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We use substituted polyanilines for the construction of new polymer electrodes for interaction studies with the redox protein cytochrome c (cyt c) and the enzyme sulfite oxidase (SO). For these purposes four different polyaniline copolymers are chemically synthesized. Three of them are copolymers, containing 2-methoxyaniline-5-sulfonic acid with variable ratios of aniline; the fourth copolymer consists of 3-amino-benzoic acid and aniline. The results show that all polymers are suitable for being immobilized as thin stable films on gold wire and indium tin oxide (ITO) electrode surfaces from DMSO solution. This can be demonstrated by cyclic voltammetry and UV-Vis spectroscopy measurements. Moreover, cyt c can be electrochemically detected not only in solution, but also immobilized on top of the polymer films. Furthermore, the appearance of a significant catalytic current has been demonstrated for the sulfonated polyanilines, when the polymer-coated protein electrode is being measured upon addition of sulfite oxidase, confirming the establishment of a bioanalytical signal chain. Best results have been obtained for the polymer with highest sulfonation grade. The redox switching of the polymer by the enzymatic reaction can also be analyzed by following the spectral properties of the polymer electrode.
Febuxostat, a drug recently approved in the US, European Union and Japan for treatment of gout, inhibits xanthine oxidoreductase (XOR)-mediated generation of uric acid during purine catabolism. It inhibits bovine milk XOR with a K-i in the picomolar-order, but we found that it is a much weaker inhibitor of Rhodobacter capsulatus XOR, even though the substrate-binding pockets of mammalian and bacterial XOR are well-conserved as regards to catalytically important residues and three-dimensional structure, and both permit the inhibitor to be accommodated in the active site, as indicated by computational docking studies. To clarify the reason for the difference of inhibitory potency towards the two XORs, we performed molecular dynamics simulations. The results indicate that differences in mobility of hydrophobic residues that do not directly interact with the substrate account for the difference in inhibitory potency.
Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.
We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are beta-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.
We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.
To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.
The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 mu M with a high sensitivity (1.85 nA mu M-1). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.
Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.