Heme Binding Induces Dimerization and Nitration of Truncated β‐Amyloid Peptide Aβ16 Under Oxidative Stress

Abstract

Several observations suggest a link between (ferric) heme and Alzheimers disease (AD), for example, an increase in heme oxygenase 1, [1,2] a loss of complex IV, [3][4][5][6] and abnormal iron metabolism. These observations suggest that heme metabolism is altered in age-related disorders. As a possible explanation, hemin has been proposed to interact with b-amyloid peptides (Ab), [7] and it is now well established that Ab peptides bind ferric heme. [8] The adducts of hemin-Ab peptides also exhibit increased peroxidase activity with respect to free hemin, [9] but these complexes have not been characterized. Herein we provide thermodynamic and spectroscopic data showing that hemin b can coordinate two Ab16 molecules to form the low-spin, six-coordinated complex [hemin(Ab16) 2 ], and this is in equilibrium with the high-spin [hemin(Ab16)] species, the relative amounts of which strongly depend on peptide concentration and temperature. In the presence of H 2 O 2 , hemin-Ab16 complexes produce dimerization of the peptide through dityrosine cross-linking, and in addition the peptide undergoes endogenous nitration at Tyr10 when nitrite is also present in solution. This result is important because both cross-linking and nitration at Tyr10 critically enhance Ab aggregation and plaque formation, [10] thus showing that the binding of heme to Ab can indeed be a factor contributing to neuroinflammation and Ab aggregation. The endogenous peroxidative (H 2 O 2 ) and nitrative (H 2 O 2 /NO 2 À ) activities exhibited by the hemin-Ab16 peptide add to, and are likely more deleterious than, the oxidation [9] and nitration [11] of external substrates which have been previously reported.Upon adding Ab16 to a 50 mm aqueous phosphate buffer solution of hemin b at pH 7.4 and 300 K, the broad Soret band of the complex near l = 385 nm progressively changes to give a sharp, red-shifted band at l = 414 nm, with additional visible bands at l = 535 and 566 nm (Figure 1 C, and see the Supporting Information). The conversion from the high-spin to the low-spin hemin species is complete after addition of 20 equivalents of Ab16, and spectrophotometric data analysis (see the Supporting Information) confirms a 2:1 Ab16/hemin stoichiometry, thus indicating the formation of the [hemin-(Ab16) 2 ] complex. The Mçssbauer and 1 H NMR spectra are consistent with a low-spin (S = 1/2) iron(III) center coordinated by two axial imidazole ligands. However, below saturating amounts of Ab16, the Mçssbauer spectrum clearly indicates the presence of a minor high-spin species (Figure 2). Figure 1. UV/vis spectra recorded in 50 mm phosphate buffer solution at pH 7.4. A) Hemin b (1.2 10 À5 m). B) Hemin b in the presence of an excess (20 equiv) of Ab16 peptide at 319 K. C) The same as (B) but at 300 K, the spectral changes are fully reversible. The D symbol indicates an increase in temperature.Figure 2. Mçssbauer spectrum of hemin b (2 mm), in the presence of 6 equivalents of the Ab16 peptide, recorded at 78 K. Quadrupole doublet simulation reveals the presence of a major...

Haem-assisted dityrosine-cross-linking of fibrinogen under non-thermal plasma exposure: one important mechanism of facilitated blood coagulation

“…It has been reported that Aβ can interact with haem to form five or six-coordinated complex which shows peroxidase activity and can lead to the formation of peptide oligomers containing dityrosine cross-linking in the presence of H 2 O 2 2122. The observed cross-linking process in this work resembles the Aβ cross-linking reported previously21.…”

“…Therefore, we have confirmed that the mechanism of fibrinogen cross-linking via dityrosine formation triggered by haem molecule under non-thermal plasma exposure is similar to the case of Aβ peptides induced by haem molecule under H 2 O 2 stress21. Coordinated fibrinogen-hematin complex shows peroxidase activity and in the presence of plasma-generated H 2 O 2 the complex leads to the cross-linking of the peptides via dityrosine formation.…”

“…Considering the requisite of hematin for the fibrinogen cross-linking described above, it reminded us to associate this to the covalent cross-linking as occurring in β-amyloid peptides (Aβ) via dityrosine triggered by haem under oxidative stress2122. To testify whether the cross-linking of fibrinogen was due to dityrosine in our case, fluorescence measurement of the layer in Fig.…”

“…With fibrinogen added to hematin solution, the broad Soret band of hematin near λ = 385 nm is red-shifted to 396 nm, with additional visible bands at λ = 535 nm and the shoulder band at 410 nm (Fig. 4a), suggesting the formation of coordinated fibrinogen-hematin complex21. The vibrational frequencies in Raman spectra sensitive to the coordination number and spin state of hematin macromolecules are ν 10 , ν 2 , and ν 3 and their assignment are listed in the inset of Fig.…”

Alzheimer’s Disease: A Heme–Aβ Perspective

“…34 The transfer of an electron from Tyr10 to O 2 will subsequently lead to the formation of a tyrosyl radical that can cause dimerization of the peptides cross-linked via the tyrosine residue, which are characteristic features of AD. 35 The formation of an Fe−O 2 intermediate ( Figure 3C, blue) is the first step involved in the PROS generation pathway by the Fe(II) center, and this intermediate has been trapped and characterized in numerous heme proteins and their synthetic analogues. 36 The Fe−O 2 intermediate involved in the O 2 reduction pathway by heme−Aβ was trapped using picket Figure 4A).…”