X-ray structure of the metcyano form of dehaloperoxidase from Amphitrite ornata: evidence for photoreductive dissociation of the iron-cyanide bond.

X-ray crystal structures of the metcyano form of dehaloperoxidase-hemoglobin (DHP A) from Amphitrite ornata (DHPCN) and the C73S mutant of DHP A (C73SCN) were determined using synchrotron radiation in order to further investigate the geometry of diatomic ligands coordinated to the heme iron. The DHPCN structure was also determined using a rotating-anode source. The structures show evidence of photoreduction of the iron accompanied by dissociation of bound cyanide ion (CN(-)) that depend on the intensity of the X-ray radiation and the exposure time. The electron density is consistent with diatomic molecules located in two sites in the distal pocket of DHPCN. However, the identities of the diatomic ligands at these two sites are not uniquely determined by the electron-density map. Consequently, density functional theory calculations were conducted in order to determine whether the bond lengths, angles and dissociation energies are consistent with bound CN(-) or O(2) in the iron-bound site. In addition, molecular-dynamics simulations were carried out in order to determine whether the dynamics are consistent with trapped CN(-) or O(2) in the second site of the distal pocket. Based on these calculations and comparison with a previously determined X-ray crystal structure of the C73S-O(2) form of DHP [de Serrano et al. (2007), Acta Cryst. D63, 1094-1101], it is concluded that CN(-) is gradually replaced by O(2) as crystalline DHP is photoreduced at 100 K. The ease of photoreduction of DHP A is consistent with the reduction potential, but suggests an alternative activation mechanism for DHP A compared with other peroxidases, which typically have reduction potentials that are 0.5 V more negative. The lability of CN(-) at 100 K suggests that the distal pocket of DHP A has greater flexibility than most other hemoglobins.

3-Hydroxykynurenine and 3-hydroxyanthranilic acid generate hydrogen peroxide and promote alpha-crystallin cross-linking by metal ion reduction.

The kynurenine pathway catabolite 3-hydroxykynurenine (3HK) and redox-active metals such as copper and iron are implicated in cataractogenesis. Here we investigate the reaction of kynurenine pathway catabolites with copper and iron, as well as interactions with the major lenticular structural proteins, the alpha-crystallins. The o-aminophenol kynurenine catabolites 3HK and 3-hydroxyanthranilic acid (3HAA) reduced Cu(II)>Fe(III) to Cu(I) and Fe(II), respectively, whereas quinolinic acid and the nonphenolic kynurenine catabolites kynurenine and anthranilic acid did not reduce either metal. Both 3HK and 3HAA generated superoxide and hydrogen peroxide in a copper-dependent manner. In addition, 3HK and 3HAA fostered copper-dependent alpha-crystallin cross-linking. 3HK- or 3HAA-modifed alpha-crystallin showed enhanced redox activity in comparison to unmodified alpha-crystallin or ascorbate-modified alpha-crystallin. These data support the possibility that 3HK and 3HAA may be cofactors in the oxidative damage of proteins, such as alpha-crystallin, through interactions with redox-active metals and especially copper. These findings may have relevance for understanding cataractogenesis and other degenerative conditions in which the kynurenine pathway is activated.

Cu(II) potentiation of alzheimer abeta neurotoxicity. Correlation with cell-free hydrogen peroxide production and metal reduction.

Oxidative stress markers as well as high concentrations of copper are found in the vicinity of Abeta amyloid deposits in Alzheimer's disease. The neurotoxicity of Abeta in cell culture has been linked to H(2)O(2) generation by an unknown mechanism. We now report that Cu(II) markedly potentiates the neurotoxicity exhibited by Abeta in cell culture. The potentiation of toxicity is greatest for Abeta1-42 > Abeta1-40 >> mouse/rat Abeta1-40, corresponding to their relative capacities to reduce Cu(II) to Cu(I), form H(2)O(2) in cell-free assays and to exhibit amyloid pathology. The copper complex of Abeta1-42 has a highly positive formal reduction potential ( approximately +500-550 mV versus Ag/AgCl) characteristic of strongly reducing cuproproteins. These findings suggest that certain redox active metal ions may be important in exacerbating and perhaps facilitating Abeta-mediated oxidative damage in Alzheimer's disease.

Dramatic stabilization of ferricytochrome c upon reduction.

By combining measurements of the free energy of denaturation of the C102T variant of Saccharomyces cerevisiae iso-1-ferricytochrome c with determination of the formal potentials for the native and chemically-denatured states we have determined the free energy of denaturation of the ferro form of the protein. We report that the simplest of all chemical modifications, addition of an electron, increases the stability of ferricytochrome c by approximately 10 kcal mol-1 at 300 K, pH 4.6. This makes reduced cytochrome c one of the most stable proteins yet investigated.

Reductive detoxification as a mechanism of fungal resistance to singlet oxygen-generating photosensitizers.

Fungi that are resistant or sensitive to the singlet oxygen-generating toxin cercosporin were assayed for their ability to detoxify it by reduction. Cercosporin reduction was assayed microscopically by using bandpass filters to differentiate between fluorescence emission from cercosporin and reduced cercosporin. Hyphae of the resistant Cercospora and Alternaria species emitted a green fluorescence, indicative of reduced cercosporin. Hyphae of nonviable cultures and of cercosporin-sensitive fungi did not reduce cercosporin. Sensitive fungi occasionally reduced cercosporin when incubated with reducing agents that protect against cercosporin toxicity. Cercosporin could not be efficiently photoreduced in the absence of the fungus. Cercospora species were also resistant to eosin Y but were sensitive to rose bengal. Microscopic observation demonstrated that Cercospora species were not capable of reducing rose bengal but were capable of reducing eosin Y. These observations were supported by in vitro electrochemical measurements that revealed the following order with respect to ease of reduction: cercosporin >> eosin Y > rose bengal. The formal redox potential (E 0') of cercosporin at pH 7.5 was found to be -0.14 V vs. the normal hydrogen electrode. We conclude that Cercospora species protect themselves against cercosporin by the reduction and detoxification of the toxin molecule.

The electrode reaction of Euglena gracilis cytochrome c-552 at edge-oriented pyrolytic graphite.

The direct electron transfer reaction of Euglena gracilis cytochrome c-552 at edge-oriented pyrolytic graphite electrodes was determined by cyclic voltammetry to be quasi-reversible, stable and reproducible. The presence of a persistent layer of irreversibly adsorbed cytochrome c-552 on the electrode surface was detected in these experiments. Heterogeneous electron transfer rate constants for both the diffusing and adsorbed forms of the protein are reported, and mechanistic aspects are addressed. The applicability of cytochrome c-552 as a complementarily charged analog of eucaryotic cytochrome c in interfacial bioelectrochemical studies is discussed.

In vivo measurement of tubular fluid ferrocyanide with carbon-fiber microelectrodes.

Techniques to construct carbon-fiber microelectrodes and to measure ferrocyanide ion concentration in single nephrons are described. The measurement involves polarizing an inert carbon-fiber microelectrode 500 mV positive with respect to a Ag-AgCl reference, while measuring the faradic current produced by the oxidation of ferrocyanide. A carbon fiber (5-7 micron diam) is heat sealed into a glass micropipette that is then sharpened, silanized, and electrochemically pretreated to minimize electrode degradation by protein. Circuit diagrams for an inexpensive voltage clamp-current monitor and a data sampling device are presented. The electrodes show a linear response to changes in ferrocyanide concentration in large and very small (20 nl) volumes in vitro. The electrodes were used in an electrochemical microassay to determine tubular fluid-to-plasma ferrocyanide concentration ratios and nephron filtration rates with proximal micropuncture samples. The results show excellent agreement with paired determinations using [3H]inulin. In vivo proximal tubule perfusion experiments show a rapid linear response to changes in tubular fluid ferrocyanide concentration. These electrodes permit rapid quantitative measurements of ferrocyanide concentration and water transport in the proximal tubule and may be useful in other biological systems.

Electron transfer reaction of cytochrome c peroxidase at tin oxide electrodes.

The unmediated one-electron reduction and reoxidation of ferric cytochrome c peroxidase at fluoride-doped tin oxide electrodes is reported. A long range interfacial electron transfer distance of 17 A is postulated by analogy to the Poulos/Kraut model for the cytochrome c peroxidase/cytochrome c electron transfer complex. The utility of the interfacial electrochemical approach for investigating cytochrome c peroxidase electron transfer behavior is discussed.