Formation of compound I in heme bound Aß-peptides relevant to Alzheimer's disease.

Proteolysis of Amyloid Precursor Protein, APP, results in the formation of amyloid ß (Aß) peptides, which have been associated with Alzheimer's disease (AD). Recently the failure of therapeutic agents that prohibit Aß aggregation and sequester Cu/Zn in providing symptomatic relief to AD patients has questioned the amyloid and metal ion hypothesis. Alternatively, abnormal heme homeostasis and reduced levels of neurotransmitters in the brain are hallmark features of AD. Heme can bind Aß peptides forming a peroxidase type active site which can oxidatively degrade neurotransmitters like serotonin. To date the reactive species responsible for this activity has not been identified. Using rapid kinetics and freeze quenching, we show that heme bound Aß forms a highly reactive intermediate, compound I. Thus, compound I provides a basis for elucidating the oxidative degradation of neurotransmitters like serotonin, resulting in abnormal neurotransmission, a key pathological feature of AD. Site directed mutants indicate that the Arg5 and Tyr10 residues, unique to human Aß, affect the rates of formation and decay of compound I providing insight into their roles in the oxidative degradation of neurotransmitters. Tyr10 can potentially play a natural protective role against the highly reactive oxidant, compound I, in AD.

Nitrite reductase activity of heme and copper bound Aß peptides.

A significant abundance of copper (Cu) and iron in amyloid ß (Aß) plaques, and several heme related metabolic disorders are directly correlated with Alzheimer's disease (AD), and these together with co-localization of Aß plaques with heme rich deposits in the brains of AD sufferers indicates a possible association of the said metals with the disease. Recently, the Aß peptides have been found to bind heme and Cu individually as well as simultaneously. Another significant finding relevant to this is the lower levels of nitrite and nitrate found in the brains of patients suffering from AD. In this study, a combination of absorption and electron paramagnetic resonance spectroscopy and kinetic assays have been used to study the interaction of nitrite with the metal bound Aß complexes. The data indicate that heme(III)-Cu(i)-Aß, heme(II)-Cu(i)-Aß, heme(II)-Aß and Cu(i)-Aß can reduce nitrite to nitric oxide (NO), an important biological messenger also related to AD, and thus behave as nitrite reductases. However these complexes reduce nitrite at different rates with heme(III)-Cu(i)-Aß being the fastest following an inner sphere electron transfer mechanism. The rest of the metal-Aß adducts follow an outer sphere electron transfer mechanism during nitrite reduction. Protonation from the Arg5 residue triggering the N-O bond heterolysis in heme(III) bound nitrite with a simultaneous electron transfer from the Cu(i) center to produce NO is the rate determining step, indicating a proton transfer followed by electron transfer (PTET) mechanism.

Copper induced spin state change of heme-Aß associated with Alzheimer's disease.

Heme binds Aß to give a mixture of a mono-histidine bound high spin peroxidase type active site and a bis-histidine bound low spin cytochrome b type active site present in an equilibrium at physiological pH. Of these, the high spin mono-histidine bound complexes produce significant amounts of partially reduced oxygen species (PROS), catalyze the degradation of neurotransmitters and oxidize cytochrome c, with potentially detrimental effects. The presence of excess Aß could lower these effects by creating a low spin bis-histidine cytochrome b type active site which exerts less oxidative stress by producing a much smaller amount of PROS. The presence of Cu(ii) reverses this effect and can convert the benign low spin heme-Aß complex to the detrimental high spin form, even in the presence of excess Aß. Data suggest that the histidine needed to form the bis-histidine site in the low spin heme-Aß complex is likely to be involved in the high affinity Cu binding site in the heme-Cu-Aß complex.

Cytochrome c peroxidase activity of heme bound amyloid ß peptides.

Heme bound amyloid ß (Aß) peptides, which have been associated with Alzheimer's disease (AD), can catalytically oxidize ferrocytochrome c (Cyt c(II)) in the presence of hydrogen peroxide (H2O2). The rate of catalytic oxidation of Cyt(II) c has been found to be dependent on several factors, such as concentration of heme(III)-Aß, Cyt(II) c, H2O2, pH, ionic strength of the solution, and peptide chain length of Aß. The above features resemble the naturally occurring enzyme cytochrome c peroxidase (CCP) which is known to catalytically oxidize Cyt(II) c in the presence of H2O2. In the absence of heme(III)-Aß, the oxidation of Cyt(II) c is not catalytic. Thus, heme-Aß complex behaves as CCP.

Peroxidase to Cytochrome b Type Transition in the Active Site of Heme-Bound Amyloid ß Peptides Relevant to Alzheimer's Disease.

Recent evidence has established the colocalization of amyloid-rich plaques and heme-rich deposits in the human cerebral cortex as a common postmortem feature in Alzheimer's disease (AD). The amyloid ß (Aß) peptides have been shown to bind heme, and the resultant heme-Aß complexes can generate toxic partially reduced oxygen species (PROS) and exhibit peroxidase activity. The heme-Aß active site exhibits a concentration-dependent equilibrium between a high-spin mono-His-bound species similar to a peroxidase-type active site and a bis-His-bound six-coordinate low-spin species similar to that of a cytochrome b type active site. The ν(Fe-His) (241 cm(-1)) vibration has been identified in the high-spin heme-Aß active site by resonance Raman spectroscopy. The formation of the low-spin heme-Aß species is promoted by the His14 and noncoordinating second-sphere Arg5 residues. The high-spin state produces more PROS than the low-spin species. Nonbiological constructs modeling different forms of Aß (oligomers, fibrils, etc.) suggest that the detrimental high-spin state is likely to dominate under most physiological conditions.

Alzheimer's Disease: A Heme-Aß Perspective.

Redox active iron is utilized in biology for various electron transfer and catalytic reactions essential for life, yet this same chemistry mediates the formation of partially reduced oxygen species (PROS). Oxidative stress derived from the iron accumulated in the amyloid plaques originating from amyloid ß (Aß) peptides and neurofibrillary tangles derived from hyperphosphorylated tau proteins has been implicated in the pathogenesis of Alzheimer's disease (AD). Altered heme homeostasis leading to dysregulation of expression of heme proteins and heme deposits in the amyloid plaques are characteristic of the AD brain. However, the pathogenic significance of heme in neurodegeneration in AD has been unappreciated due to the lack of detailed understanding of the chemistry of the interaction of heme and Aß peptides. As a result, the biochemistry and biophysics of heme complexes of Aß peptides (heme-Aß) remained largely unexplored. In this Account, we discuss the active site environment of heme bound Aß complexes, which involves three amino acid residues unique in mammalian Aß (Arg5, Tyr10, and His13) and missing in Aß from rodents, which do not get affected by AD. The histidine residue binds heme, while the arginine and the tyrosine act as key second sphere residues of the heme-Aß active site that play a crucial role in its reactivity. Generation of PROS, enhanced peroxidase activity, and oxidation of neurotransmitters such as serotonin (5-HT) are all found to be catalyzed by heme-Aß in in vitro assays, and these reactivities can potentially be linked to the observed neuropathologies in AD brain. Association of Cu with heme-Aß leads to the formation of heme-Cu-Aß. The heme-Cu-Aß complex produces a greater amount of PROS than reduced heme-Aß or Cu-Aß alone. Nitric oxide (NO), a signaling molecule, is found to ameliorate the detrimental effects of heme-Aß and Cu bound heme-Aß complexes by detaching heme from the heme-Aß complex and releasing it into the environment solution. Heme-Aß complexes show fast electron transfer with oxidized cytochrome c and rapid heme transfer with apomyoglobin and aponeuroglobin. NO, cytochrome c, and apoglobins can all lead to reduction in PROS generated by reduced heme-Aß. Synthetic analogues of heme, offering a hydrophobic distal environment, have been used to trap oxygen bound intermediates, which provides insight into the mechanism of PROS generation by reduced heme-Aß. Artificial constructs of Aß on nonbiological platforms are used not only to stabilize metastable and physiologically relevant large and small amyloid aggregates but also to monitor the interaction of various drug candidates with heme and Cu bound Aß aggregates, representing a tractable avenue for testing therapeutic agents targeting metals and cofactors in AD.

Direct electron transfer between Cyt c and heme-Aß relevant to Alzheimer's disease.

Partially reduced oxygen species (PROS), produced by reduced heme bound Aß peptides, can cause oxidative stress and synaptic damage in the brain, which is one of the key pathological features of Alzheimer's disease. In situ oxidation of the heme center by a physiological redox agent like Cytochrome c (Cyt c) can significantly suppress neurotoxic PROS formation. Thus, Cyt c can potentially act as a neuroprotective agent against AD.

Ligand-field and ligand-binding analysis of the active site of copper-bound Aß associated with Alzheimer's disease.

Alzheimer's disease (AD) patients show abnormally high concentrations of Cu(2+) in the amyloid ß plaques. This invokes that Cu(2+) might play a crucial role in the onset of AD. The last few decades of research have also shown that Cu(2+) plays a significant role in the aggregation of Aß plaques in the brain and the generation of oxidative stress. Because the crystal structures of Cu-Aß are yet to be obtained, there are various proposed models for the Cu(2+) coordination environment of Aß peptides. In this study, we have used the truncated hydrophilic part of the native Aß peptide to probe the Cu(2+) coordination site of the peptide, using a combination of spectroscopy and exogenous ligand-binding studies. It is evident from our study that Aß(1-16) binds 1 equiv of Cu(2+) and yet shows an equilibrium between two species with a pK(a) of ~8.1. Ligand-field analysis of absorption and circular dichroism spectroscopy data indicates five-coordinate geometry for both components. We investigate the effect of azide and 8-hydroxyquinoline binding to Cu-Aß and demonstrate the presence of a water-derived ligand and a second exchangeable ligand coordinated to copper at physiological pH, along the equatorial plane of a square-pyramidal active site.

Interaction of NO with Cu and heme-bound Aß peptides associated with Alzheimer's disease.

Reduced Cu and heme has been invoked to be involved in Alzheimer's disease (AD). Recently the Aß peptides have been demonstrated to bind heme and Cu simultaneously, and this complex produces significantly more toxic partially reduced oxygen species (PROS) than the Cu or heme-bound Aß peptides. Here a combination of absorption, EPR, and resonance Raman spectroscopy along with kinetic assays are used to investigate the interaction of nitric oxide (NO) with the physiologically relevant form of Cu and heme-bound Aß peptides, since a down-regulation of nitric oxide synthase activity is observed in patients suffering from AD. The data indicate that NO oxidizes the Cu(I) sites, making them less toxic toward PROS generation and releases heme from the Aß peptides ameliorating the effects of heme binding to Aß peptides associated with AD. This process involves a tyrosine-mediated electron transfer between the Cu and heme sites. These results provide a mechanistic pathway for the possible protective role of NO in AD.

Heme-Cu bound aß peptides: spectroscopic characterization, reactivity, and relevance to Alzheimer's disease.

Recently, it has been shown that heme binds to Aß peptides which may play a major role in Alzheimer's disease (AD). This study illustrates that Aß peptides can bind both Cu and heme cofactors at the same time. Both cofactors have unique spectroscopic and electrochemical features which are unaffected in the presence of the other, implying that they are electronically, chemically, and electrochemically uncoupled. These data clearly indicate that Cu cannot bind to three histidine residues simultaneously in Cu-Aß complexes as previously proposed, since one of the histidines is involved in binding heme. The heme-Aß and the heme-Cu-Aß peptide complexes function as peroxidases. Interestingly, the Cu-Aß complex also exhibits peroxidase activity, which may have significant implications in AD. Both Cu(+)-Aß and heme (Fe(2+))-Aß complexes reduce O(2) to H(2)O(2) quantitatively. Only one of the two electrons that are required for the reduction of O(2) to H(2)O(2) is derived from the reduced metal site, while the Tyr(10) residue of the native Aß peptide donates the second electron. This Tyr(10) residue, the source of electron for the generation of partially reduced oxygen species (PROS, e.g., H(2)O(2)) is absent in rodents, which do not get affected by AD. When both heme and Cu are bound to the Aß peptides, which is likely to happen physiologically, the amount of toxic PROS generated is maximum, implying that heme-Cu-Aß complexes could potentially be most toxic for AD.