Reinhard Kissner

Human peroxiredoxin 5 is a peroxynitrite reductase.

Peroxiredoxins are an ubiquitous family of peroxidases widely distributed among prokaryotes and eukaryotes. Peroxiredoxin 5, which is the last discovered mammalian member, was previously shown to reduce peroxides with the use of reducing equivalents derived from thioredoxin. We report here that human peroxiredoxin 5 is also a peroxynitrite reductase. Analysis of peroxiredoxin 5 mutants, in which each of the cysteine residues was mutated, suggests that the nucleophilic attack on the O–O bond of peroxynitrite is performed by the N‐terminal peroxidatic Cys47. Moreover, with the use of pulse radiolysis, we show that human peroxiredoxin 5 reduces peroxynitrite with an unequalled high rate constant of (7 ± 3) × 107 M−1 s−1.

Kinetic study of the reaction of ebselen with peroxynitrite

The second‐order rate constant for the reaction of ebselen with peroxynitrite (ONOO−) is (2.0±0.1) × 106 M−1s−1 at pH ≥ 8 and 25°C, 3–4 orders of magnitude higher than the rate constants observed for cysteine, ascorbate, or methionine. The activation energy is relatively low, 12.8 kJ/mol. This is the fastest reaction of peroxynitrite observed so far. It may allow Secontaining compounds to play a novel role in the defense against peroxynitrite, one of the important reactive species generated during inflammatory processes.

Aerobic Epoxidation of Olefins Catalyzed by the Cobalt‐Based Metal–Organic Framework STA‐12(Co)

A Co-based metal-organic framework (MOF) was investigated as a catalytic material in the aerobic epoxidation of olefins in DMF and exhibited, based on catalyst mass, a remarkably high catalytic activity compared with the Co-doped zeolite catalysts that are typically used in this reaction. The structure of STA-12(Co) is similar to that of STA-12(Ni), as shown by XRD Rietveld refinement and is stable up to 270 °C. For the epoxidation reaction, significantly different selectivities were obtained depending on the substrate. Although styrene was epoxidized with low selectivity due to oligomerization, (E)-stilbene was converted with high selectivities between 80 and 90 %. Leaching of Co was low and the reaction was found to proceed mainly heterogeneously. The catalyst was reusable with only a small loss of activity. The catalytic epoxidation of stilbene with the MOF featured an induction period, which was, interestingly, considerably reduced by styrene/stilbene co-epoxidation. This could be traced back to the formation of benzaldehyde promoting the reaction. Detailed parameter and catalytic studies, including in situ EPR and EXAFS spectroscopy, were performed to obtain an initial insight into the reaction mechanism.

Vesicles as Soft Templates for the Enzymatic Polymerization of Aniline

The feasibility of using surfactant vesicles as soft templates for the peroxidase-triggered polymerization of aniline was investigated. It was found that mixed anionic vesicles (diameter approximately 80 nm) composed of sodium dodecylbenzenesulfonate (SDBS) and decanoic acid (1:1, molar ratio) are promising templates. In the presence of the vesicles and horseradish peroxidase/hydrogen peroxide (H2O2) as initiator system, aniline polymerizes under optimized conditions at pH=4.3 to the desired conductive emeraldine form of polyaniline (PANI). The optimal polymerization conditions were elaborated, and some of the chemical and physicochemical aspects of the reaction system were investigated. After addition of aniline and peroxidase to the vesicles, aniline is only loosely associated with the vesicles, as shown by NOESY-NMR and zeta potential measurements. In contrast, the peroxidase strongly binds to the vesicle surface, as shown by fluorescence measurements using TNS (2-(p-toluidino)naphthalene-6-sulfonate) as vesicle membrane probe. This binding of the enzyme to the vesicle surface indicates that the polymerization reaction is initiated predominantly on the surface of the vesicles. Cryo-transmission electron microscopy indicates that the polymerization product remains associated with the vesicles on their surface. For short reaction times (30 s<t<60 s), it is shown that oligoanilines containing an excess of oxidized units are obtained, as shown by VIS/NIR spectroscopy and MALDI-TOF mass spectrometry. For longer reaction times (1 min<t<30 min), the relative amount of over oxidized units in PANI decreases until polymers are obtained which have a VIS/NIR spectrum that is typical for the emeraldine salt form of PANI (lambdamax approximately 1000 nm). The appearance of stable unpaired electrons during the reaction was demonstrated by EPR measurements, in full support of the in situ formation of the conductive emeraldine salt form of PANI. At the end of the reaction (after 1 h), the PANI formed remains homogenously dispersed in the aqueous solution thanks to the presence of the vesicles. No precipitation occurs on a time scale of at least several weeks. FTIR and 13C NMR measurements of the product isolated from the reaction mixture confirm the formation of the emeraldine form of PANI. If the polymerization reaction is carried out in the absence of vesicles but under otherwise identical reaction conditions, the outcome of the reaction is very different, i.e., no indication at all for the formation of the conductive form of PANI.

Intermediates in the autoxidation of nitrogen monoxide.

We have identified two intermediates in the autoxidation of NO*: ONOO*, which was detected by EPR spectroscopy at 295 K and atmospheric pressure in the gas phase, and ONOONO, a red substance produced at 113 K in 2-methylbutane. The red compound is diamagnetic and absorbs maximally at 500 nm. The ONOONO intermediate is unstable above the melting point of 2-methylbutane and rapidly converts to O2NNO2. From the semiquantitative determination of mole fractions present in the gas phase by EPR spectroscopy, we estimated the rate constants for the steps that lead to ONOO* and ONOONO, from the known overall rate constant of the autoxidation reaction, by assuming that a quasi-stationary mechanism applies. The rate constant for the rate-determining formation of ONOO* is about 3.1 x 10(-18) cm3 molecule(-1) s(-1) (or 80 s(-1) in mole fractions), the dissociation rate constant of ONOO* is about 6.5 x 10(3) s(-1), and ONOONO is formed with a rate constant of k=7.7 x 10(-14) cm3 molecule(-1) s(-1) (1.9 x 10(6) s(-1) in mole fractions). From these constants, we estimate that the equilibrium constant for the formation of ONOO* from NO* and O2 (K(ONOO*)) is 4.8 x 10(-22) cm3 molecule(-1) (1.2 x 10(-2)), and, therefore, DeltaG=+11.0 kJ mol(-1). In water, the Gibbs energy change is close to zero. The presence of ONOO* at steady-state concentrations under dioxygen excess may be important not only for reactions in the atmosphere, but especially for reactions in aerosols and biological environments, because the rate constant for formation in solution is higher than that in the gas phase, and, therefore, the half-life of ONOO* is longer.

Mechanistic aspects of the horseradish peroxidase-catalysed polymerisation of aniline in the presence of AOT vesicles as templates

The mechanism of the horseradish peroxidase (HRP)–H2O2-catalysed polymerisation of aniline in the presence of AOT vesicles was investigated. AOT (= bis-(2-ethylhexyl)sulfosuccinate) served as vesicle-forming surfactant and dopant for obtaining at pH = 4.3 and room temperature within 24 h under optimal reaction conditions the green emeraldine salt form of polyaniline in 90–95% yield. Based on UV/VIS/NIR and EPR measurements carried out during the polymerisation reaction, and based on changes in aniline and H2O2 concentrations and HRP activity, a mechanism is proposed. According to this “radical cation mechanism” chain growth occurs on the vesicle surface through addition of aniline radical cations to the growing polymer chain. H2O2 plays two essential roles, to oxidise the heme group of HRP, and to oxidise the growing polymer chain for allowing the stepwise addition of new aniline radical cations. The entire reaction can be divided into three kinetically distinct phases. In the first rapid phase (5–10 min), the actual polymer formation takes place to yield the emeraldine salt form of polyaniline in its bipolaron state. In the second and third slower phases (1–2 days) the bipolarons transform into polarons with unpaired electrons. During the reaction, the HRP activity is decreasing until the enzyme becomes inactive after polymer formation. Reactions carried out with partially deuterated anilines were analysed by 2H magic-angle spinning (MAS) NMR spectroscopy to demonstrate the regioselectivity of the chain growth: para-coupling of the aniline units clearly dominates. Association of the formed polyaniline with the vesicle membrane is evident from cryo-TEM and SANS measurements.

Peroxynitrous Acid - Where is the Hydroxyl Radical?

Peroxynitrite is an inorganic toxin of physiological interest, formed from the diffusion‐controlled reaction of superoxide and nitrogen monoxide with a rate constant of (1.6 ± 0.3) × 1010 M ‐ 1 s ‐ 1. On the basis of three experiments we conclude that homolysis of the O‐O bond in peroxynitrous acid is unlikely: (1) the yield of nitrite from the decomposition of peroxynitrite shows a dependence on the peroxynitrite concentration and is lower than expected for homolysis; (2) the yield of [15N]nitrate from the reaction of [15N]nitrite with peroxynitrous acid predicted by homolysis does not correspond to that found experimentally, and (3) the reaction of peroxynitrous acid with monohydroascorbate does not yield ascorbyl radicals. Activation volumes determined from high‐pressure kinetic studies are inconclusive. IUBMB Life, 55: 567‐572, 2003

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis

RATIONALE Hemolysis occurs not only in conditions such as sickle cell disease and malaria but also during transfusion of stored blood, extracorporeal circulation, and sepsis. Cell-free Hb depletes nitric oxide (NO) in the vasculature, causing vasoconstriction and eventually cardiovascular complications. We hypothesize that Hb-binding proteins may preserve vascular NO signaling during hemolysis. OBJECTIVES Characterization of an archetypical function by which Hb scavenger proteins could preserve NO signaling during hemolysis. METHODS We investigated NO reaction kinetics, effects on arterial NO signaling, and tissue distribution of cell-free Hb and its scavenger protein complexes. MEASUREMENTS AND MAIN RESULTS Extravascular translocation of cell-free Hb into interstitial spaces, including the vascular smooth muscle cell layer of rat and pig coronary arteries, promotes vascular NO resistance. This critical disease process is blocked by haptoglobin. Haptoglobin does not change NO dioxygenation rates of Hb; rather, the large size of the Hb:haptoglobin complex prevents Hb extravasation, which uncouples NO/Hb interaction and vasoconstriction. Size-selective compartmentalization of Hb functions as a substitute for red blood cells after hemolysis and preserves NO signaling in the vasculature. We found that evolutionarily and structurally unrelated Hb-binding proteins, such as PIT54 found in avian species, functionally converged with haptoglobin to protect NO signaling by sequestering cell-free Hb in large protein complexes. CONCLUSIONS Sequential compartmentalization of Hb by erythrocytes and scavenger protein complexes is an archetypical mechanism, which may have supported coevolution of hemolysis and normal vascular function. Therapeutic supplementation of Hb scavengers may restore vascular NO signaling and attenuate disease complications in patients with hemolysis.

Decomposition kinetics of peroxynitrite: influence of pH and buffer.

The decay of ONOOH near neutral pH has been examined as a function of isomerization to H(+) and NO3(-), and decomposition to NO2(-) and O2via O2NOO(-). We find that in phosphate buffer k(isomerization) = 1.11 ± 0.01 s(-1) and k(disproportionation) = (1.3 ± 0.1) × 10(3) M(-1) s(-1) at 25 °C and I = 0.2 M. In the presence of 0.1 M tris(hydroxymethyl)aminomethane (Tris), the decay proceeds more rapidly: k(disproportionation) = 9 × 10(3) M(-1) s(-1). The measured first half-life of the absorbance of peroxynitrite correlates with [Tris]0·([ONOO(-)]0 + [ONOOH]0)(2), where the subscript 0 indicates initial concentrations; if this function exceeds 6.3 × 10(-12) M(3), then Tris significantly accelerates the decomposition of peroxynitrite.

The use of Trametes versicolor laccase for the polymerization of aniline in the presence of vesicles as templates

The enzymatic polymerization of aniline to polyaniline (PANI) with Trametes versicolor laccase (TvL) as catalyst and dioxygen (O₂) as oxidant was investigated in an aqueous medium containing unilamellar vesicles with an average diameter of about 80 nm formed from AOT (=sodium bis(2-ethylhexyl) sulfosuccinate). Compared to the same reaction carried out with horseradish peroxidase isoenzyme C (HRPC) as catalyst and hydrogen peroxide (H₂O₂) as oxidant, notable differences were found in the kinetics of the reaction, as well as in the characteristics of the PANI obtained. Under comparable optimal conditions, which are pH 3.5 for TvL/O₂ and pH 4.3 for HRPC/H₂O₂, the reaction with TvL/O₂ was much slower than with HRPC/H₂O₂, i.e. ≈27 days vs. 1 day reaction time to reach equilibrium with >90% yield at 25 °C. Although in both cases, aniline monomer coupling occurred mainly via the carbon atom in para position of aniline, UV-vis-NIR absorption and EPR measurements indicate that the reaction with TvL/O₂ yielded mainly overoxidized products (with λ(max)=730 nm). These products had a lower amount of unpaired electrons if compared with the products obtained with HRPC/H₂O₂ (with λ(max)≈1000 nm, which is characteristic for the polaron state of PANI-ES, the emeraldine salt form of PANI). Similarly to previous findings with HRPC/H₂O₂, enzyme inactivation occurred during the polymerization also in the case of TvL/O₂. Since the aqueous PANI-vesicle suspensions obtained are of high colloidal stability, they can be used directly as ink in a conventional thermal inkjet printer for printing on paper or on surface treated polyimide films. Printed PANI-ES patterns on paper changed colour from green (emeraldine salt) to blue (emeraldine base) upon exposure to ammonia gas, demonstrating the expected ammonia sensing properties.