Non-heme Iron Protein Research Articles

Characterization of the active site in the thiocyanate-forming protein from Thlaspi arvense (TaTFP) using EPR spectroscopy.

Glucosinolates are plant thioglucosides, which act as chemical defenses. Upon tissue damage, their myrosinase-catalyzed hydrolysis yields aglucones that rearrange to toxic isothiocyanates. Specifier proteins such as thiocyanate-forming protein from Thlaspi arvense (TaTFP) are non-heme iron proteins, which capture the aglucone to form alternative products, e.g. nitriles or thiocyanates. To resolve the electronic state of the bound iron cofactor in TaTFP, we applied continuous wave electron paramagnetic resonance (CW EPR) spectroscopy at X-and Q-band frequencies (∼9.4 and ∼34 GHz). We found characteristic features of high spin and low spin states of a d 5 electronic configuration and local rhombic symmetry during catalysis. We monitored the oxidation states of bound iron during conversion of allylglucosinolate by myrosinase and TaTFP in presence and absence of supplemented Fe2+. Without added Fe2+, most high spin features of bound Fe3+ were preserved, while different g'-values of the low spin part indicated slight rearrangements in the coordination sphere and/or structural geometry. We also examined involvement of the redox pair Fe3+/Fe2 in samples with supplemented Fe2+. The absence of any EPR signal related to Fe3+ or Fe2+ using an iron-binding deficient TaTFP variant allowed us to conclude that recorded EPR signals originated from the bound iron cofactor.

Immunization of broiler chickens with five newly identified surface-exposed proteins unique to Clostridium perfringens causing necrotic enteritis

Since the ban or reduction on the use of antibiotic growth promoters (AGPs) in commercial broiler chickens in many countries, avian necrotic enteritis (NE) caused by Clostridium perfringens has re-emerged as one of the biggest threats for the poultry industry worldwide. While the toolbox for controlling NE in the absence of antibiotics consists of a limited number of alternatives for which the overall effectiveness has yet proven to be suboptimal, an effective vaccine would represent the best control strategy for this often-deadly disease. Using a comparative and subtractive reverse vaccinology approach, we previously identified 14 putative antigenic proteins unique to NE-causing strains of C. perfringens. In the current work, the in silico findings were confirmed by PCR and sequencing, and five vaccine candidate proteins were produced and purified subsequently. Among them, two candidates were hypothetical proteins, two candidates were prepilin proteins which are predicted to form the subunits of a pilus structure, and one candidate was a non-heme iron protein. Western blotting and ELISA results showed that immunization of broiler chickens with five of these proteins raised antibodies which can specifically recognize both the recombinant and native forms of the protein in pathogenic C. perfringens.

Review of a Few Selected Examples of Intermolecular Dioxygenases Involving Molecular Oxygen and Non-Heme Iron Proteins

Molecular oxygen and non-heme iron proteins (NHIPs) are proteins that have an iron atom bound to one or more oxygen atoms, and are found in bacteria, archaea, and some eukaryotes. NHIPs are involved in a variety of biological processes, such as respiration, electron transfer, and catalysis. They can be categorized into two classes: cytochrome proteins, which are involved in electron transfer, and oxygenases, which catalyze the oxidation of substrates by utilizing molecular oxygen. NHIPs are critical for multiple biological processes, and their impairment has been associated with diseases such as anemia, cancer, and neurological disorders.

Iron deficiency exacerbates cisplatin- or rhabdomyolysis-induced acute kidney injury through promoting iron-catalyzed oxidative damage

Iron deficiency is the most common micronutrient deficiency worldwide. While iron deficiency is known to suppress embryonic organogenesis, its effect on the adult organ in the context of clinically relevant damage has not been considered. Here we report that iron deficiency is a risk factor for nephrotoxic intrinsic acute kidney injury of the nephron (iAKI). Iron deficiency exacerbated cisplatin-induced iAKI by markedly increasing non-heme catalytic iron and Nox4 protein which together catalyze production of hydroxyl radicals followed by protein and DNA oxidation, apoptosis and ferroptosis. Crosstalk between non-heme catalytic iron/Nox4 and downstream oxidative damage generated a mutual amplification cycle that facilitated rapid progression of cisplatin-induced iAKI. Iron deficiency also exacerbated a second model of iAKI, rhabdomyolysis, via increasing catalytic heme-iron. Heme-iron induced lipid peroxidation and DNA oxidation by interacting with Nox4-independent mechanisms, promoting p53/p21 activity and cellular senescence. Our data suggests that correcting iron deficiency and/or targeting specific catalytic iron species are strategies to mitigate iAKI in a wide range of patients with diverse forms of kidney injury.

Stepwise nitrosylation of the nonheme iron site in an engineered azurin and a molecular basis for nitric oxide signaling mediated by nonheme iron proteins.

Mononitrosyl and dinitrosyl iron species, such as {FeNO}7, {FeNO}8 and {Fe(NO)2}9, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO}7 and {Fe(NO)2}9 species was achieved, and the elusive {FeNO}8 species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO}7 to {Fe(NO)2}9 species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO}7 → {FeNO}8 → {Fe(NO)2}9, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins.

Characterization of phthalate reductase from Ralstonia eutropha CH34 and in silico study of phthalate dioxygenase and phthalate reductase interaction

Microbial degradation is a natural means to combat ubiquitous, harmful and non-covalently bound plasticizers, phthalic acid esters (PAEs), due to their slower photo-degradation and hydrolysis rates. In microbial aerobic degradation of phthalates, phthalate dioxygenase system (PDS) belongs to a large family of Rieske non-heme iron oxygenases with type1A electron-transfer system. Two important proteins of this system include: phthalate dioxygenase reductase (PDR), a flavo-iron-sulfur protein having NADH-dependent oxidoreductase activity; and phthalate dioxygenase oxygenase (PDO), a non-heme iron protein with oxygenase activity. In the present study, phthalate dioxygenase reductase (RePDR) from Ralstonia eutropha CH34, has been cloned, expressed and purified. Further, in order to understand the interactions between RePDO and RePDR, protein-protein docking has been carried out. The homology model structures of the individual proteins served as an input for protein-protein interaction studies and helps in the prediction of complex structure. Important interface residues from both the individual counterparts, i.e. RePDO and RePDR have been identified and the evolutionary relationship between these important interface residues has also been established. Moreover, the stability of these interactions involving highly conserved interface residues, highlights their involvement in structurally conserved interactions and play significant role in phthalate degrading system.

Renal Iron Accumulation and Oxidative Injury With Aging: Effects of Treatment With an Iron Chelator.

Dysregulation of iron metabolism in the kidney may contribute to age-related increases in renal oxidative stress and dysfunction. This study assessed the effects of short-term iron chelation on markers of iron status, oxidative stress, inflammation, and autophagy in the kidneys of old rats. Old Fischer 344 rats (24 months) were treated with deferoxamine (DFO; 200 mg/kg, twice daily for 4.5 days); saline-treated young (6 months) and old rats served as controls. Renal nonheme iron was significantly higher in the old rats, with iron localized in the renal cortex. Ferritin levels were elevated in the kidneys of old rats, while expression of several antioxidant enzymes and mitochondrial proteins were reduced and protein carbonyls increased compared to young rats. DFO treatment significantly reduced ferritin levels, and increased transferrin receptor-1 protein, but did not affect nonheme iron content or protein carbonyls, nor did it reverse age-related changes in antioxidant enzymes and mitochondrial proteins. Although short-term DFO treatment did not mitigate the age-related increase in iron content and oxidative damage, this work demonstrates that old rats respond appropriately to DFO, suggesting that optimization of iron chelation regimens could be useful in improving renal homeostasis with aging.

Direct and Mediated Spectro-Electrochemistry of Highly Oxidized Heme Species in Horseradish Peroxidase

Highly oxidized iron species play key role in the catalysis of many heme and non-heme enzymes due to their ability to activate substrates via electron or hydrogen atom transfer. Their high reactivity also makes them unstable and typically transient in nature, which hinders their structural and mechanistic investigation. Furthermore, generation of ferryloxo states typically requires addition of either oxygen and reductants or partially reduced oxygen equivalents, followed by irreversible oxygen activation. This unidirectional reaction effectively excludes investigation of associated structural changes in the protein moiety by such sensitive techniques as infrared spectroelectrochemistry, for example. Here we report on our effort to develop novel approaches toward reversible generation of ferryloxo species in horseradish peroxidase and other heme and non-heme iron proteins. We demonstrate reversible, sequential formation and quenching of Compounds I and II of HRP using several mediators in optically transparent thin layer electrochemical cell and a novel bulk optical transmission electrochemical cell. We also show that pre-conditioning of carbon-based electrodes enables generation of highly oxidized species without the use of mediators or oxygen. Following predictions of kinetically limited pre-equilibrium computational modelling and taking advantage of our chemical-free approach, we investigated the mechanisms of spontaneous quenching of highly oxidized species in HRP, the role of solvent, and the effect of pH, which yielded an unexpected result. General rules and potential pitfalls in electrochemistry of highly oxidized species in metalloenzymes will be outlined. Figure 1

Phylogenomic analysis of integral diiron membrane histidine motif-containing enzymes in ciliates provides insights into their function and evolutionary relationships

The Integral Membrane Histidine Motif-containing Enzymes (IMHME) are a class of binuclear non-heme iron proteins widely distributed among prokaryotes and eukaryotes. They are characterized by a conserved tripartite motif consisting of eight to ten histidine residues. Their known function is the activation of the dioxygen moiety to serve as efficient catalysts for reactions of hydroxylation, desaturation or reduction. To date most studies on IMHME were carried out in metazoan, phototrophic or parasitic organisms, whereas genome-wide analysis in heterotrophic free living protozoa, such as the Ciliophora phylum, has not been undertaken. In the seven fully sequenced genomes available we retrieved 118 putative sequences of the IMHME type, albeit with large differences in number among the ciliates: 11 sequences in Euplotes octocarinatus, 7 in Ichthyophthirius multifiliis, 13 in Oxytricha trifallax, 18 in Stylonychia lemnae, 25 in Tetrahymena thermophila, 31 in Paramecium tetraurelia and 13 in Pseudocohnilembus persalinus.The pool of putative sequences was classified in 16 orthologous groups from which 11 were related to fatty acid desaturase (FAD) and 5 to the fatty acid hydroxylase (FAH) superfamilies. Noteworthy, a large diversity on the number and type of FAD / FAH proteins were found among the ciliates, a feature that, in principle, may be attributed to peculiarities of the evolutionary process, such as gene expansion and reduction, but also to horizontal gene transfer, as we demonstrate in this work. We identified twelve putative enzymatic activities, from which four were newly assigned activities: sphingolipid Δ4-desaturase, ω3/Δ15 fatty acid desaturase, a large group of alkane 1-monooxygenases, and acylamide-delta-3(E)-desaturase, although unequivocal allocation would require additional experiments. We also combined the phylogenetics analysis with lipids analysis, thereby allowing the detection of two enzymatic activities not previously reported: a C-5 sterol desaturase in P. tetraurelia and a delta-9 fatty acid desaturase in Cohnilembus reniformis.The analysis revealed a significant lower number of FAD’s sequences in the spirotrichea ciliates than in the oligohymenophorea, emphasizing the importance of fatty acids trophic transfer among aquatic organisms as a source of variation in metabolic activity, individual and population growth rates, and reproduction.

Hepatic iron storage is related to body adiposity and hepatic inflammation

BackgroundObesity has been reported to be associated with iron deficiency. However, few studies have investigated iron status in low adiposity. To investigate whether body adiposity was associated with altered hepatic iron status, we compared liver iron levels and markers involved in inflammation and iron absorption in obese, control, and mildly calorie restricted mice.MethodsSeven week old C57BL/6 mice were fed control (10% kcal fat, Control) or high fat (60% kcal fat, HFD) diets, or reduced amount of control diet to achieve 15% calorie restriction (CR) for 16 weeks. Hepatic non-heme iron content and ferritin protein level, and hematocrit and hemoglobin levels were determined to assess iron status. Hepatic expression of Mcp-1 and Tnf-α were measured as hepatic inflammatory markers. Hepatic hepcidin (Hamp) and Bmp6, and duodenal Dmt1, Dcyt1b, hephaestin (Heph) and ferroportin mRNA levels were measured as factors involved in regulation of iron absorption.ResultsHepatic non-heme iron and ferritin protein levels were significantly higher in the CR group compared with the Control group, and significantly lower in the HFD group. These two iron status markers showed significantly negative correlations with the amount of white adipose tissue (r = -0.689 for hepatic non-heme iron and r = -0.740 for ferritin). Hepatic Mcp-1 and Tnf-α mRNA levels were significantly lower in the CR compared with the HFD (74 and 47% lower) and showed significantly negative correlations with hepatic non-heme iron levels (Mcp-1: r = -0.557, P < 0.05; Tnf-α: r = -0.464, P < 0.05). Hepatic Hamp mRNA levels were lower in the HFD and higher in the CR groups compared with the Control group, which could be a response to maintain iron homeostasis. Duodenal Dcyt1b mRNA levels were higher in the CR group compared with the HFD group and duodenal Heph mRNA levels were higher in the CR group than the Control group.ConclusionWe showed that body adiposity was inversely correlated with liver iron status. Low inflammation levels in hepatic milieu and enhanced expression of duodenal oxidoreductases induced by calorie restriction could have contributed to higher iron status.

Determinants of Fe2+ over M2+ (M = Mg, Mn, Zn) Selectivity in Non-Heme Iron Proteins.

Iron cations are indispensable players in a number of vital biological processes such as respiration, cell division, nitrogen fixation, oxygen transport, nucleotide synthesis, oxidant protection, O2 activation in the metabolism of various organic substrates, gene regulation, and protein structure stabilization. The basic mechanisms and factors governing the competition between Fe2+ and other metal species from the cellular fluids such as Mg2+, Mn2+, and Zn2+ are, however, not well understood, and several outstanding questions remain. (i) How does the Fe2+ binding site select the "right" cation and protect itself from attacks by other biogenic cations present in the surrounding milieu? (ii) Do the iron binding sites employ different selectivity strategies toward metal cations possessing different ligand affinities and cytosolic concentrations? (iii) What are the key determinants of metal selectivity in Fe2+ proteins? In this study, by employing density functional theory calculations combined with polarizable continuum model computations, we endeavor to address these questions by evaluating the thermodynamic outcome of the competition between Fe2+ and Mg2+/Mn2+/Zn2+ in model non-heme mononuclear metal binding sites of various compositions and charge states. The present calculations, which are in line with available experimental data, shed light on the mechanism of Fe2+-Mg2+/Mn2+/Zn2+ competition in non-heme iron proteins and disclose the key factors governing the process.

57 - Succinate Is a Mitochondrial Retrograde Signal That Links MnSOD Depletion to Loss of Epigenetic Control and Aberrant Genomic DNA Methylation

Mitochondrial superoxide dismutase activity MnSOD is necessary for optimal function of the electron transport chain (ETC) and certain functions of the tricarboxylic acid (TCA) cycle. Alterations in the expression of MnSOD have been reported in association with a variety of pathophysiological conditions including cancer, cardiovascular disease, diabetes and aging. These diseases are often accompanied by aberrant epigenetic control of normal gene expression patterns, however to date there has not been a clear mechanistic link between alterations in MnSOD expression/activity and disruption of epigenetic control. Our results and those of others indicate that loss of MnSOD inhibits not only complex I activity, but also complex II activity, likely by O 2 ●– disruption of the Fe-S cluster(s) in the SDHB subunit. Complex II is the intersection point of the ETC and TCA since it also serves as succinate dehydrogenase (SDH), converting succinate to fumarate in the TCA cycle in the mitochondrial matrix. Several enzymes that alter the epigenome, particularly histone and DNA demethylases including the TET family of 5-methyl cytosine (5mC) hydroxylases, are α-ketoglutarate dependent dioxygenases and non-heme iron proteins. Notably, these enzymes are product-inhibited by excess succinate as well as by hypoxia. Thus, we hypothesized that alterations in nuclear epigenetic regulatory mechanisms subsequent to mitochondrial MnSOD depletion in human cells were mediated by cellular succinate accumulation and inhibition of nuclear demethylases. In both HEK293 (human embryonic kidney) and HEL (human erythroid leukemia) cells depleted of MnSOD activity using CRISPR/Cas9 compared to their parent control cells we observed ~90% inhibition of SDH activity, ~5-fold increases in succinate levels, ~50% inhibition of TET activity, significantly decreased levels of genomic 5hmC (5-hydroxymethyl cytosine), and ~30% increases in total genomic 5mC. Taken together, these findings provide a clear mechanistic link between alterations of MnSOD activity that occur in cancer and other human diseases with aberrant regulation of nuclear epigenetic programming and a DNA hypermethylator phenotype.

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase

Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.

Iron management and production of electricity by microorganisms.

The increasing dependency on fossil fuels has driven researchers to seek for alternative energy sources. Renewable energy sources such as sunlight, wind, or water are the most common. However, since the 1990s, other sources for energy production have been studied. The use of microorganisms such as bacteria or archaea to produce energy is currently in great progress. These present several advantages even when compared with other renewable energy sources. Besides the energy production, they are also involved in bioremediation such as the removal of heavy metal contaminants from soils or wastewaters. Several research groups have demonstrated that these organisms are able to interact with electrodes via heme and non-heme iron proteins. Therefore, the role of iron as well as iron metabolism in these species must be of enormous relevance. Recently, the influence of cellular iron regulation by Fur in the Geobacter sulfurreducens growth and ability to produce energy was demonstrated. In this review, we aim to briefly describe the most relevant proteins involved in the iron metabolism of bacteria and archaea and relate them and their biological function with the ability of selected organisms to produce energy.

Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin.

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

Formation of high-valent iron-oxo species in superoxide reductase: characterization by resonance Raman spectroscopy.

Superoxide reductase (SOR), a non-heme mononuclear iron protein that is involved in superoxide detoxification in microorganisms, can be used as an unprecedented model to study the mechanisms of O2 activation and of the formation of high-valent iron-oxo species in metalloenzymes. By using resonance Raman spectroscopy, it was shown that the mutation of two residues in the second coordination sphere of the SOR iron active site, K48 and I118, led to the formation of a high-valent iron-oxo species when the mutant proteins were reacted with H2O2. These data demonstrate that these residues in the second coordination sphere tightly control the evolution and the cleavage of the O-O bond of the ferric iron hydroperoxide intermediate that is formed in the SOR active site.

Regulation of CuZnSOD and Its Redox Signaling Potential: Implications for Amyotrophic Lateral Sclerosis

Molecular oxygen is a Janus-faced electron acceptor for biological systems, serving as a reductant for respiration, or as the genesis for oxygen-derived free radicals that damage macromolecules. Superoxide is well known to perturb nonheme iron proteins, including Fe/S proteins such as aconitase and succinate dehydrogenase, as well as other enzymes containing labile iron such as the prolyl hydroxylase domain-containing family of enzymes; whereas hydrogen peroxide is more specific for two-electron reactions with thiols on glutathione, glutaredoxin, thioredoxin, and the peroxiredoxins. Over the past two decades, familial cases of amyotrophic lateral sclerosis (ALS) have been shown to have an association with commonly altered superoxide dismutase 1 (SOD1) activity, expression, and protein structure. This has led to speculation that an altered redox balance may have a role in creating the ALS phenotype. While SOD1 alterations in familial ALS are manifold, they generally create perturbations in the flux of electrons. The nexus of SOD1 between one- and two-electron signaling processes places it at a key signaling regulatory checkpoint for governing cellular responses to physiological and environmental cues. The manner in which ALS-associated mutations adjust SOD1's role in controlling the flow of electrons between one- and two-electron signaling processes remains obscure. Here, we discuss the ways in which SOD1 mutations influence the form and function of copper zinc SOD, the consequences of these alterations on free radical biology, and how these alterations might influence cell signaling during the onset of ALS.

Cysteine Dioxygenase Structures from pH 4 to 9: Consistent Cys-Persulfenate Formation at Intermediate pH and a Cys-Bound Enzyme at Higher pH

Mammalian cysteine dioxygenase (CDO) is a mononuclear non-heme iron protein that catalyzes the conversion of cysteine (Cys) to cysteine sulfinic acid by an unclarified mechanism. One structural study revealed that a Cys-persulfenate (or Cys-persulfenic acid) formed in the active site, but quantum mechanical calculations have been used to support arguments that it is not an energetically feasible reaction intermediate. Here, we report a series of high-resolution structures of CDO soaked with Cys at pH values from 4 to 9. Cys binding is minimal at pH≤5 and persulfenate formation is consistently seen at pH values between 5.5 and 7. Also, a structure determined using laboratory-based X-ray diffraction shows that the persulfenate, with an apparent average O–O separation distance of ~1.8Å, is not an artifact of synchrotron radiation. At pH≥8, the active-site iron shifts from 4- to 5-coordinate, and Cys soaks reveal a complex with Cys, but no dioxygen, bound. This ‘Cys-only’ complex differs in detail from a previously published ‘Cys-only’ complex, which we reevaluate and conclude is not reliable. The high-resolution structures presented here do not resolve the CDO mechanism but do imply that an iron-bound persulfenate (or persulfenic acid) is energetically accessible in the CDO active site, and that CDO active-site chemistry in the crystals is influenced by protonation/deprotonation events with effective pKa values near ~5.5 and ~7.5 that influence Cys binding and oxygen binding/reactivity, respectively. Furthermore, this work provides reliable ligand-bound models for guiding future mechanistic considerations.

Inhibition of Soybean Lipoxygenase‐1 by N‐oleoyl‐Dtryptophan

Soybean lipoxygenase‐1 (SBLO‐1) is a nonheme iron protein that catalyzes the oxygenation of polyunsaturated fatty acids using molecular oxygen, resulting in conjugated diene hydroperoxides. Although the three‐dimensional structure of SBLO‐1 is known, it is not certain how substrates bind. Following the discovery that N‐linoleoyl‐ D‐tryptophan is an excellent substrate for SBLO‐1, N‐oleoyl‐ D‐tryptophan (ODT) was synthesized as a potential competitive inhibitor. Potent inhibition was observed at micromolar concentrations of ODT. Kinetic analysis demonstrated that ODT had only small effects on Vmax, indicating that the inhibition is nearly competitive; however, the inhibition is strikingly non‐linear, which implies that OTD and substrate can bind simultaneously. Similar behavior was observed with N‐oleoyl‐ L‐tryptophan and N‐oleoyl‐D‐phenylalanine. Slow oxygenation of ODT was detected by HPLC, which indicates that ODT can access the active site. However, the inhibition kinetics imply that ODT binds more effectively somewhere else. Funded by NSF grant CHE‐1213262.

A Novel Enzymatic System against Oxidative Stress in the Thermophilic Hydrogen-Oxidizing Bacterium Hydrogenobacter thermophilus

Rubrerythrin (Rbr) is a non-heme iron protein composed of two distinctive domains and functions as a peroxidase in anaerobic organisms. A novel Rbr-like protein, ferriperoxin (Fpx), was identified in Hydrogenobacter thermophilus and was found not to possess the rubredoxin-like domain that is present in typical Rbrs. Although this protein is widely distributed among aerobic organisms, its function remains unknown. In this study, Fpx exhibited ferredoxin:NADPH oxidoreductase (FNR)-dependent peroxidase activity and reduced both hydrogen peroxide (H2O2) and organic hydroperoxide in the presence of NADPH and FNR as electron donors. The calculated Km and V max values of Fpx for organic hydroperoxides were comparable to that for H2O2, demonstrating a multiple reactivity of Fpx towards hydroperoxides. An fpx gene disruptant was unable to grow under aerobic conditions, whereas its growth profiles were comparable to those of the wild-type strain under anaerobic and microaerobic conditions, clearly indicating the indispensability of Fpx as an antioxidant of H. thermophilus in aerobic environments. Structural analysis suggested that domain-swapping occurs in Fpx, and this domain-swapped structure is well conserved among thermophiles, implying the importance of structural stability of domain-swapped conformation for thermal environments. In addition, Fpx was located on a deep branch of the phylogenetic tree of Rbr and Rbr-like proteins. This finding, taken together with the wide distribution of Fpx among Bacteria and Archaea, suggests that Fpx is an ancestral type of Rbr homolog that functions as an essential antioxidant and may be part of an ancestral peroxide-detoxification system.