Diiron Center Research Articles

Diverse YqeK Diadenosine Tetraphosphate Hydrolases Control Biofilm Formation in an Iron-Dependent Manner

YqeK is a bacterial HD-domain metalloprotein that hydrolyzes the putative second messenger diadenosine tetraphosphate (Ap4A). Elevated Ap4A levels are primarily observed upon exposure of bacteria to factors such as heat or oxidative stress and cause pleiotropic effects, including antibiotic sensitivity and disrupted biofilm formation. Ap4A thus plays a central role in bacterial physiology and metabolism, and its hydrolysis by YqeK is intimately linked to the ability of these microbes to cope with stress. Although YqeK is reported to hydrolyze Ap4A under aerobic conditions, all four existing crystal structures reveal an active site that consists of a diiron center, portraying a cryptic chemical nature for the active metallocofactor. This study examines two YqeK proteins from two ecologically diverse parent organisms: the obligate anaerobe Clostridium acetobutylicum and the facultative aerobe Bacillus halodurans. Both enzymes utilize Fe-based cofactors for catalysis, while under ambient or oxidative conditions, Bh YqeK hydrolyzes Ap4A more efficiently compared to Ca YqeK. This redox-dependent activity difference stems from the following two molecular mechanisms: the incorporation of mixed-metal, Fe-based bimetallic cofactors, in which the second metal is redox inert (i.e., Fe–Zn) and the upshift of the Fe–Fe cofactor reduction potentials. In addition, three strictly conserved, positively charged residues vicinal to the active site are critical for tuning Ap4A hydrolysis. In conclusion, YqeK is an Fe-dependent phosphohydrolase that appears to have evolved to permit Ap4A hydrolysis under different environmental niches (aerobic vs. anaerobic) by expanding its cofactor configuration and O2 tolerance.

Tertiary phosphine‐supported diiron model complexes relevant to [FeFe]‐hydrogenases

In an effort to better probe the structure–function relationship on the biological [Fe4S4] cluster of [FeFe]‐hydrogenases, a new library of tertiary phosphine‐supported diiron dithiolate complexes [Fe2(μ‐adtNR)(CO)5{P(C6H4X)3}] (1–3) with various substituents (X = F vs. H vs. Me), wherein adtNR is denoted as abbreviation for the (SCH2)2N(C6H4C2H4OC(O)CH2C6H4Me) unit, is prepared as the active site mimics of [FeFe]‐hydrogenases. Notably, the substituent influences of tertiary phosphines on the structures and electrochemical behaviors of 1–3 are studied by spectroscopic and crystallographic techniques as well as cyclic voltammetry. For example, the electronic effect of tertiary phosphines [P(C6H4X)3] is responsible for the electron density around diiron center and the key Fe–Fe as well as Fe–P lengths in 1–3 as revealed by IR spectroscopy and crystallographic analysis. Further, complexes 1–3 show the electrochemical activity for proton reduction into molecular hydrogen (H2) using acetic acid (AcOH) as a proton source. Noticeably, F‐substituent‐containing diiron model complex 1 exhibits greater turnover frequency (i.e., turnover frequency value) and similar overpotential for electrocatalytic H2 evolution relative to H‐ or Me‐substituent‐bearing diiron homologs 2 or 3.

Construction and Function of Thiolate-Bridged Diiron NxHy Nitrogenase Model Complexes.

ConspectusBiological nitrogen fixation mediated by nitrogenases has garnered significant research interest due to its critical importance to the development of efficient catalysts for mild ammonia synthesis. Although the active center of the most studied FeMo-nitrogenases has been determined to be a complicated [Fe7S9MoC] hetero-multinuclear metal-sulfur cluster known as the FeMo-cofactor, the exact binding site and reduction pathway of N2 remain a subject of debate. Over the past decades, the majority of studies have focused on mononuclear molybdenum or iron centers as potential reaction sites. In stark contrast, cooperative activation of N2 through bi- or multimetallic centers has been largely overlooked and underexplored, despite the renewed interest sparked by recent biochemical and computational studies. Consequently, constructing bioinspired bi- or multinuclear metallic model complexes presents an intriguing yet challenging prospect. In this Account, we detail our long-standing research on the design and synthesis of novel thiolate-bridged diiron complexes as nitrogenase models and their application to chemical simulations of potential biological N2 reduction pathways.Inspired by the structural and electronic features of the potential diiron active center in the belt region of the FeMo-cofactor, we have designed and synthesized a series of new thiolate-bridged diiron nitrogenase model complexes, wherein iron centers with +2 or +3 oxidation states are coordinated by Cp* as carbon-based donors and thiolate ligands as sulfur donors. Through the synergistic interaction between the two iron centers, unstable diazene (NH═NH) species can be trapped to generate the first example of a [Fe2S2]-type complex bearing a cis-μ-η1:η1-NH═NH subunit. Significantly, this species can not only catalyze the reductive N-N bond cleavage of hydrazine to ammonia but also trigger a stepwise reduction sequence NH═NH → [NH2-NH]- → [NH]2-(+NH3) → [NH2]- → NH3. Furthermore, an unprecedented thiolate-bridged diiron μ-nitride featuring a bent Fe-N-Fe moiety was successfully isolated and structurally characterized. Importantly, this diiron μ-nitride can undergo successive proton-coupled electron transfer processes to efficiently release ammonia in the presence of separate protons and electrons and can even be directly hydrogenated using H2 as a combination of protons and electrons for high-yield ammonia formation. Based on combined experimental and computational studies, we proposed two distinct reductive transformation sequences on the diiron centers, which involve a series of crucial NxHy intermediates. Moreover, we also achieved catalytic N2 reduction to silylamines with [Fe2S2]-type complexes by ligand modulation.Our bioinspired diiron cooperative scaffold may provide a suitable model for probing the potential N2 stepwise reduction pathways from the molecular level. Different from the traditional alternating and distal pathways dominated by mononuclear iron or molybdenum complexes, our proposed alternating transformation route based on the diiron centers may not involve the N2H4 intermediate, and the convergence point of the alternating and terminal pathways is imide, not amide. Our research strategy could inform the design and development of new types of bioinspired catalysts for mild and efficient nitrogen reduction in the future.

Pseudomonas aeruginosa gene PA4880 encodes a Dps-like protein with a Dps fold, bacterioferritin-type ferroxidase centers, and endonuclease activity.

We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases.

Origin of Unprecedented Formation and Reactivity of FeIV═O Species via Oxygen Activation: Role of Noncovalent Interactions and Magnetic Coupling.

Emulating the capabilities of the soluble methane monooxygenase (sMMO) enzymes, which effortlessly activate oxygen at diiron(II) centers to form a reactive diiron(IV) intermediate Q, which then performs the challenging oxidation of methane to methanol, poses a significant challenge. Very recently, one of us reported the mononuclear complex [(cyclam)FeII(CH3CN)2]2+ (1), which performed a rare bimolecular activation of the molecule of O2 to generate two molecules of FeIV═O without the requirement of external proton or electron sources, similar to sMMO. In the present study, we employed the density functional theory (DFT) calculations to investigate this unique mechanism of O2 activation. We show that secondary hydrogen-bonding interactions between ligand N-H groups and O2 play a vital role in reducing the energy barrier associated with the initial O2 binding at 1 and O-O bond cleavage to form the FeIV═O complex. Further, the unique reactivity of FeIV═O species toward simultaneous C-H and O-H bond activation process has been demonstrated. Our study unveils that the nature of the magnetic coupling between the diiron centers is also crucial. Given that the influence of magnetic coupling and noncovalent interactions in catalysis remains largely unexplored, this unexplored realm presents numerous avenues for experimental chemists to develop novel structural and functional analogues of sMMO.

A selective discussion on oxo-bridged diiron (III,III) centers in chemistry related to bio-functional activity

Diiron (III, III) oxo- bridged core is playing significant role in various biologically active compounds. Several synthetic diiron compounds are prepared which are the structural and functional models of these bio active centers. Extensive study over these compounds helps us to gain an idea about the mechanistic pathways adopted in the bio active centers. In vivo studies are highly expensive and critical. Analysis of these complexes outside the bio-environment is comparatively simple but give us an idea about the mechanistic overview of the metal centers which guide us for further proceedings.

A Review on Biologically Active oxo-bridged diiron (III,III) centers

Particular metal ions played vital role in functioning of enzymes. In this following discussion we concentrate over oxo bridged diiron III,III centers. Fe–O–Fe is playing important role in many enzymes like hemerythrin, ribonucleotide reductase, methane monooxygenase etc. Presence of metal ions in active centers may facilitate the electron transfer process.

Structural Insights into the Reaction between Hydrogen Peroxide and Di-iron Complexes at the Ferroxidase Center of Ferritin.

The Fe(II) oxidation mechanism in the ferroxidase center of heavy chain ferritin has been studied extensively. However, the actual production of H2O2 was found to be substantially lower than expected at low flux of Fe(II) to ferritin subunits. Here, we demonstrated that H2O2 could interact with the di-iron nuclear center, leading to the production of hydroxyl radicals and oxygen. Two reaction intermediates were captured in the ferroxidase center by using the time-lapse crystallographic techniques in a shellfish ferritin. The crystal structures revealed the binding of H2O2 as a μ -1,2-peroxo-diferric species and the binding of O2 to the diferric structure. This investigation sheds light on the reaction between the di-iron nuclear center and H2O2 and provides insights for the exploitation of metalloenzymes.

Structural insights into the initiation of free radical formation in the Class Ib ribonucleotide reductases in Mycobacteria

Class I ribonucleotide reductases consisting of α and β subunits convert ribonucleoside diphosphates to deoxyribonucleoside diphosphates involving an intricate free radical mechanism. The generation of free radicals in the Class Ib ribonucleotide reductases is mediated by di-manganese ions in the β subunits and is externally assisted by flavodoxin-like NrdI subunit. This is unlike Class Ia ribonucleotide reductases, where the free radical generation is initiated at its di-iron centre in the β subunits with no external support from another subunit. Class 1b ribonucleotide reductase complex is an essential enzyme complex in the human pathogen Mycobacterium tuberculosis and its structural details are largely unknown. In this study we have determined the crystal structures of Mycobacterial NrdI in oxidised and reduced forms, and similarly those of NrdF2:NrdI complexes. These structures provide detailed atomic view of the mechanism of free radical generation in the β subunit in this pathogen. We observe a well-formed channel in NrdI from the surface leading to the buried FMN moiety and propose that oxygen molecule accesses FMN through it. The oxygen molecule is further converted to a superoxide ion upon electron transfer at the FMN moiety. Similarly, a path for superoxide radical transfer between NrdI and NrdF2 is also observed. The oxidation of Mn(II) in NrdF2I to high valent oxidation state (either Mn(III) or Mn(IV) assisted by the reduced FMN site was evidently confirmed by EPR studies. SEC-MALS and low resolution cryo-EM map indicate unusual stoichiometry of 2:1 in the M. tuberculosis NrdF2I complex. A density close to Tyr 110 at a distance <2.3 Å is observed, which we interpret as OH group. Overall, the study therefore provides important clues on the initiation of free radical generation in the β subunit of the ribonucleotide reductase complex in M. tuberculosis.

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

Methanol on the rocks: green rust transformation promotes the oxidation of methane.

Shared coordination geometries between metal ions within reactive minerals and enzymatic metal cofactors hints at mechanistic and possibly evolutionary homology between particular abiotic chemical mineralogies and biological metabolism. The octahedral coordination of reactive Fe2+/3+ minerals such as green rusts, endemic to anoxic sediments and the early Earth's oceans, mirrors the di-iron reaction centre of soluble methane monooxygenase (sMMO), responsible for methane oxidation in methanotrophy. We show that methane oxidation occurs in tandem with the oxidation of green rust to lepidocrocite and magnetite, mimicking radical-mediated methane oxidation found in sMMO to yield not only methanol but also halogenated hydrocarbons in the presence of seawater. This naturally occurring geochemical pathway for CH4 oxidation elucidates a previously unidentified carbon cycling mechanism in modern and ancient environments and reveals clues into mineral-mediated reactions in the synthesis of organic compounds necessary for the emergence of life.

Bacterial nitric oxide reductase (NorBC) models employing click chemistry

Bacterial NO Reductase (NorBC or cNOR) is a membrane-bound enzyme found in denitrifying bacteria that catalyzes the two-electron reduction of NO to N2O and water. The mechanism by which NorBC operates is highly debated, due to the fact that this enzyme is difficult to work with, and no intermediates of the NO reduction reaction could have been identified so far. The unique active site of NorBC consists of a heme b3/non-heme FeB diiron center. Synthetic model complexes provide the opportunity to obtain insight into possible mechanistic alternatives for this enzyme. In this paper, we present three new synthetic model systems for NorBC, consisting of a tetraphenylporphyrin-derivative clicked to modified BMPA-based ligands (BMPA = bis(methylpyridyl)amine) that model the non-heme site in the enzyme. These complexes have been characterized by EPR, IR and UV–Vis spectroscopy. The reactivity with NO was then investigated, and it was found that the complex with the BMPA-carboxylate ligand as the non-heme component has a very low affinity for NO at the non-heme iron site. If the carboxylate functional group is replaced with a phenolate or pyridine group, reactivity is restored and formation of a diiron dinitrosyl complex was observed. Upon one-electron reduction of the nitrosylated complexes, following the semireduced pathway for NO reduction, formation of dinitrosyl iron complexes (DNICs) was observed in all three cases, but no N2O could be detected.

Free ferrous ions sustain activity of mammalian stearoyl-CoA desaturase-1

Mammalian stearoyl-CoA desaturase-1 (SCD1) introduces a double-bond to a saturated long-chain fatty acid in a reaction catalyzed by a diiron center. The diiron center is well-coordinated by conserved histidine residues and is thought to remain with the enzyme. However, we find here that SCD1 progressively loses its activity during catalysis and becomes fully inactive after about nine turnovers. Further studies show that the inactivation of SCD1 is due to the loss of an iron (Fe) ion in the diiron center and that the addition of free ferrous ions (Fe2+) sustains the enzymatic activity. Using SCD1 labeled with Fe isotope, we further show that free Fe2+ is incorporated into the diiron center only during catalysis. We also discover that the diiron center in SCD1 has prominent electron paramagnetic resonance signals in its diferric state, indicative of distinct coupling between the two ferric ions. These results reveal that the diiron center in SCD1 is structurally dynamic during catalysis and that labile Fe2+ in cells could regulate SCD1 activity and hence lipid metabolism.

Structure and mechanism of the alkane-oxidizing enzyme AlkB

Alkanes are the most energy-rich form of carbon and are widely dispersed in the environment. Their transformation by microbes represents a key step in the global carbon cycle. Alkane monooxygenase (AlkB), a membrane-spanning metalloenzyme, converts straight chain alkanes to alcohols in the first step of the microbially-mediated degradation of alkanes, thereby playing a critical role in the global cycling of carbon and the bioremediation of oil. AlkB biodiversity is attributed to its ability to oxidize alkanes of various chain lengths, while individual AlkBs target a relatively narrow range. Mechanisms of substrate selectivity and catalytic activity remain elusive. Here we report the cryo-EM structure of AlkB, which provides a distinct architecture for membrane enzymes. Our structure and functional studies reveal an unexpected diiron center configuration and identify molecular determinants for substrate selectivity. These findings provide insight into the catalytic mechanism of AlkB and shed light on its function in alkane-degrading microorganisms.

Structural basis for enzymatic terminal C–H bond functionalization of alkanes

Alkane monooxygenase (AlkB) is a widely occurring integral membrane metalloenzyme that catalyzes the initial step in the functionalization of recalcitrant alkanes with high terminal selectivity. AlkB enables diverse microorganisms to use alkanes as their sole carbon and energy source. Here we present the 48.6-kDa cryo‐electron microscopy structure of a natural fusion from Fontimonas thermophila between AlkB and its electron donor AlkG at 2.76 Å resolution. The AlkB portion contains six transmembrane helices with an alkane entry tunnel within its transmembrane domain. A dodecane substrate is oriented by hydrophobic tunnel-lining residues to present a terminal C–H bond toward a diiron active site. AlkG, an [Fe–4S] rubredoxin, docks via electrostatic interactions and sequentially transfers electrons to the diiron center. The archetypal structural complex presented reveals the basis for terminal C–H selectivity and functionalization within this broadly distributed evolutionary class of enzymes.

Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase.

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining the protein structure. Despite recent technical advances, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air-water interface, the presence of preferred orientations, nonuniform ice layers, etc. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently gained attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Here, we introduce a reliable, easily implemented, and reproducible method to produce 36 graphene-coated grids within 1.5 days. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.5 Å using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages, including a smaller amount of protein required and avoiding protein denaturation at the air-water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we identified subtle yet significant geometrical changes at the nonheme diiron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity.

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps.

Lipid extract derived from newly isolated Rhodotorula toruloides LAB-07 for cosmetic applications

Rhodotorula toruloides is a non-conventional yeast with a natural carotenoid pathway. In particular, R. toruloides is an oleaginous yeast that can accumulate lipids in high content, thereby gaining interest as a promising industrial host. In this study, we isolated and taxonomically identified a new R. toruloides LAB-07 strain. De novo genome assembly using PacBio and Illumina hybrid platforms yielded 27 contigs with a 20.78 Mb genome size. Subsequent genome annotation analysis based on RNA-seq predicted 5296 protein-coding genes, including the fatty acid production pathway. We compared lipid production under different media; it was highest in the yeast extract salt medium with glycerol as a carbon source. Polyunsaturated α-linolenic acid was detected among the fatty acids, and docking phosphatidylcholine as a substrate to modeled Fad2, which annotated as Δ12-fatty acid desaturase showed bifunctional Δ12, 15-desaturation is structurally possible in that the distances between the diiron center and the carbon-carbon bond in which desaturation occurs were similar to those of structurally identified mouse stearoyl-CoA desaturase. Finally, the applicability of the extracted total lipid fraction of R. toruloides was investigated, demonstrating an increase in filaggrin expression and suppression of heat-induced MMP-1 expression when applied to keratinocytes, along with the additional antioxidant activity. This work presents a new R. toruloides LAB-07 strain with genomic and lipidomic data, which would help understand the physiology of R. toruloides. Also, the various skin-related effect of R. toruloides lipid extract indicates its potential usage as a promising cosmetic ingredient.

Molecular insights into two ferritin subunits from red-lip mullet (Liza haematocheila): Detectable antibacterial activity with its expressional response against immune stimulants

Iron (Fe) is considered as an essential micronutrient due to its diverse functions in living systems. However, regulation of free iron levels is essential because free Fe ions, in excess, induce biological toxicity through different routes, including production of reactive oxygen species. Ferritin proteins play a vital role in controlling free Fe ion homeostasis by sequestering excess iron in the body. Ferritins comprise an H subunit with a ferroxidase center and an L subunit with a Fe nucleation site. However, lower vertebrates such as fish harbor an additional subunit termed ferritin M, which shows the characteristic features of both H and L. In this study, two ferritin subunits (H and M) with ferroxidase centers were identified and characterized from red-lip mullet (Liza haematocheila). The open reading frames of red-lip mullet ferritin H (LhFerH) and ferritin M−like (LhFerM) subunits comprise 534 and 531 bps, which encode for putative polypeptides of 177 and 176 amino acids, respectively. LhFerH and LhFerM were found to retain well-conserved residues, including seven ferroxidase di-iron centers, characteristic domains, and signatures of their known homologs. We cloned the open reading frames of the two ferritin subunits to overexpress the corresponding proteins in Escherichia coli and subsequently demonstrated their iron sequestration activity along with antibacterial activity against E. coli using respective purified recombinant proteins in vitro. A basal expression analysis of two LhFer genes in selected tissues using qPCR assays showed pronounced expression in blood cells with respect to both genes. A relatively high expression level of LhFerH was also detected in muscle tissues. The expression level of LhFer in the head kidney was significantly up-regulated following lipopolysaccharides (LPS) and Lactococcus garvieae injection. The resulting gene expression pattern upon immune stimulation suggests that ferritin may contribute to the defense against harmful pathogen infection. Collectively, our results indicate that both LhFerH and LhFerM potentially participate in the homeostasis of free Fe ions and in the host immune defense of red-lip mullet.

Spectroscopic Characterization of a Diferric Mycobacterial Hemerythrin-Like Protein with Unprecedented Reactivity toward Nitric Oxide.

Hemerythrin-like proteins (HLPs) are broadly distributed across taxonomic groups and appear to play highly diverse functional roles in prokaryotes. Mycobacterial HLPs contribute to the survival of these pathogenic bacteria in mammalian macrophages, but their modes of action remain unclear. A recent crystallographic characterization of Mycobacterium kansasii HLP (Mka-HLP) revealed the unexpected presence of a tyrosine sidechain (Tyr54) near the coordination sphere of one of the two iron centers. Here, we show that Tyr54 is a true ligand to the Fe2(III) ion which, in conjunction with the presence of a μ-oxo group bridging the two iron(III), brings unique reactivity toward nitric oxide (NO). Monitoring the titration of Mka-HLP with NO by Fourier-transform infrared and electron paramagnetic resonance spectroscopies shows that both diferric and diferrous forms of Mka-HLP accumulate an uncoupled high-spin and low-spin {FeNO}7 pair. We assign the reactivity of the diferric protein to an initial radical reaction between NO and the μ-oxo bridge to form nitrite and a mixed-valent diiron center that can react further with NO. Amperometric measurements of NO consumption by Mka-HLP confirm that this reactivity can proceed at low micromolar concentrations of NO, before additional NO consumption, supporting a NO scavenging role for mycobacterial HLPs.