Bis-Fe(IV): nature’s sniper for long-range oxidation

Iron-dependent enzymes are prevalent in nature and participate in a wide range of biological redox activities. Frequently, high-valence iron intermediates are involved in the catalytic events of iron-dependent enzymes, especially when the activation of peroxide or molecular oxygen is involved. Building on the fundamental framework of iron–oxygen chemistry, these reactive intermediates constantly attract significant attention from the enzymology community. During the past few decades, tremendous efforts from a number of laboratories have been dedicated to the capture and characterization of these intermediates to improve mechanistic understandings. In 2008, an unprecedented bis-Fe(IV) intermediate was reported in a c-type diheme enzyme, MauG, which is involved in the maturation of a tryptophan tryptophylquinone cofactor of methylamine dehydrogenase. This intermediate, although chemically equivalent to well-characterized high-valence iron intermediates, such as compound I, compound ES, and intermediate Q in methane monooxygenase, as well as the hypothetical Fe(V) species in Rieske non-heme oxygenases, is orders of magnitude more stable than these other high-valence species in the absence of its primary substrate. It has recently been discovered that the bis-Fe(IV) intermediate exhibits a unique near-IR absorption feature which has been attributed to a novel charge-resonance phenomenon. This review compares the properties of MauG with structurally related enzymes, summarizes the current knowledge of this new high-valence iron intermediate, including its chemical origin and structural basis, explores the formation and consequences of charge resonance, and recounts the long-range catalytic mechanism in which bis-Fe(IV) participates. Biological strategies for storing oxidizing equivalents with iron ions are also discussed.     

Non-heme iron oxygenases

Our understanding of the biological significance and chemical properties of non-heme iron oxygenases has increased dramatically in recent years. New group members have emerged from genome sequences and biochemical analyses. Spectroscopic and crystallographic studies have provided critical insights into catalysis. Self-hydroxylation reactions, commonplace in these proteins, reveal important features of metallocenter reactivity.     

Non-heme Iron as Ferrous Sulfate Does Not Interact with Heme Iron Absorption in Humans

The absorption of heme iron has been described as distinctly different from that of non-heme iron. Moreover, whether heme and non-heme iron compete for absorption has not been well established. Our objective was to investigate the potential competition between heme and non-heme iron as ferrous sulfate for absorption, when both iron forms are ingested on an empty stomach. Twenty-six healthy nonpregnant women were selected to participate in two iron absorption studies using iron radioactive tracers. We obtained the dose?Cresponse curve for absorption of 0.5, 10, 20, and 50?mg heme iron doses, as concentrated red blood cells. Then, we evaluated the absorption of the same doses, but additionally we added non-heme iron, as ferrous sulfate, at constant heme/non-heme iron molar ratio (1:1). Finally, we compare the two curves by a two-way ANOVA. Iron sources were administered on an empty stomach. One factor analysis showed that heme iron absorption was diminished just by increasing total heme iron (P?<?0.0001). The addition of non-heme iron as ferrous sulfate did not have any effect on heme iron absorption (P?=?NS). We reported evidence that heme and non-heme iron as ferrous sulfate does not compete for absorption. The mechanism behind the absorption of these iron sources is not clear.     

Transition metal homeostasis: from yeast to human disease

Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer??s and Parkinson??s disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.     

Amphipatic molecules affect the kinetic profile of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Dioxygenases are nonheme iron enzymes that biodegrade recalcitrant compounds, such as catechol and derivatives, released into the environment by modern industry. Intradiol dioxygenases have attracted much attention due to the interest in their use for bioremediation, which has demanded efforts towards understanding their action mechanism and also how to control it. The role of unexpected amphipatic molecules, observed in crystal structures of intradiol dioxygenases, during catalysis has been poorly explored. We report results obtained with the intradiol enzyme chlorocatechol 1,2-dioxygenase (1,2-CCD) from Pseudomonas putida subjected to delipidation. The delipidated enzyme is more stable and shows more cooperative thermal denaturation. The kinetics changes from Michaelis–Menten to a cooperative scheme, indicating that conformational changes propagate between monomers in the absence of amphipatic molecules. Furthermore, these molecules inhibit catalysis, yielding lower v _max values. To the best of our knowledge, this is the first report concerning the effects of amphipatic molecules on 1,2-CCD function.     

Post-translational self-hydroxylation: a probe for oxygen activation mechanisms in non-heme iron enzymes

Recent years have seen considerable evolution in our understanding of the mechanisms of oxygen activation by non-heme iron enzymes, with high-valent iron-oxo intermediates coming to the forefront as formidably potent oxidants. In the absence of substrate, the generation of vividly colored chromophores deriving from the self-hydroxylation of a nearby aromatic amino acid for a number of these enzymes has afforded an opportunity to discern the conditions under which O2 activation occurs to generate a high-valent iron intermediate, and has provided a basis for a rigorous mechanistic examination of the oxygenation process. Here, we summarize the current evidence for self-hydroxylation processes in both mononuclear non-heme iron enzymes and in mutant forms of ribonucleotide reductase, and place it within the context of our developing understanding of the oxidative transformations accomplished by non-heme iron centers.     

Comparison of Structurally-Related Alkoxide, Amine, and Thiolate-Ligated M (M= Fe, Co) Complexes: the Influence of Thiolates on the Properties of Biologically Relevant Metal Complexes

Mechanistic pathways of metalloenzymes are controlled by the metal ion’s electronic and magnetic properties, which are tuned by the coordinated ligands. The functional advantage gained by incorporating cysteinates into the active site of non-heme iron enzymes such as superoxide reductase (SOR) is not entirely understood. Herein, we compare the structural and redox properties of a series of structurally-related thiolate, alkoxide, and amine-ligated Fe(II) complexes in order to determine how the thiolate influences properties critical to function. Thiolates are shown to reduce metal ion Lewis acidity relative to alkoxides and amines, and have a strong trans influence thereby helping to maintain an open coordination site. Comparison of the redox potentials of the structurally analogous compounds described herein shows that alkoxide ligands favor the higher-valent Fe³⁺ oxidation state, amine ligands favor the reduced Fe²⁺ oxidation state, and thiolates fall somewhere in between. These properties provide a functional advantage for substrate reducing enzymes in that they provide a site at the metal ion for substrate to bind, and a moderate potential that facilitates both substrate reduction and regeneration of the catalytically active reduced state. Redox potentials for structurally-related Co(II) complexes are shown to be cathodically-shifted relative to their Fe(II) analogues, making them ineffective reducing agents for substrates such as superoxide.     

Emerging roles of secreted phospholipase A2 enzymes: Lessons from transgenic and knockout mice

Among the emerging phospholipase A₂ (PLA₂) superfamily, the secreted PLA₂ (sPLA₂) family consists of low-molecular-mass, Ca²⁺-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA₂ enzymes have been identified in mammals. Individual sPLA₂s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA₂ enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA₂ isoforms fueled by studies employing transgenic and knockout mice for several sPLA₂s.     

Structural and Functional Characterization of Three DsbA Paralogues from Salmonella enterica Serovar Typhimurium

In prototypic Escherichia coli K-12 the introduction of disulfide bonds into folding proteins is mediated by the Dsb family of enzymes, primarily through the actions of the highly oxidizing protein EcDsbA. Homologues of the Dsb catalysts are found in most bacteria. Interestingly, pathogens have developed distinct Dsb machineries that play a pivotal role in the biogenesis of virulence factors, hence contributing to their pathogenicity. Salmonella enterica serovar (sv.) Typhimurium encodes an extended number of sulfhydryl oxidases, namely SeDsbA, SeDsbL, and SeSrgA. Here we report a comprehensive analysis of the sv. Typhimurium thiol oxidative system through the structural and functional characterization of the three Salmonella DsbA paralogues. The three proteins share low sequence identity, which results in several unique three-dimensional characteristics, principally in areas involved in substrate binding and disulfide catalysis. Furthermore, the Salmonella DsbA-like proteins also have different redox properties. Whereas functional characterization revealed some degree of redundancy, the properties of SeDsbA, SeDsbL, and SeSrgA and their expression pattern in sv. Typhimurium indicate a diverse role for these enzymes in virulence.     

Coupling of collective motions of the protein matrix to vibrations of the non-heme iron in bacterial photosynthetic reaction centers

Non-heme iron is a conservative component of type II photosynthetic reaction centers of unknown function. We found that in the reaction center from Rba. sphaeroides it exists in two forms, high and low spin ferrous states, whereas in Rsp. rubrum mostly in a low spin state, in line with our earlier finding of its low spin state in the algal photosystem II reaction center (Burda et al., 2003). The temperature dependence of the non-heme iron displacement studied by Mössbauer spectroscopy shows that the surrounding of the high spin iron is more flexible (Debye temperature ~ 165 K) than that of the low spin atom (~ 207 K). Nuclear inelastic scattering measurements of the collective motions in the Rba. sphaeroides reaction center show that the density of vibrational states, originating from non-heme iron, has well-separated modes between lower (4-17 meV) and higher (17-25 meV) energies while in the one from Rsp. rubrum its distribution is more uniform with only little contribution of low energy (~ 6 meV) vibrations. It is the first experimental evidence that the fluctuations of the protein matrix in type II reaction center are correlated to the spin state of non-heme iron. We propose a simple mechanism in which the spin state of non-heme iron directly determines the strength of coupling between the two quinone acceptors (Q_A and Q_B) and fast collective motions of protein matrix that play a crucial role in activation and regulation of the electron and proton transfer between these two quinones. We suggest that hydrogen bond network on the acceptor side of reaction center is responsible for stabilization of non-heme iron in different spin states.     

Dioxygen activation by copper, heme and non-heme iron enzymes: comparison of electronic structures and reactivities

Enzymes containing heme, non-heme iron and copper active sites play important roles in the activation of dioxygen for substrate oxidation. One key reaction step is CH bond cleavage through H-atom abstraction. On the basis of the ligand environment and the redox properties of the metal, these enzymes employ different methods of dioxygen activation. Heme enzymes are able to stabilize the very reactive iron(IV)-oxo porphyrin-radical intermediate. This is generally not accessible for non-heme iron systems, which can instead use low-spin ferric-hydroperoxo and iron(IV)-oxo species as reactive oxidants. Copper enzymes employ still a different strategy and achieve H-atom abstraction potentially through a superoxo intermediate. This review compares and contrasts the electronic structures and reactivities of these various oxygen intermediates.     

X-ray Crystal Structure of Michaelis Complex of Aldoxime Dehydratase

Aldoxime dehydratase (Oxd) catalyzes the dehydration of aldoximes (R–CH=N–OH) to their corresponding nitrile (R–CN). Oxd is a heme-containing enzyme that catalyzes the dehydration reaction as its physiological function. We have determined the first two structures of Oxd: the substrate-free OxdRE at 1.8 Å resolution and the n-butyraldoxime- and propionaldoxime-bound OxdREs at 1.8 and 1.6 Å resolutions, respectively. Unlike other heme enzymes, the organic substrate is directly bound to the heme iron in OxdRE. We determined the structure of the Michaelis complex of OxdRE by using the unique substrate binding and activity regulation properties of Oxd. The Michaelis complex was prepared by x-ray cryoradiolytic reduction of the ferric dead-end complex in which Oxd contains a Fe³⁺ heme form. The crystal structures reveal the mechanism of substrate recognition and the catalysis of OxdRE.     

Purification and Characterization of the Alkene Monooxygenase from Nocardia corallina B-276

Alkene monooxygenase from the propene utilizer Nocardia corallina B-276 was separated into three components, and all components were purified to homogeneity and their properties were examined. The epoxidase, with a molecular mass of 95 kDa, was considered to catalyze the oxidation of the substrate propene to propylene oxide. It consisted of 53- and 35-kDa subunits, which contained approximately 2-mol of non-heme iron per mole of protein. The reductase, molecular mass 40 kDa, was found to contain an FAD and an Fe₂ S₂ cluster. A third protein, which we have called the coupling protein, with a mass of 14 kDa, appears to function as a regulator of activity. The purified AMO system required NADH as an electron donor, and catalyzed alkene epoxidation only. Acetylene, a specific inhibitor for methane monooxygenase, did not inhibit the AMO activity.     

On the redox properties of three bisulfite reductases from the sulfate-reducing bacteria.

The redox properties of purified bisulfite reductases from Desulfovibrio gigas, D. desulfuricans (Norway) and Desulfotomaculum ruminis, containing non-heme iron and siroheme have been studied by EPR spectroscopy. Each enzyme shows ferric siroheme EPR signals which are not completely reduced by dithionite after 20 min, but are readily reduced within 1 min by dithionite plus methyl viologen. With the latter reducing system, each reductase also reveals a variable Beinert “g=1.94” type iron-sulfur signal. Reaction of each reductase with reduced methyl viologen results in reduction of only the siroheme. These results suggest different redox potentials for the iron-sulfur and siroheme moieties, and indicate that their functional properties are similar for each reductase.     

Iron homeostasis: recently identified proteins provide insight into novel control mechanisms

Iron is an essential nutrient required for a variety of biochemical processes. It is a vital component of the heme in hemoglobin, myoglobin, and cytochromes and is also an essential cofactor for non-heme enzymes such as ribonucleotide reductase, the limiting enzyme for DNA synthesis. When in excess, iron is toxic because it generates superoxide anions and hydroxyl radicals that react readily with biological molecules, including proteins, lipids, and DNA. As a result, humans possess elegant control mechanisms to maintain iron homeostasis by coordinately regulating iron absorption, iron recycling, and mobilization of stored iron. Disruption of these processes causes either iron-deficient anemia or iron overload disorders. In this minireview, we focus on the roles of recently identified proteins in the regulation of iron homeostasis.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

Various physicochemical and surface properties controlling the bioactivity of cerium oxide nanoparticles

Amidst numerous emerging nanoparticles, cerium oxide nanoparticles (CNPs) possess fascinating pharmacological potential as they can be used as a therapeutic for various oxidative stress-associated chronic diseases such as cancer, inflammation and neurodegeneration due to unique redox cycling between Ce³⁺ and Ce⁴⁺ oxidation states on their surface. Lattice defects generated by the formation of Ce³⁺ ions and compensation by oxygen vacancies on CNPs surface has led to switching between CeO₂ and CeO_2–x during redox reactions making CNPs a lucrative catalytic nanoparticle capable of mimicking key natural antioxidant enzymes such as superoxide dismutase and catalase. Eventually, most of the reactive oxygen species and nitrogen species in biological system are scavenged by CNPs via an auto-regenerative mechanism in which a minimum dose can exhibit catalytic activity for a longer duration. Due to the controversial outcomes on CNPs toxicity, considerable attention has recently been drawn towards establishing relationships between the physicochemical properties of CNPs obtained by different synthesis methods and biological effects ranging from toxicity to therapeutics. Unlike non-redox active nanoparticles, variations in physicochemical properties and the surface properties of CNPs obtained from different synthesis methods can significantly affect their biological activity (inactive, antioxidant, or pro-oxidant). Moreover, these properties can influence the biological identity, cellular interactions, cellular uptake, biodistribution, and therapeutic efficiency. This review aims to highlight the critical role of various physicochemical and the surface properties of CNPs controlling their biological activity based on 165 cited references.     

Proteome wide identification of iron binding proteins of <Emphasis Type="Italic">Xanthomonas translucens</Emphasis> pv. <Emphasis Type="Italic">undulosa</Emphasis>: focus on secretory virulent proteins

Xanthomonas translucens pv. undulosa (Xtu) causes Bacterial Leaf Streak disease in the staple food crops such as wheat and barley. The survival strategies of pathogen and host are determined by the complex interactions occurring between the host plants and the pathogenic microbes. Iron binding proteins are important in the plant–microbe interactions as they are indulged in enzyme catalysis, virulence, metabolic and transport activities. In the presented study, we have identified that ~9.8% of Xtu proteome possess iron binding sequence motifs. Further, the analysis of Xtu proteome for secretory iron binding virulent proteins (IBVPs) revealed the fact that iron co-regulate the function of secretory proteins in virulence. We have found 26 secretory IBVPs and observed that these proteins are diverse in their biological functions ranging from transport to antimicrobial resistance, Reactive oxygen species detoxification and carbohydrate catabolism. The inferences may instigate to design the new strategies to combat and control the microbial diseases of staple food crops.