These findings are in contrast to previous results obtained with beef heart cytochrome c oxidase which triggers the formation of a new conformational state of cytochrome c assumed to be involved in the biological electron transfer process. (+info)
Resonance Raman spectroscopic study of the tryptic 39-kDa fragment of phytochrome.
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The 39-kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state P(r) reveals far-reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the P(r) phototransformation, denoted as P(bl), is more closely related to that of the P(fr) precursor of the native phytochrome, i.e. meta-R(C), rather than to that of P(fr) itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in P(bl) so that, unlike in P(fr), the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of P(fr), cannot be established in the 39-kDa fragment due to the lack of the major C-terminal part of the protein. These findings, furthermore, support the view that the meta-R(C)-->P(fr) transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome. (+info)