Kinetic stability of the peroxidase activity of unfolded cytochrome c: heme degradation and catalyst inactivation by hydrogen peroxide

Unfolding converts Paracoccus versutus cytochrome c-550 into a potent peroxidase (Diederix, R. E. M.; Ubbink, M.; Canters, G. W. ChemBioChem 2002, 3, 110-112). The catalytic activity is accompanied by peroxide-driven inactivation that is prevented, in part, by reducing substrate. Here, the kinetics of inactivation are described, and evidence is presented for the occurrence of a labile intermediate on the catalytic peroxidase pathway of unfolded cytochrome c-550. This intermediate represents a branching point, whereby the protein proceeds along either the productive pathway or self-inactivates. Reducing substrate suppresses inactivation by decreasing the steady-state concentration of the labile intermediate. Inactivation is accompanied by heme degradation. Its chemical reactivity, UV-vis, and EPR properties identify the first intermediate as hydroxyheme-cytochrome c-550, i.e. with heme hydroxylated at one of the heme meso positions. The occurrence of this species argues for the peroxo-iron species in the peroxidase mechanism as the labile intermediate leading to inactivated cytochrome c-550.     

Tyrosine or Tryptophan? Modifying a Metalloradical Catalytic Site by Removal of the Cys–Tyr Cross-Link in the Galactose 6-Oxidase Homologue GlxA

The concerted redox action of a metal ion and an organic cofactor is a unique way to maximize the catalytic power of an enzyme. An example of such synergy is the fungal galactose 6-oxidase, which has inspired the creation of biomimetic copper oxidation catalysts. Galactose 6-oxidase and its bacterial homologue, GlxA, possess a metalloradical catalytic site that contains a free radical on a covalently linked Cys–Tyr and a copper atom. Such a catalytic site enables for the two-electron oxidation of alcohols to aldehydes. When the ability to form the Cys–Tyr in GlxA is disrupted, a radical can still be formed. Surprisingly, the radical species is not the Tyr residue but rather a copper second-coordination sphere Trp residue. This is demonstrated through the introduction of a new algorithm for Trp-radical EPR spectra simulation. Our findings suggest a new mechanism of free-radical transfer between aromatic residues and that the Cys–Tyr cross-link prevents radical migration away from the catalytic site.     

Laser flash photolysis study of electron transfer processes of adsorbed anthracene on titania–silica surfaces

We have studied the photophysics of anthracene adsorbed to photocatalytic silica–titania mixed oxide systems prepared by two different methods: a sol–gel synthesis and an impregnation route. The observed photophysics depend upon the method of synthesis, with the sol–gel prepared samples resulting in enhanced radical cation formation via static excited singlet state quenching. This mechanism, whilst operative, does not give rise to long-lived charge separation in the case of the impregnated samples. These results are discussed in terms of differing sample morphologies.     

Oxidative addition reactions of group III metals in low oxidation states. Reactions of Ga2Br4 with alkyl bromides

The oxidative addition reactions of gallium “dibromide” with several primary, secondary and tertiary alkyl bromides, and bromides of the type (CH₂)_nBr₂ have been examined. Whilst all the organic halides readily react with Ga₂Br₄, only in two reactions, namely with methyl and ethyl bromides are isolatable compounds produced and in these cases oxidative addition may be represented as: Ga₂Br₄+RBr→RGa₂Br₅.     

Spectroscopy and photophysics of flavin-related compounds: 3-ethyl-lumiflavin

Electronic structure and singlet–singlet and triplet–triplet absorption spectra of 3-ethyl-lumiflavin were calculated using time-dependent density functional theory (TD-DFT) methods. The measured lower-energy transitions are well reproduced in calculations, which are limited by the neglect of the solvent interactions. All the observable singlet–singlet and triplet–triplet transitions have π–π^* character. Singlet oxygen production by the studied compound demonstrated that, similar to other lumiflavins, it is an efficient singlet oxygen sensitizer (_Δ = 0.55). Radiationless deactivation of the S₁ state in solutions was shown to result in the T₁ state formation.     

Quantitative rate constants for radical reactions in the nanopores of cotton

The understanding of radical reactions in nanostructured materials is important for developing new synthetic procedures and controlling degradation reactions. To develop this area, an easy method for measuring quantitative rate constants of some radical reactions in nanostructures is required. A simple method for measuring the rate constant of dye bleaching, kdye, by organic radicals in such materials is introduced, involving the measurement of microsecond bleaching kinetics by diffuse reflectance spectroscopy, following laser flash creation of the radicals. Using wet and dry cotton as model substrates, we obtained kdye of 2-hydroxy-2-propyl and 1-hydroxy-1-cyclohexyl radicals with reactive red 3 and reactive orange 4 and compared them to solution-phase values. Surprisingly, the reactions in cotton follow simple liquid-phase kinetics and are diffusion-controlled. A cage effect in cotton is also found.     

The CrIIL reduction of [2Fe-2S] ferredoxins and site of attachment of CrIII using 1H NMR and site-directed mutagenesis

The recently reported NMR solution structure of FeIIIFeIII parsley FdI has made possible 2D NOESY NMR studies to determine the point of attachment of CrIIIL in FeIIIFeIII...CrIIIL. The latter Cr-modified product was obtained by reduction of FeIIIFeIII parsley and spinach FdI forms with [Cr(15-aneN4) (H2O)2]2+ (15-aneN4 = 1,4,8,12-tetraazacyclopentadecane), referred to here as CrIIL, followed by air oxidation and chromatographic purification. From a comparison of NMR cross-peak intensities of native and Cr-modified proteins, two surface sites designated A and B, giving large paramagnetic CrIIIL broadening of a number of amino acid peaks, have been identified. The effects at site A (residues 19-22, 27, and 30) are greater than those at site B (residues 92-94 and 96), which is on the opposite side of the protein. From metal (ICP-AES) and electrospray ionization mass spectrometry (EIMS) analyses on the Cr-modified protein, attachment of a single CrIIIL only is confirmed for both parsley and spinach FdI and FdII proteins. Electrostatic interaction of the 3+ CrIIIL center covalently attached to one protein molecule (charge approximately -18) with a second (like) molecule provides an explanation for the involvement of two regions. Thus for 3-4 mM FeIIIFeIII...CrIIIL solutions used in NMR studies (CrIIIL attached at A), broadening effects due to electrostatic interactions at B on a second molecule are observed. Experiments with the Cys18Ala spinach FdI variant have confirmed that the previously suggested Cys-18 at site A is not the site of CrIIIL attachment. Line broadening at Val-22 of A gives the largest effect, and CrIIIL attachment at one or more adjacent (conserved) acidic residues in this region is indicated. The ability of CrIIL to bind in some (parsley and spinach) but not all cases (Anabaena variabilis) suggests that intramolecular H-bonding of acidic residues at A is relevant. The parsley and spinach FeIIFeIII...CrIIIL products undergo a second stage of reduction with the formation of FeIIFeII...CrIIIL. However, the spinach Glu92Ala (site B) variant undergoes only the first stage of reduction, and it appears that Glu-92 is required for the second stage of reduction to occur. A sample of CrIIIL-modified parsley FeIIIFeIII Fd is fully active as an electron carrier in the NADPH-cytochrome c reductase reaction catalyzed by ferredoxin-NADP+ reductase.