Intermediates involved in the reduction of SO2: insight into the mechanism of sulfite reductases.

Sulfite reductases (SiRs) catalyze the reduction of SO32- to H2S in biosynthetic sulfur assimilation and dissimilation of sulfate. The mechanism of the 6e-/6H+ reduction of SO32- at the siroheme cofactor is debated, and proposed intermediates involved in this 6e- reduction are yet to be spectroscopically characterized. The reaction of SO2 with a ferrous iron porphyrin is investigated, and two intermediates are trapped and characterized: an initial Fe(III)-SO22- species, which undergoes proton-assisted S-O bond cleavage to form an Fe(III)-SO species. These species are characterized using a combination of resonance Raman (with 34S-labelled SO2), EPR and DFT calculations. Results obtained help reconcile the different proposed mechanisms for the SiRs.

Unraveling the Endocannabinoid System: Exploring Its Therapeutic Potential in Autism Spectrum Disorder.

The salient features of autism spectrum disorder (ASD) encompass persistent difficulties in social communication, as well as the presence of restricted and repetitive facets of behavior, hobbies, or pursuits, which are often accompanied with cognitive limitations. Over the past few decades, a sizable number of studies have been conducted to enhance our understanding of the pathophysiology of ASD. Preclinical rat models have proven to be extremely valuable in simulating and analyzing the roles of a wide range of established environmental and genetic factors. Recent research has also demonstrated the significant involvement of the endocannabinoid system (ECS) in the pathogenesis of several neuropsychiatric diseases, including ASD. In fact, the ECS has the potential to regulate a multitude of metabolic and cellular pathways associated with autism, including the immune system. Moreover, the ECS has emerged as a promising target for intervention with high predictive validity. Particularly noteworthy are resent preclinical studies in rodents, which describe the onset of ASD-like symptoms after various genetic or pharmacological interventions targeting the ECS, providing encouraging evidence for further exploration in this area.

Homologous acetone carboxylases select Fe(II) or Mn(II) as the catalytic cofactor.

Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design.

Reduction of Sulfur Dioxide to Sulfur Monoxide by Ferrous Porphyrin.

The reduction of SO2 to fixed forms of sulfur can address the growing concerns regarding its detrimental effect on health and the environment as well as enable its valorization into valuable chemicals. The naturally occurring heme enzyme sulfite reductase (SiR) is known to reduce SO2 to H2 S and is an integral part of the global sulfur cycle. However, its action has not yet been mimicked in artificial systems outside of the protein matrix even after several decades of structural elucidation of the enzyme. While the coordination of SO2 to transition metals is documented, its reduction using molecular catalysts has remained elusive. Herein reduction of SO2 by iron(II) tetraphenylporphyrin is demonstrated. A combination of spectroscopic data backed up by theoretical calculations indicate that FeII TPP reduces SO2 by 2e- /2H+ to form an intermediate [FeIII -SO]+ species, also proposed for SiR, which releases SO. The SO obtained from the chemical reduction of SO2 could be evidenced in the form of a cheletropic adduct of butadiene resulting in an organic sulfoxide.

Spin state dependent peroxidase activity of heme bound amyloid ß peptides relevant to Alzheimer's disease.

The colocalization of heme rich deposits in the senile plaque of Aß in the cerebral cortex of the Alzheimer's disease (AD) brain along with altered heme homeostasis and heme deficiency symptoms in AD patients has invoked the association of heme in AD pathology. Heme bound Aß complexes, depending on the concentration of the complex or peptide to heme ratio, exhibit an equilibrium between a high-spin mono-His bound peroxidase-type active site and a low-spin bis-His bound cytochrome b type active site. The high-spin heme-Aß complex shows higher peroxidase activity than free heme, where compound I is the reactive oxidant. It is also capable of oxidizing neurotransmitters like serotonin in the presence of peroxide, owing to the formation of compound I. The low-spin bis-His heme-Aß complex on the other hand shows enhanced peroxidase activity relative to high-spin heme-Aß. It reacts with H2O2 to produce two stable intermediates, compound 0 and compound I, which are characterized by absorption, EPR and resonance Raman spectroscopy. The stability of compound I of low-spin heme-Aß is accountable for its enhanced peroxidase activity and oxidation of the neurotransmitter serotonin. The effect of the second sphere Tyr10 residue of Aß on the formation and stability of the intermediates of low-spin heme-Aß has also been investigated. The higher stability of compound I for low-spin heme-Aß is likely due to H-bonding interactions involving Tyr10 in the distal pocket.

Second Sphere Interactions in Amyloidogenic Diseases.

Amyloids are protein aggregates bearing a highly ordered cross ß structural motif, which may be functional but are mostly pathogenic. Their formation, deposition in tissues and consequent organ dysfunction is the central event in amyloidogenic diseases. Such protein aggregation may be brought about by conformational changes, and much attention has been directed toward factors like metal binding, post-translational modifications, mutations of protein etc., which eventually affect the reactivity and cytotoxicity of the associated proteins. Over the past decade, a global effort from different groups working on these misfolded/unfolded proteins/peptides has revealed that the amino acid residues in the second coordination sphere of the active sites of amyloidogenic proteins/peptides cause changes in H-bonding pattern or protein-protein interactions, which dramatically alter the structure and reactivity of these proteins/peptides. These second sphere effects not only determine the binding of transition metals and cofactors, which define the pathology of some of these diseases, but also change the mechanism of redox reactions catalyzed by these proteins/peptides and form the basis of oxidative damage associated with these amyloidogenic diseases. The present review seeks to discuss such second sphere modifications and their ramifications in the etiopathology of some representative amyloidogenic diseases like Alzheimer's disease (AD), type 2 diabetes mellitus (T2Dm), Parkinson's disease (PD), Huntington's disease (HD), and prion diseases.

Simultaneous binding of heme and Cu with amyloid ß peptides: active site and reactivities.

Amyloid imbalance and Aß plaque formation are key histopathological features of Alzheimer's disease (AD). These amyloid plaques observed in post-mortem AD brains have been found to contain increased levels of Cu and deposition of the heme cofactor. The increased Cu concentration and heme co-localization together with other heme related dysfunctions hint towards the likely association of the metal and cofactor in the pathology of the disease. Heme and Cu bind with Aß separately to form heme-Aß and Cu-Aß complexes, respectively. In addition, the metal and cofactor can simultaneously bind with the peptide to generate a physiologically relevant heme-Cu-Aß complex. In this review, we discuss the active site environment, electronic structure, spectroscopic and electrochemical properties, and some interesting reactivities exhibited by the heme-Cu-Aß complex with small molecules, such as oxygen (O2), nitric oxide (NO) and nitrite (NO2-).

Peroxidase activity of heme bound amyloid ß peptides associated with Alzheimer's disease.

The amyloid cascade hypothesis attributes the neurodegeneration observed in Alzheimer's disease (AD) to the deposition of the amyloid ß (Aß) peptide into plaques and fibrils in the AD brain. The metal ion hypothesis which implicates several metal ions, viz. Zn2+, Cu2+ and Fe3+, in the AD pathology on account of their abnormal accumulation in the Aß plaques along with an overall dyshomeostasis of these metals in the AD brain was proposed a while back. Metal ion chelators and ionophores, put forward as possible drug candidates for AD, are yet to succeed in clinical trials. Heme, which is widely distributed in the mammalian body as the prosthetic group of several important proteins and enzymes, has been thought to be associated with AD by virtue of its colocalization in the Aß plaques along with the similarity of several heme deficiency symptoms with those of AD and most importantly, due to its ability to bind Aß. This feature article illustrates the active site environment of heme-Aß which resembles those of peroxidases. It also discusses the peroxidase activity of heme-Aß, its ability to effect oxidative degradation of neurotransmitters like serotonin and also the identification of the highly reactive high-valent intermediate, compound I. The effect of second sphere residues on the formation and peroxidase activity of heme-Aß along with the generation and decay of compound I is highlighted throughout the article. The reactivities of heme bound Aß peptides give an alternative theory to understand the possible cause of this disease.

Intermediates involved in serotonin oxidation catalyzed by Cu bound Aß peptides.

The degradation of neurotransmitters is a hallmark feature of Alzheimer's disease (AD). Copper bound Aß peptides, invoked to be involved in the pathology of AD, are found to catalyze the oxidation of serotonin (5-HT) by H2O2. A combination of EPR and resonance Raman spectroscopy reveals the formation of a Cu(ii)-OOH species and a dimeric, EPR silent, Cu2O2 bis-µ-oxo species under the reaction conditions. The Cu(ii)-OOH species, which can be selectively formed in the presence of excess H2O2, is the reactive intermediate responsible for 5-HT oxidation. H2O2 produced by the reaction of O2 with reduced Cu(i)-Aß species can also oxidize 5-HT. Both these pathways are physiologically relevant and may be involved in the observed decay of neurotransmitters as observed in AD patients.

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.