Sergio Brochsztain

Spectroscopic, structural, and functional characterization of the alternative low-spin state of horse heart cytochrome C.

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.

Stabilization of naphthalene-1,8:4,5-dicarboximide radicals in zirconium phosphonate solid materials and thin films

Solid zirconium phosphonate (ZP) materials were prepared by direct precipitation of N,N′-bis(2-phosphonoethyl)naphthalene-1,8:4,5-dicarboximide (NDI-BP) from aqueous solution by addition of a ZrOCl2 solution. Slow precipitation in the presence of HF gave crystalline NDI-BP/ZP materials with a layered structure, whereas rapid precipitation in the absence of HF gave mainly amorphous materials. When ZrOCl2 solutions were added to photolyzed NDI-BP solutions, radical-doped solid materials with intense EPR response were obtained. Multilayer thin films of NDI-BP/ZP were grown on gold substrates in a layer-by-layer fashion, by exposing the phosphonate-rich modified surface to solutions of ZrOCl2 and NDI-BP, alternately. Electrochemical reduction of 20-layer films resulted in the formation of stable NDI-BP radical anions and dianions.

Photochemical Reduction of Cytochrome c by a 1,4,5,8‐Naphthalenediimide Radical Anion¶

Abstract Steady-state UV irradiation of aqueous solutions containing cytochrome c (cyt c) and N,N′-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (BPNDI), a water-soluble aromatic imide, resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) oxidation state. The reaction kinetics were followed by the increase of the ferrocytochrome c absorbance band at 549 nm. The rate of the photochemical reaction was pH dependent, reaching its maximum values over the pH range 4–7. Addition of electrolyte (NaCl) at pH 5 resulted in a decrease in the reaction rate, as expected for reactions between oppositely charged species. Flash photolysis studies revealed that the actual reductant in the reaction was a photogenerated BPNDI radical anion, which transferred an electron to the cyt c heme iron. The participation of imide radicals in the process was confirmed by the ready reduction of cyt c by BPNDI radicals chemically generated with sodium dithionite.

Reaction route control by microperoxidase-9/CTAB micelle ratios

Microperoxidases (MP) as water-soluble models attract interest to studying the reaction mechanism of peroxidases because these heme peptides are able to form the same enzyme intermediates during the reaction with peroxides. In this work we have demonstrated that the association of Fe(III)MP-9 and Fe(III)MP-11 with CTAB micelles (MP-9/CTAB and MP11/CTAB) provides a microenvironment with an alkaline interface and a hydrophobic core that exhibits peroxidase behavior. This microenvironment shifts positively the redox potential of microperoxidases by approximately 100 mV. tert-Butylhydroperoxide (t-BuOOH) when added to the medium, converted Fe(III)MP-9/CTAB to MP-9/CTAB Compound II, a high valence oxidized intermediate of the heme peptide. Subsequent addition of diphenylacetaldehyde (DPAA) to MP-9/CTAB Compound II regenerated the native form of the enzyme, Fe(III)MP-9/CTAB, what characterizes the occurrence of a peroxidase cycle. Fe(III)MP-9/CTAB regenerated during the peroxidase cycle reacted with residual DPAA in the medium to form Fe(II)MP-9/CTAB, which indicates that both Fe(III)MP-9/CTAB and its oxyferryl form can use aldehydes as reducing agents. According to the determined reduction potential, Fe(III)MP-9 and Fe(III)MP-9/CTAB should be able to oxidize DPAA (reduction potential -630 mV). The reaction of MP-9/CTAB with DPAA produced benzophenone as final product, detected by infrared spectroscopy and mass spectrometry. Interestingly, a significant difference was observed in the benzophenone yield according to the micelle/MP-9 molar ratio.

Photoinduced electron transfer in silica-supported self-assembled thin films containing a 1,4,5,8-naphthalenetetracarboxylic diimide and cytochrome c

Self-assembled thin films containing an inner monolayer of N,N′-bis(2-phosphonoethyl)-1,4,5,8-naphthalenetetracarboxylic diimide (PNDI) and an outer monolayer of the cationic protein cytochrome c n (cyt c) were prepared on the surface of non-porous silica gel particles. Silica particles modified with PNDI alone or with cyt c alone were also prepared for comparison. The materials were characterized by elemental and thermogravimetric analysis, as well as UV-visible absorption, fluorescence and infrared spectroscopies. Binding of PNDI to the silica resulted in a low density, sub-monolayer coverage, in contrast to cyt c adsorption which gave a rather compact protein layer. The loading of cyt c found in the new materials (ca. 12 µmol g−1) was higher than that found in the literature for porous silica gel. Irradiation of the materials at the imide absorption band resulted in the reduction of the cyt c heme iron, showing that the protein retained its activity upon binding to the modified silica gel surface. The proposed mechanism involves electron transfer from a photogenerated imide radical anion to the heme iron of the protein in the adjacent layer.