Inhibition of the biosynthesis ofN-acetylneuraminic acid by metal ions and seleniumin vitro

In liver homogenate the biosynthesis ofN-acetylneuraminic acid usingN-acetylglucosamine as precursor can be followed stepwise by applying different chromatographic procedures. In this cell-free system 16 metal ions (Zn²⁺, Mn²⁺, La³⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Ce³⁺, Cd²⁺, Fe²⁺, Fe³⁺, Al³⁺, Sn²⁺, Cs⁺ and Li⁺) and the selenium compounds, selenium(IV) oxide and sodium selenite, have been checked with respect to their ability to influence a single or possible several steps of the biosynthesis ofN-acetylneuraminic acid. It could be shown that the following enzymes are sensitive to these metal ions (usually applied at a concentration of 1 mmoll^–1):N-acetylglucosamine kinase (inhibited by Zn²⁺ and vandate), UDP-N-acetylglucosamine-2-epimerase (inhibited by zn²⁺, Co²⁺, Cu²⁺, Hg²⁺, VO ₃ ^– , Pb²⁺, Cd²⁺, Fe³⁺, Cs⁺, Li⁺, selenium(IV) oxide and selenite), andN-acetylmannosamine kinase (inhibited by Zn²⁺, Cu²⁺, Cd²⁺, and Co²⁺). Dose dependent measurements have shown that Zn²⁺, Cu²⁺ and selenite are more efficient inhibitors of UDP-N-acetylglucosamine-2-epimerase than vanadate. As for theN-acetylmannosamine kinase inhibition, a decreasing inhibitory effect exists in the following order Zn²⁺, Cd²⁺, Co²⁺ and Cu²⁺. In contrast, La³⁺, Al³⁺ and Mn²⁺ (1 mmoll^–1) did not interfere with the biosynthesis ofN-acetylneuraminic acid. Thus, the conclusion that the inhibitory effect of the metal ions investigated cannot be regarded as simply unspecific is justified.Dedicated to Professor Theodor Günther on the occasion of his 60th birthday     

Inhibitory effects of copper on bacteria related to the free ion concentration

Cu²⁺ ion determinations were carried out in complex and in inorganic salts-glycerol media, to which increasing amounts of Cu(II) had been added, with the ion-specific Cu(II)-Selectrode. Likewise, complexing capacity of bacterial suspensions was estimated by titration with CuSO₄.Copper-sensitive bacteria, e.g.,Klebsiella aerogenes, were inhibited in their growth and survival in the range of 10^–8–10^–6 M Cu²⁺ ion concentrations. In copper-buffered complex media, high copper loads could be tolerated, as growth proceeded with most of the copper bound to medium components. In low-complexing mineral salts media, in which high Cu²⁺ ion concentrations exist at low copper loads, there was competition of Cu²⁺ for binding sites of the cells. Total allowed copper was then determined by the ratio of copper to biomass.Copper-resistant bacteria could be isolated from a stock solution of CuSO₄, containing 100 ppm Cu(II). They were of thePseudomonas type and showed a much higher tolerance towards Cu²⁺, up to 10^–3 M.     

Purification and characterisation of NAD+-dependent xylitol dehydrogenase from Fusarium oxysporum

An NAD⁺-dependent xylitol dehydrogenase (XDH) from Fusarium oxysporum, a key enzyme in the conversion of xylose to ethanol, was purified to homogeneity and characterised. It was homodimeric with a subunit of M _r 48 000, and pI 3.6. It was optimally active at 45 °C and pH 9–10. It was fully stable at pH 6–7 for 24 h and 30 °C. K _m values for d-xylitol and NAD⁺ were 94 mM and 0.14 mM, respectively. Mn²⁺ at 10 mM increased XDH activity 2-fold and Cu²⁺ at 10 mM inhibited activity completely.     

Throughfall chemistry in a loblolly pine plantationunder elevated atmospheric CO2 concentrations

Accelerated tree growth under elevatedatmospheric CO₂ concentrations may influencenutrient cycling in forests by (i) increasingthe total leaf area, (ii) increasing the supplyof soluble carbohydrate in leaf tissue, and (iii) increasing nutrient-use efficiency. Here wereport the results of intensive sampling andlaboratory analyses of NH ₄ ⁺ , NO ₃ ^– , PO ₄ ^3– , H⁺, K⁺, Na⁺,Ca²⁺, Mg²⁺, Cl^–, SO ₄ ^2– , and dissolved organic carbon (DOC) in throughfallprecipitation during the first 2.5+ years of the DukeUniversity Free-Air CO₂ Enrichment (FACE)experiment. After two growing seasons, a largeincrease (i.e., 48%) in throughfall deposition of DOCand significant trends in throughfall volume and inthe deposition of NH ₄ ⁺ , NO ₃ ^– , H⁺, and K⁺ can be attributed to the elevatedCO₂ treatment. The substantial increase indeposition of DOC is most likely associated withincreased availability of soluble C in plant foliage,whereas accelerated canopy growth may account forsignificant trends toward decreasing throughfallvolume, decreasing deposition of NH ₄ ⁺ ,NO ₃ ^– , and H⁺, and increasing deposition of K⁺ under elevated CO₂. Despiteconsiderable year-to-year variability, there wereseasonal trends in net deposition of NO ₃ ^– ,H⁺, cations, and DOC associated with plant growthand leaf senescence. The altered chemical fluxes inthroughfall suggest that soil solution chemistry mayalso be substantially altered with continued increasesin atmospheric CO₂ concentrations in the future.     

L-<Emphasis Type="Italic">myo</Emphasis>-inositol-1-phosphate synthase: partial purification and characterisation from <Emphasis Type="Italic">Gleichenia glauca</Emphasis>

A screening for the enzyme L-myo-inositol-1-phosphate synthase [EC 5.5.1.4] has been made first time in both vegetative and reproductive parts of the representative members of pteridophytes: Lycopodium, Selaginella, Equisetum, Polypodium, Dryopteris, and Gleichenia. The enzyme has been partially purified following low-speed centrifugation, streptomycin sulphate precipitation, ammonium sulphate fractionation, chromatography on DEAE-cellulose and gel-filtration through Sephadex G-200, and characterised from the reproductive pinnules of Gleichenia glauca Smith. The enzyme has a pH optimum at 7.5. The K_m for glucose-6-P and NAD⁺ were 0.922 × 10^–3 M and 0.9 × 10^–4 M, respectively. A basal activity of the enzyme has been recorded in absence of exogenous NAD⁺. The enzyme activity was augmented with NH₄Cl, but heavy metals like Hg²⁺, Cu²⁺ and Zn²⁺ inactivated it.     

Changes in haemolymph ion concentrations of Astacus astacus L. and Pacifastacus leniusculus (Dana) after exposure to low pH and aluminium

During exposure to soft water, acidified to pH 4.0, the haemolymph concentrations of Na⁺, K⁺, and Cl^– decreased whereas the Ca²⁺ concentration fluctuated in Astacus astacus. The haemocyte content of K⁺ decreased from 9% to 2% of the total haemolymph K⁺ content after exposure to pH 3.7 for 3 days. Within 14 days, 250 µg Al³⁺ l^–1, as Al₂(SO₄)₃ at pH 5.0, reduced the haemolymph Na⁺ content in Astacus astacus and Pacifastacus leniusculus, however, the effects were less pronounced than earlier reported for fish. Disturbed ion regulation, mainly depending on low pH, is thought to contribute to the absence of these species in acid waters.     

The effect of copper on frog skin. The role of sulphydryl groups

1. 1. Cu²⁺ at a concentration of 10⁻⁴ M, when applied to the external side of the frog skin produces an increase in the short-circuit current (I_sc).

2. 2. This effect was studied in skins of Rana temporaria adapted to cold (5°C) and room temperature (20°C), skins of Rana pipiens adapted to cold, and the results compared with those obtained previously with Rana ribibunda.

3. 3. The observed effect is less dependent upon the adaptation to cold than upon the functional state of the skin: skins with low short circuit currents have a bigger response to Cu²⁺ than skins with high I_sc.

4. 4. A species difference cannot be ruled out since skins of Rana ribibunda exhibiting high I_sc give good responses to Cu²⁺.

5. 5. 5,5′-dithiobis(2-nitrobenzoic acid), a sulphydryl-oxidizing reagent, produces an effect similar to that of Cu²⁺, and dithiothreitol an SH-reducing agent, reverses the effect of this ion.

6. 6. Cu²⁺ also induces an increase in the unidirectional K⁺ fluxes and unmasks a net outward potassium flux.

7. 7. The outward K⁺ flux induced by Cu²⁺ is sensitive to ouabain.

8. 8. It is concluded that Cu²⁺ increases the permeability of the external barrier of the frog skin to Na⁺ and K⁺, probably by reacting with SH groups.

Medium optimization for a methanol utilizing bacterium based on chemostat theory

Summary In the productions of biomass and vitamin B₁₂ using methanol as the sole carbon source, it is necessary to use a medium in which methanol is the growth limiting substrate. Other inorganic salts should be in slight excess so that the yield of cells and the intracellular content of vitamin B₁₂ do not vary. From basic principles of chemostat culture, a medium was optimized for Pseudomonas AM-1 a methanol utilizing bacterium, for the concentrations of various inorganic salts. This was done in a series of chemostat cultures at a dilution rate of 0.1 h^–1. Optimum amounts of NH₄ ⁺, PO₄ ^3- and Mg²⁺ were estimated from the minimum concentration of the salt at which methanol became growth limiting. The optimum concentrations of Ca²⁺, Fe²⁺, Mn²⁺, and Zn²⁺ as a group were determined in the same way. Cu²⁺, Mo⁶⁺, Co²⁺ and B³⁺ are required at concentrations of g/l and they were not studied as these very low level can be introduced as contaminants from other salts. The optimum medium composition (in g/l) was as follows: (NH₄)₂SO₄, 1.0; H₃PO₄, 75×10^–3; MgSO₄ · 7H₂O, 30×10^–3; CaCl₂ · 2H₂O, 3.3×10^–3; FeSO₄ · 7H₂O, 1.3×10^–3, MnSO₄ · 4H₂O, 0.13×10^–3; ZnSO₄ · 7H₂O, 0.13×10^–3; CuSO₄ · 5H₂O, 40×10^–6; Na₂MoO₄, 40×10^–6; CoCl₂ · 6H₂O, 40×10^–6; H₃BO₃, 30×10^–6 and methanol 4.     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.     

Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica

Summary High levels of glutamine synthetase, detected using both a biosynthetic assay (P _i release from ATP) and a -glutamyl transferase assay, are present in aerobically grown N₂-fixing cultures of Anabaena cylindrica. The enzyme is soluble, has a pH optimum of 6.5–7.5, with a peak at 7.1–7.2 (biosynthetic activity) or 6.9 (transferase activity), and a temperature optimum at 30°C–40°C. Partially purified preparations are stable in air at 5°C for at least 3 days. Mg²⁺, Mn²⁺, Co²⁺ and Ca²⁺ support high rates of biosynthetic activity, Zn²⁺ is less effective and Cu²⁺ and Ba²⁺ are ineffective.Enzyme activity is regulated at several levels: possibly by repression and derepression of the enzyme in response to NH₄ ⁺ level; by variation in the Mn²⁺: ATP ratio with optimum activity at a 1:1 ratio; by feed-back inhibition which may be of a cumulative type. The consensus of the evidence suggests the absence of a covalent enzyme modification of the type found in E. coli. Glutamine synthetase levels are almost twice as high on a protein basis in the heterocysts as in the vegetative cells. Apparent K _m values for whole filaments for NH₄ ⁺ and glutamate in the biosynthetic reactions are 1 mM and 2 mM respectively.     

Accumulation of technetium by cyanobacteria

Accumulation of pertechnetate ions (⁹⁹TcO₄ ^–) by the cyanobacterial speciesSynechocystis PCC 6803,Synechococcus PCC 6301,Plectonema boryanum,Anabaena variabilis and a redOscillatoria sp. consisted solely of a single rapid energy-independent phase (biosorption); no energy-dependent uptake was detected. Biosorption of TcO₄ ^– was concentration-dependent and could be described by a Freundlich adsorption isotherm for each cyanobacterial species examined. Decreasing pH increased the accumulation of TcO₄ ^– by all the species as did an increase in external NaCl concentration. Accumulation of TcO₄ ^– was also increased inA. variabilis, P. boryanum and the redOscillatoria by an increased external osmotic potential. Concentrations of cations affected TcO₄ ^– accumulation; K⁺ increased accumulation in all the species, Mg²⁺, Ca²⁺, Sr²⁺ and Cs⁺ increased accumulation inSynechococcus PCC 6301 and Ca²⁺ increased accumulation by the redOscillatoria. Some anions decreased TcO₄ ^– accumulation; CO₃ ^2– inA. variabilis and the redOscillatoria, SO₄ ^2– inSynechocystis PCC 6803, and HCO₃ ^– inP. boryanum. The majority of TcO₄ ^– accumulated by all the cyanobacteria was easily desorbed, with no difference in the amounts desorbed between desorption agents of different pH or cation concentration.(*author for correspondence)     

Biosorption of chromium to fungi

Eighteen fungal strains were isolated from water and soil samples and tested for their ability to enrich chromium. The microorganism with the highest enrichment capacity, a zygomycete (Mucor hiemalis MP/92/3/4), was chosen for detailed investigations. Some basic tests such as the pH-dependence, the kinetics of the enrichment and the metal selectivity were carried out with the two most frequent oxidation states of chromium, the trivalent cation (Cr³⁺) and the hexavalent anion (CrO₄ ^2–). With Cr³⁺ the enrichment showed a saturation kinetic reaching 70% of the maximum capacity after about 30 min, whereas with CrO₄ ^2– a linear time course with a much lower metal enrichment was observed. The highest level of enrichment for Cr³⁺ was observed at pH 5.5 (21.4 mg/g dry wt), and for CrO₄ ^2– at pH 1 (4.3 mg/g dry wt). Investigations concerning the metal enrichment selectivity resulted in the following series of decreasing ion uptake: Cr³⁺ > Cu²⁺ > Pb²⁺ > Ag⁺ > Al³⁺ > Co²⁺ > Zn²⁺ > Ni²⁺ > Fe²⁺ > Mo⁵⁺ > Cd²⁺ > ^2– > CrO₄ ^2– > VO^3–, calculated on a molar basis. Trivalent chromium caused a staining of the outer cell wall region in transmission electron microscopy. The localization of chromium in the stained outer layers of the cell wall could be verified by electron energy loss spectroscopy. The enrichment of Cr³⁺ by M. hiemalis seemed to be mainly a passive biosorption to the cell wall, whereas for the uptake of CrO₄ ^2– intracellular accumulation as well as biosorption is possible.     

Electrogenic proton secretion in the Hindgut of the desert locust,Schistocerca gregaria

Summary The cellular mechanisms responsible for rectal acidification in the desert locust, Schistocerca gregaria, were investigated in isolated recta mounted as flat sheets in modified Ussing chambers. Previous studies conducted in the nominal absence of exogenous CO₂ and HCO ₃ ^– suggested that the acidification was due to a proton-secretory rather than bicarbonate-reabsorptive mechanism (Thomson, R.B., Speight, J.D., Phillips, J.E. 1988. J. Insect Physiol. 34:829–837). This conclusion was confirmed in the present study by demonstrating that metabolic CO₂ could not contribute sufficient HCO ₃ ^– to the lumen to account for the rates of rectal acidification observed under the nominally CO₂/ HCO ₃ ^– -free conditions used in these investigations.Rates of luminal acidification (J _H ⁺) were completely unaffected by changes in contraluminal pH, but could be progressively reduced (and eventually abolished) by imposition of either transepithelial pH gradients (lumen acid) or transepithelial electrical gradients (lumen positive). Under short-circuit current conditions, the bulk of J _H ⁺ was not dependent on Na⁺, K⁺, Cl^–,Mg²⁺, or Ca²⁺ and was due to a primary electrogenic proton translocating mechanism located on the apical membrane. A small component (10–16%) of J _H ⁺ measured under these conditions could be attributed to an apical amiloride-inhibitable Na⁺/H⁺ exchange mechanism.This work was supported by operating grants to J.E.P. and postgraduate scholarships to R.B.T. from Natural Sciences & Engineering Research Council, Canada.     

Na<Superscript>+</Superscript>-mediated piezoprotection in<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis>

Sodium concentrations as low as 2 mM exerted a significant protective effect on the high-pressure inactivation (160–210 MPa) of Rhodotorula rubra at pH 6.5, but not on two other yeasts tested (Shizosaccharomyces pombe and Saccharomyces cerevisiae). A piezoprotective effect of similar magnitude was observed with Li⁺ (2 and 10 mM), and at elevated pH (8.0–9.0), but no effect was seen with K⁺, Ca²⁺, Mg²⁺, Mn²⁺, or NH₄ ⁺. Intracellular Na⁺ levels in cells exposed to low concentrations of Na⁺ or to pH 8.0–9.0 provided evidence for the involvement of a plasma membrane Na⁺/H⁺ antiporter and a correlation between intracellular Na⁺ levels and pressure resistance. The results support the hypothesis that moderate high pressure causes indirect cell death in R. rubra by inducing cytosolic acidification.Communicated by K. Horikoshi     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Influence of inorganic microelements on the production of camptothecin with suspension cultures of <Emphasis Type="Italic">Camptotheca acuminata</Emphasis>

The effects of eight microelements (I^–, BO₃ ^3–, MoO₄ ^2–, Co²⁺, Cu²⁺, Mn²⁺, Fe²⁺, Zn²⁺) on the biosynthesis of camptothecin and the growth of suspension cultures of Camptotheca acuminata were studied. The increase of I^– to 25 M l^–1, Cu²⁺ to 1 M l^–1, Co²⁺ to 2 M l^–1 and MoO₄ ^2– to 10 M l^–1 in Murashige and Skoog (MS) medium resulted in 1.66, 2.84, 2.53 and 2.04 times higher of camptothecin yield than that in standard MS medium respectively. Combined treatment of I^– (25 M l^–1), Cu²⁺ (1 M l^–1), Co²⁺ (2 M l^–1) and MoO₄ ^2– (10 M l^–1) lead to improve cell dry weight, camptothecin content, and camptothecin yield to 30.56 g l^–1, 0.0299%, and 9.15 mg l^–1, respectively, which were 20.2, 208.9 and 273.8% increment respectively when compared with those of control.     

Calcium absorption by fish intestine: The involvement of ATP-and sodium-dependent calcium extrusion mechanisms

Summary Measurements of unidirectional calcium fluxes in stripped intestinal epithelium of the tilapia,Oreochromis mossambicus, in the presence of ouabain or in the absence of sodium indicated that calcium absorption via the fish intestine is sodium dependent. Active Ca²⁺ transport mechanisms in the enterocyte plasma membrane were analyzed. The maximum capacity of the ATP-dependent Ca²⁺ pump (V _m :0.63 nmol·min^–1 mg^–1,K _m : 27nm Ca²⁺) is calculated to be 2.17 nmol·min^–1·mg^–1, correcting for 29% inside-out oriented vesicles in the membrane preparation. The maximum capacity of the Na⁺/Ca²⁺ exchanger with high affinity for Ca²⁺ (V _m :7.2 nmol·min^–1·mg^–1,K _m : 181nm Ca²⁺) is calculated to be 13.6 nmol·min^–1·mg^–1, correcting for 53% resealed vesicles and assuming symmetrical behavior of the Na⁺/Ca²⁺ exchanger. The high affinity for Ca²⁺ and the sixfold higher capacity of the exchanger compared to the ATPase suggest strongly that the Na⁺/Ca²⁺ exchanger will contribute substantially to Ca²⁺ extrusion in the fish enterocyte. Further evidence for an important contribution of Na⁺/Ca²⁺ exchange to Ca²⁺ extrusion was obtained from studies in which the simultaneous operation of ATP-and Na⁺-gradient-driven Ca²⁺ pumps in inside-out vesicles was evaluated. The fish enterocyte appears to present a model for a Ca²⁺ transporting cell, in which Na⁺/Ca²⁺ exchange activity with high affinity for Ca²⁺ extrudes Ca²⁺ from the cell.     

Capturing a Reactive State of Amyloid Aggregates: NMR-BASED CHARACTERIZATION OF COPPER-BOUND ALZHEIMER DISEASE AMYLOID β-FIBRILS IN A REDOX CYCLE*

The interaction of redox-active copper ions with misfolded amyloid β (Aβ) is linked to production of reactive oxygen species (ROS), which has been associated with oxidative stress and neuronal damages in Alzheimer disease. Despite intensive studies, it is still not conclusive how the interaction of Cu⁺/Cu²⁺ with Aβ aggregates leads to ROS production even at the in vitro level. In this study, we examined the interaction between Cu⁺/Cu²⁺ and Aβ fibrils by solid-state NMR (SSNMR) and other spectroscopic methods. Our photometric studies confirmed the production of ∼60 μm hydrogen peroxide (H₂O₂) from a solution of 20 μm Cu²⁺ ions in complex with Aβ(1–40) in fibrils ([Cu²⁺]/[Aβ] = 0.4) within 2 h of incubation after addition of biological reducing agent ascorbate at the physiological concentration (∼1 mm). Furthermore, SSNMR ¹H T₁ measurements demonstrated that during ROS production the conversion of paramagnetic Cu²⁺ into diamagnetic Cu⁺ occurs while the reactive Cu⁺ ions remain bound to the amyloid fibrils. The results also suggest that O₂ is required for rapid recycling of Cu⁺ bound to Aβ back to Cu²⁺, which allows for continuous production of H₂O₂. Both ¹³C and ¹⁵N SSNMR results show that Cu⁺ coordinates to Aβ(1–40) fibrils primarily through the side chain N_δ of both His-13 and His-14, suggesting major rearrangements from the Cu²⁺ coordination via N_ϵ in the redox cycle. ¹³C SSNMR chemical shift analysis suggests that the overall Aβ conformations are largely unaffected by Cu⁺ binding. These results present crucial site-specific evidence of how the full-length Aβ in amyloid fibrils offers catalytic Cu⁺ centers.