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.

Active Site Environment and Reactivity of Copper-Aß in Membrane Mimetic SDS Micellar Environment.

Alzheimer's disease (AD) is characterized by the abnormal aggregation of amyloid ß (Aß) peptide in extracellular deposits generated upon proteolysis of Amyloid Precursor Protein (APP). While copper (Cu(II)) binds to Aß in soluble oligomeric and aggregated forms, its interaction with membrane-bound Aß remains elusive. Investigating these interactions is crucial for understanding AD pathogenesis. Here, utilizing SDS micelles as a simplified membrane mimic, we focus on elucidating the interplay between membrane-anchored Aß and copper, given their pivotal roles in AD. We employed spectroscopic techniques including UV, CD, and EPR to characterize the active site of Cu-Aß complexes. Our findings demonstrate that copper interacts with Aß peptides in membrane-mimicking micellar environments similarly to aqueous buffer solutions. Cu-Aß complexes in this medium also induce higher hydrogen peroxide (H2O2) production, potentially contributing to AD-related oxidative stress. Moreover, we observe an increased oxidation rate of neurotransmitter such as dopamine by Cu-Aß complexes. These results enhance our understanding of Cu-Aß interactions in AD pathology and offer insights into potential therapeutic interventions targeting this interaction.

Facile electrocatalytic proton reduction by a [Fe-Fe]-hydrogenase bio-inspired synthetic model bearing a terminal CN- ligand.

An azadithiolate bridged CN- bound pentacarbonyl bis-iron complex, mimicking the active site of [Fe-Fe] H2ase is synthesized. The geometric and electronic structure of this complex is elucidated using a combination of EXAFS analysis, infrared and Mössbauer spectroscopy and DFT calculations. The electrochemical investigations show that complex 1 effectively reduces H+ to H2 between pH 0-3 at diffusion-controlled rates (1011 M-1 s-1) i.e. 108 s-1 at pH 3 with an overpotential of 140 mV. Electrochemical analysis and DFT calculations suggests that a CN- ligand increases the pKa of the cluster enabling hydrogen production from its Fe(i)-Fe(0) state at pHs much higher and overpotential much lower than its precursor bis-iron hexacarbonyl model which is active in its Fe(0)-Fe(0) state. The formation of a terminal Fe-H species, evidenced by spectroelectrochemistry in organic solvent, via a rate determining proton coupled electron transfer step and protonation of the adjacent azadithiolate, lowers the kinetic barrier leading to diffusion controlled rates of H2 evolution. The stereo-electronic factors enhance its catalytic rate by 3 order of magnitude relative to a bis-iron hexacarbonyl precursor at the same pH and potential.

Rapid autoxidation of ferrous heme-Aß complexes relevant to Alzheimer's disease.

Heme bound Aß peptides have been reported to reduce O2 by 2e- to H2O2 which may result in oxidative stress commonly encountered in Alzheimer's disease. In this study we report the first instance of rapid freeze quench trapping and characterizing the heme(III)-O2˙- intermediate involved in the heme-Aß induced formation of partially reduced oxygen species (PROS) in physiologically relevant aqueous medium using absorption and resonance Raman spectroscopy. The kinetics of this process indicates a key role of the Tyr10 residue, unique to human Aß, in the generation of H2O2 from O2.

Highly regioselective oxidation of C-H bonds in water using hydrogen peroxide by a cytochrome P450 mimicking iron complex.

Cytochrome P450, one of nature's oxidative workhorses, catalyzes the oxidation of C-H bonds in complex biological settings. Extensive research has been conducted over the past five decades to develop a fully functional mimic that activates O2 or H2O2 in water to oxidize strong C-H bonds. We report the first example of a synthetic iron complex that functionally mimics cytochrome P450 in 100% water using H2O2 as the oxidant. This iron complex, in which one methyl group is replaced with a phenyl group in either wing of the macrocycle, oxidized unactivated C-H bonds in small organic molecules with very high selectivity in water (pH 8.5). Several substrates (34 examples) that contained arenes, heteroaromatics, and polar functional groups were oxidized with predictable selectivity and stereoretention with moderate to high yields (50-90%), low catalyst loadings (1-4 mol%) and a small excess of H2O2 (2-3 equiv.) in water. Mechanistic studies indicated the oxoiron(v) to be the active intermediate in water and displayed unprecedented selectivity towards 3° C-H bonds. Under single-turnover conditions, the reactivity of this oxoiron(v) intermediate in water was found to be around 300 fold higher than that in CH3CN, thus implying the role water plays in enzymatic systems.

Heme-Aß in SDS micellar environment: Active site environment and reactivity.

Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder that causes brain cell death. Oxidative stress derived from the accumulation of redox cofactors like heme in amyloid plaques originating from amyloid ß (Aß) peptides has been implicated in the pathogenesis of AD. In the past our group has studied the interactions and reactivities of heme with soluble oligomeric and aggregated forms of Aß. In this manuscript we report the interaction of heme with Aß that remains membrane bound using membrane mimetic SDS (sodium dodecyl sulfate) micellar medium. Employing different spectroscopic techniques viz. circular dichroism (CD), absorption (UV-Vis), electron paramagnetic resonance (EPR) and resonance Raman (rR) we find that Aß binds heme using one of its three His (preferentially His13) in SDS micellar medium. We also find that Arg5 is an essential distal residue responsible for higher peroxidase activity of heme bound Aß in this membrane mimetic environment than free heme. This peroxidase activity exerted by even membrane bound heme-Aß can potentially be more detrimental as the active site remains close to membranes and can hence oxidise the lipid bilayer of the neuronal cell, which can induce cell apoptosis. Thus, heme-Aß in solution as well as in membrane-bound form are detrimental.

The Role of Heme and Copper in Alzheimer's Disease and Type 2 Diabetes Mellitus.

Beyond the well-explored proposition of protein aggregation or amyloidosis as the central event in amyloidogenic diseases like Alzheimer's Disease (AD), and Type 2 Diabetes Mellitus (T2Dm); there are alternative hypotheses, now becoming increasingly evident, which suggest that the small biomolecules like redox noninnocent metals (Fe, Cu, Zn, etc.) and cofactors (Heme) have a definite influence in the onset and extent of such degenerative maladies. Dyshomeostasis of these components remains as one of the common features in both AD and T2Dm etiology. Recent advances in this course reveal that the metal/cofactor-peptide interactions and covalent binding can alarmingly enhance and modify the toxic reactivities, oxidize vital biomolecules, significantly contribute to the oxidative stress leading to cell apoptosis, and may precede the amyloid fibrils formation by altering their native folds. This perspective highlights this aspect of amyloidogenic pathology which revolves around the impact of the metals and cofactors in the pathogenic courses of AD and T2Dm including the active site environments, altered reactivities, and the probable mechanisms involving some highly reactive intermediates as well. It also discusses some in vitro metal chelation or heme sequestration strategies which might serve as a possible remedy. These findings might open up a new paradigm in our conventional understanding of amyloidogenic diseases. Moreover, the interaction of the active sites with small molecules elucidates potential biochemical reactivities that can inspire designing of drug candidates for such pathologies.

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 Hydrogen-Bond Donors and Acceptors Affect the Rate and Selectivity of Electrochemical Oxygen Reduction by Iron Porphyrins Differently.

The factors that control the rate and selectivity of 4e-/4H+ O2 reduction are important for efficient energy transformation as well as for understanding the terminal step of respiration in aerobic organisms. Inspired by the design of naturally occurring enzymes which are efficient catalysts for O2 and H2O2 reduction, several artificial systems have been generated where different second-sphere residues have been installed to enhance the rate and efficiency of the 4e-/4H+ O2 reduction. These include hydrogen-bonding residues like amines, carboxylates, ethers, amides, phenols, etc. In some cases, improvements in the catalysis were recorded, whereas in some cases improvements were marginal or nonexistent. In this work, we use an iron porphyrin complex with pendant 1,10-phenanthroline residues which show a pH-dependent variation of the rate of the electrochemical O2 reduction reaction (ORR) over 2 orders of magnitude. In-situ surface-enhanced resonance Raman spectroscopy reveals the presence of different intermediates at different pH's reflecting different rate-determining steps at different pH's. These data in conjunction with density functional theory calculations reveal that when the distal 1,10-phenanthroline is neutral it acts as a hydrogen-bond acceptor which stabilizes H2O (product) binding to the active FeII state and retards the reaction. However, when the 1,10-phenanthroline is protonated, it acts as a hydrogen-bond donor which enhances O2 reduction by stabilizing FeIII-O2.- and FeIII-OOH intermediates and activating the O-O bond for cleavage. On the basis of these data, general guidelines for controlling the different possible rate-determining steps in the complex multistep 4e-/4H+ ORR are developed and a bioinspired principle-based design of an efficient electrochemical ORR is presented.

Bioinorganic Chemistry on Electrodes: Methods to Functional Modeling.

One of the major goals of bioinorganic chemistry has been to mimic the function of elegant metalloenzymes. Such functional modeling has been difficult to attain in solution, in particular, for reactions that require multiple protons and multiple electrons (nH+/ne-). Using a combination of heterogeneous electrochemistry, electrode and molecule design one may control both electron transfer (ET) and proton transfer (PT) of these nH+/ne- reactions. Such control can allow functional modeling of hydrogenases (H+ + e- → 1/2 H2), cytochrome c oxidase (O2 + 4 e- + 4 H+ → 2 H2O), monooxygenases (RR'CH2 + O2 + 2 e- + 2 H+ → RR'CHOH + H2O) and dioxygenases (S + O2 → SO2; S = organic substrate) in aqueous medium and at room temperatures. In addition, these heterogeneous constructs allow probing unnatural bioinspired reactions and estimation of the inner- and outer-sphere reorganization energy of small molecules and proteins.

Insights from Self-Assembled Aggregates of Amyloid ß Peptides on Gold Surfaces.

Amyloid ß (Aß) peptides mutated at different positions using a cysteine moiety assemble on Au electrodes using the thiol functionality of cysteine. Self-assembled monolayers (SAMs) of Aß on Au surfaces can act as abiological platforms that allow the mimicking of fibrils and oligomeric Aß via the formation of controlled large and small peptide aggregates. These Aß constructs bind with heme and Cu and exhibit different reactivities. These abiological platforms can also be used to investigate potential drugs that can interact with heme and Cu-Aß. SAM formation of Aß mutants allows the study of different morphology and structure as well as behavior changes on binding with different metals and cytochrome c (Cyt c). This review provides a detailed insight into the structure and reactivities of various Aß aggregated on Au electrodes mimicking the cell membrane.

Assembly of redox active metallo-enzymes and metallo-peptides on electrodes: Abiological constructs to probe natural processes.

Redox active metallo-proteins and metallo-peptides attached to self-assembled monolayers (SAM) of thiols on Au electrodes or constituting the SAM on Au electrodes can provide unique opportunities to investigate a range of complicated biological phenomena in controlled abiological constructs. In addition to conventional biochemical tools like site-directed mutagenesis, these constructs allow control over electron transfer (ET) processes, micro solvation (SAM design), folding/misfolding and orientation of these biological entities. This article presents a review of the work done by this group in creating abiological bio-inspired SAM on Au electrodes to probe several important biological processes where redox plays or might play a major role. These include stabilisation of different morphologies of Aß peptides and which allow investigation of the reactivity of their Cu/Zn/heme-bound forms, determination of both outer-sphere and inner-sphere reorganisation energies of cytochrome c along with deciphering the role of the fluxional methionine and finally creation of bio-chemical constructs of cytochrome c oxidase which not only reduce O2 selectively to H2O efficiently but also provide key insights in O2 reduction mechanism which has aided the development of efficient artificial catalysts.

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-).

A Bidirectional Bioinspired [FeFe]-Hydrogenase Model.

With the price-competitiveness of solar and wind power, hydrogen technologies may be game changers for a cleaner, defossilized, and sustainable energy future. H2 can indeed be produced in electrolyzers from water, stored for long periods, and converted back into power, on demand, in fuel cells. The feasibility of the latter process critically depends on the discovery of cheap and efficient catalysts able to replace platinum group metals at the anode and cathode of fuel cells. Bioinspiration can be key for designing such alternative catalysts. Here we show that a novel class of iron-based catalysts inspired from the active site of [FeFe]-hydrogenase behave as unprecedented bidirectional electrocatalysts for interconverting H2 and protons efficiently under near-neutral aqueous conditions. Such bioinspired catalysts have been implemented at the anode of a functional membrane-less H2/O2 fuel cell device.

Interaction of ApoMyoglobin with Heme-hIAPP complex.

Human Islet Amyloid Polypeptide (hIAPP) or amylin, can bind heme and the resultant complexes are prone to generate partially reduced oxygen species (PROS). The formation of PROS and the related oxidative stress highlight the importance of Heme-hIAPP in the onset and development of Type 2 Diabetes mellitus (T2Dm) in humans. In this study, the interaction of Heme-hIAPP with apomyoglobin (ApoMb) has been investigated using a combination of spectroscopic and electrophoresis techniques. Absorption, resonance Raman data and gel electrophoresis results confirm that ApoMb can uptake heme from Heme-hIAPP and constitute a six-coordinate high-spin ferric heme active site identical to that of myoglobin (Mb). The heme transfer reaction has two distinct kinetic steps. A possible mechanism of this reaction involves heme transfer to the apoprotein in the first step followed by a reorganisation of the protein chain to form the active site of native Mb. Increase in the pH of the reaction medium enhances the rate of the second step of heme transfer. This possibly corresponds to the deprotonation of a propionate side chain of the heme moiety at high pH which facilitates secondary interactions with the conserved distal Lys45 residue of horse heart Mb. Additionally, ApoMb sequesters ligand bound heme from Heme-hIAPP. After the heme transfer reaction, the amount of PROS formed by Heme-hIAPP complex diminishes significantly. This not only potentially diminishes heme-induced toxicity in the pancreatic ß-cells but also produces Mb which has well-documented functions throughout the respiratory system and can thereby likely reduce the risks associated with T2Dm.

Oxygen Reduction by Iron Porphyrins with Covalently Attached Pendent Phenol and Quinol.

Phenols and quinols participate in both proton transfer and electron transfer processes in nature either in distinct elementary steps or in a concerted fashion. Recent investigations using synthetic heme/Cu models and iron porphyrins have indicated that phenols/quinols can react with both ferric superoxide and ferric peroxide intermediates formed during O2 reduction through a proton coupled electron transfer (PCET) process as well as via hydrogen atom transfer (HAT). Oxygen reduction by iron porphyrins bearing covalently attached pendant phenol and quinol groups is investigated. The data show that both of these can electrochemically reduce O2 selectively by 4e-/4H+ to H2O with very similar rates. However, the mechanism of the reaction, investigated both using heterogeneous electrochemistry and by trapping intermediates in organic solutions, can be either PCET or HAT and is governed by the thermodynamics of these intermediates involved. The results suggest that, while the reduction of the FeIII-O2̇- species to FeIII-OOH proceeds via PCET when a pendant phenol is present, it follows a HAT pathway with a pendant quinol. In the absence of the hydroxyl group the O2 reduction proceeds via an electron transfer followed by proton transfer to the FeIII-O2̇- species. The hydrogen bonding from the pendant phenol group to FeIII-O2̇- and FeIII-OOH species provides a unique advantage to the PCET process by lowering the inner-sphere reorganization energy by limiting the elongation of the O-O bond upon reduction.

A Single Iron Porphyrin Shows pH Dependent Switch between "Push" and "Pull" Effects in Electrochemical Oxygen Reduction.

The "push-pull" effects associated with heme enzymes manifest themselves through highly evolved distal amino acid environments and axial ligands to the heme. These conserved residues enhance their reactivities by orders of magnitude relative to small molecules that mimic the primary coordination. An instance of a mononuclear iron porphyrin with covalently attached pendent phenanthroline groups is reported which exhibit reactivity indicating a pH dependent "push" to "pull" transition in the same molecule. The pendant phenanthroline residues provide proton transfer pathways into the iron site, ensuring selective 4e-/4H+ reduction of O2 to water. The protonation of these residues at lower pH mimics the pull effect of peroxidases, and a coordination of an axial hydroxide ligand at high pH emulates the push effect of P450 monooxygenases. Both effects enhance the rate of O2 reduction by orders of magnitude over its value at neutral pH while maintaining exclusive selectivity for 4e-/4H+ oxygen reduction reaction.