Diiron Center Research Articles

Hydration of Nitriles to Amides by Thiolate-Bridged Diiron Complexes

A series of nitrile-coordinating complexes [Cp*Fe(μ-SEt)RCN]2[PF6]2 (1, R = alkyl, aryl, vinyl, amine) have been obtained by the reaction of [Cp*Fe(μ-SEt)MeCN]2[PF6]2 (1a) with various nitriles in acetone. Complexes 1 can realize the hydration of a nitrile ligand under ambient conditions. Complexes [Cp*Fe(μ-SEt)2(μ-η1:η1-NH(O)CR)FeCp*][PF6] (2) were successfully isolated as intermediates during the hydration process, with 2b and 2e (R = CH2═CH and Et2N) being characterized by spectrometry and X-ray crystallography. Treatment of 2 with HBF4·Et2O in the presence of nitriles released corresponding amides 3. At the same time, the structural features of the [Fe2S2] scaffold were retained. These results confirmed that the hydration of nitriles was realized by cooperative interaction on diiron centers.

X-ray structure of a mammalian stearoyl-CoA desaturase

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl-CoAs1–4. Since the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters, and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologs (SCD1 and SCD5), while mice have four (SCD1–SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity5,6. Thus, SCD1 is a pharmacological target in the treatment of obesity, diabetes, and other metabolic diseases7. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyzes the formation of a cis-double bond between the 9th and 10th carbons of stearoyl- or palmitoyl-CoA8,9. The reaction requires molecular oxygen, which is activated by a diiron center, and cytochrome b5, which regenerates the diiron center10. To better understand the structural basis of these characteristics of SCD function, we crystallized and solved the structure of mouse SCD1 bound to stearoyl-CoA at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain, and a plausible pathway for lateral substrate access and product egress. The acyl chain of the bound stearoyl-CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel and configuration of the bound acyl chain provide a structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The dimetal center is coordinated by a unique configuration of nine conserved histidine residues that implies a potentially novel metal center and mechanism for oxygen activation. The structure also illustrates a possible route for electron transfer from cytochrome b5 to the diiron center.

Diiron azadithiolates as models for the [FeFe]-hydrogenase active site and paradigm for the role of the second coordination sphere.

The [FeFe] hydrogenases (H2ases) catalyze the redox reaction that interconverts protons and H2. This area of biocatalysis has attracted attention because the metal-based chemistry is unusual, and the reactions have practical implications. The active site consists of a [4Fe-4S] cluster bridged to a [Fe2(μ-dithiolate)(CN)2(CO)3](z) center (z = 1- and 2-). The dithiolate cofactor is [HN(CH2S)2](2-), called the azadithiolate ([adt(H)](2-)). Although many derivatives of Fe2(SR)2(CO)6-xLx are electrocatalysts for the hydrogen evolution reaction (HER), most operate by slow nonbiomimetic pathways. Biomimetic hydrogenogenesis is thought to involve intermediates, wherein the hydride substrate is adjacent to the amine of the adt(H), being bonded to only one Fe center. Formation of terminal hydride complexes is favored when the diiron carbonyl models contain azadithiolate. Although unstable in the free state, the adt cofactor is stable once it is affixed to the Fe2 center. It can be prepared by alkylation of Fe2(SH)2(CO)6 with formaldehyde in the presence of ammonia (to give adt(H) derivatives) or amines (to give adt(R) derivatives). Weak acids protonate Fe2(adt(R))(CO)2(PR3)4 to give terminal hydrido (term-H) complexes. In contrast, protonation of the related 1,3-propanedithiolate (pdt(2-)) complexes Fe2(pdt)(CO)2(PR3)4 requires strong acids. The amine in the azadithiolate is a kinetically fast base, relaying protons to and from the iron, which is a kinetically slow base. The crystal structure of the doubly protonated model [(term-H)Fe2(Hadt(H))(CO)2(dppv)2](2+) confirms the presence of both ammonium and terminal hydrido centers, which interact through a dihydrogen bond (dppv = cis-C2H2(PPh2)2). DFT calculations indicate that this H---H interaction is sensitive to the counterions and is strengthened upon reduction of the diiron center. For the monoprotonated models, the hydride [(term-H)Fe2(adt(H))(CO)2(dppv)2](+) exists in equilibrium with the ammonium tautomer [Fe2(Hadt(H))(CO)2(dppv)2](+). Both [(term-H)Fe2(Hadt(H))(CO)2(dppv)2](2+) and [(term-H)Fe2(adt(H))(CO)2(dppv)2](+) are highly active electrocatalysts for HER. Catalysis is initiated by reduction of the diferrous center, which induces coupling of the protic ammonium center and the hydride ligand. In contrast, the propanedithiolate [(term-H)Fe2(pdt)(CO)2(dppv)2](+) is a poor electrocatalyst for HER. Oxidation of H2 has been demonstrated, starting with models for the oxidized state ("Hox"), for example, [Fe2(adt(H))(CO)3(dppv)(PMe3)](+). Featuring a distorted Fe(II)Fe(I) center, this Hox model reacts slowly with high pressures of H2 to give [(μ-H)Fe2(adt(H))(CO)3(dppv)(PMe3)](+). Highlighting the role of the proton relay, the propanedithiolate [Fe2(pdt)(CO)3(dppv)(PMe3)](+) is unreactive toward H2. The Hox-model + H2 reaction is accelerated in the presence of ferrocenium salts, which simulate the role of the attached [4Fe-4S] cluster. The redox-complemented complex [Fe2(adt(Bn))(CO)3(dppv)(FcP*)](n+) catalyzes both proton reduction and hydrogen oxidation (FcP* = (C5Me5)Fe(C5Me4CH2PEt2)).

Ferritin M of Paralichthys olivaceus possesses antimicrobial and antioxidative properties.

Ferritin is an evolutionarily conserved protein that plays a vital role in maintaining iron homeostasis. In this study, we identified a ferritin M (PoFerM) from Japanese flounder (Paralichthys olivaceus) and analyzed its biological property. PoFerM is composed of 176 amino acid residues and contains the conserved ferroxidase diiron center and the ferrihydrite nucleation center typical of M ferritins. Expression of PoFerM occurred in multiple tissues and was most abundant in blood. Bacterial infection upregulated PoFerM expression in head kidney, spleen, and liver in a time-dependent manner. Recombinant PoFerM (rPoFerM) purified from Escherichia coli exhibited iron-chelating activity and inhibited bacterial growth, whereas rPoFerMM, the mutant protein that bears alanine substitution at two conserved residues of the ferroxidase center and the ferrihydrite nucleation center, failed to do so. Oxidative protection analysis showed that rPoFerM, but not rPoFerMM, was able to alleviate the deleterious effect of H2O2-induced free radicals on plasmid DNA and primary flounder cells. Together these results indicate that PoFerM is an iron chelator with antimicrobial and antioxidative properties, all which depend on the conserved ferroxidase center and the ferrihydrite nucleation site.

Regulation of plasmid-encoded isoprene metabolism in Rhodococcus, a representative of an important link in the global isoprene cycle.

Emissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation. Here, we report the genome of an isoprene degrading isolate, Rhodococcus sp. AD45, and, using mutagenesis shows that a plasmid-encoded soluble di-iron centre isoprene monooxygenase (IsoMO) is essential for isoprene metabolism. Using RNA sequencing (RNAseq) to analyse cells exposed to isoprene or epoxyisoprene in a substrate-switch time-course experiment, we show that transcripts from 22 contiguous genes, including those encoding IsoMO, were highly upregulated, becoming among the most abundant in the cell and comprising over 25% of the entire transcriptome. Analysis of gene transcription in the wild type and an IsoMO-disrupted mutant strain showed that epoxyisoprene, or a subsequent product of isoprene metabolism, rather than isoprene itself, was the inducing molecule. We provide a foundation of molecular data for future research on the environmental biological consumption of this important, climate-active compound.

Synthesis of bridging hydrides of phenyl-functionalized diiron propanedithiolate complexes with 1,2-bis(diphenylphosphine)ethylene or 1,2-bis(diphenylphosphine)ethane ligands

Reaction of 2-phenyl-1,3-dibromopropane with in situ generated (μ-SLi)2Fe2(CO)6 yielded a known complex [(μ-SCH2)2CHC6H5]Fe2(CO)6 (A). Displacement of two carbonyls from complex A by cis-1,2-bis(diphenylphosphine)ethylene (dppv) or 1,2-bis(diphenylphosphine)ethane (dppe) in the presence of Me3NO·2H2O gave two chelate complexes [(μ-SCH2)2CHC6H5]Fe2(CO)4(κ 2-dppv), [A(κ 2-dppv)] and [(μ-SCH2)2CHC6H5]Fe2(CO)4(κ 2-dppe), [A(κ 2-dppe)], respectively. Protonation of the diiron centers of [A(κ 2-dppv)] and [A(κ 2-dppe)] using an excess of HBF4·Et2O in dichloromethane at room temperature gave the bridging hydrides [(μ-H)A(κ 2-dppv)]BF4 and [(μ-H)A(κ 2-dppe)]BF4. The complexes [(μ-H)A(κ 2-dppv)]BF4, [A(κ 2-dppe)] and [(μ-H)A(κ 2-dppe)]BF4 were characterized by elemental analysis and spectroscopic methods, and [(μ-H)A(κ 2-dppv)]BF4 and [(μ-H)A(κ 2-dppe)]BF4 were also characterized by X-ray crystallography. The electrochemical behavior of [(μ-H)A(κ 2-dppv)]BF4 was investigated by cyclic voltammetry, and the catalytic electrochemical reduction in protons from trifluoroacetic acid or p-methylbenzene sulfonic acid to give hydrogen was investigated.

Crystal Structure of the Peroxo-diiron(III) Intermediate of Deoxyhypusine Hydroxylase, an Oxygenase Involved in Hypusination

Deoxyhypusine hydroxylase (DOHH) is a non-heme diiron enzyme involved in the posttranslational modification of a critical lysine residue of eukaryotic translation initiation factor 5A (eIF-5A) to yield the unusual amino acid residue hypusine. This modification is essential for the role of eIF-5A in translation and in nuclear export of a group of specific mRNAs. The diiron center of human DOHH (hDOHH) forms a peroxo-diiron(III) intermediate (hDOHHperoxo) when its reduced form reacts with O2. hDOHHperoxo has a lifetime exceeding that of the peroxo intermediates of other diiron enzymes by several orders of magnitude. Here we report the 1.7-Å crystal structures of hDOHHperoxo and a complex with glycerol. The structure of hDOHHperoxo reveals the presence of a μ-1,2-peroxo-diiron(III) species at the active site. Augmented by UV/Vis and Mössbauer spectroscopic studies, the crystal structures offer explanations for the extreme longevity of hDOHHperoxo and illustrate how the enzyme specifically recognizes its only substrate, deoxyhypusine-eIF-5A.

Controlling the Activities of the Diiron Centre in Bacterial Monooxygenases: Lessons from Mutagenesis and Biodiversity

AbstractThe soluble diiron monooxygenases (SDIMOs) are a diverse group of bacterial enzymes that possess a deeply buried binuclear iron centre that forms an oxo‐diferryl (FeIV) intermediate that is capable of oxygenating a wide range of unfunctionalised hydrocarbons and other hydrophobic organic molecules, ranging in size from methane to diaromatics such as naphthalene and substituted biphenyls. In the environment, these enzymes are important in bioremediation of hydrocarbons and chlorinated hydrocarbons and in mitigation of the greenhouse effect due to methane, and also have numerous potential applications in synthetic organic chemistry. In this review we consider how both natural variations among the enzymes and an increasing body of evidence from mutagenesis studies cast light on how the protein controls the size of substrates that can access the active site, the precision of regio‐ and stereoselectivity and the oxidising power of the active centre.

New insights into novel inhibitors against deoxyhypusine hydroxylase from plasmodium falciparum: compounds with an iron chelating potential.

Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme required for hydroxylation of the aminobutyl side chain of deoxyhypusine in eukaryotic translation initiation factor 5A (eIF-5A), the second step in hypusine biosynthesis. DOHH has been recently identified in P. falciparum and P. vivax. Both enzymes have very peculiar features including E-Z type HEAT-like repeats and a diiron centre in their active site. Both proteins share only 26 % amino acid identity to the human paralogue. Hitherto, no X-ray structure exists from either enzyme. However, structural predictions based on the amino acid sequence of the active site in comparison to the human enzyme show that four conserved histidine and glutamate residues provide the coordination sites for chelating the ferrous iron ions. Recently, we showed that P. vivax DOHH is inhibited by zileuton (N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea), a drug that is known for inhibiting human 5-lipoygenase (5-LOX) by the complexation of ferrous iron. A novel discovery program was launched to identify inhibitors of the P. falciparum DOHH from the Malaria Box, consisting of 400 chemical compounds, which are highly active in the erythrocytic stages of Malaria infections. In a first visual selection for potential ligands of ferrous iron, three compounds from different scaffold classes namely the diazonapthyl benzimidazole MMV666023 (Malaria Box plate A, position A03), the bis-benzimidazole MMV007384 (plate A, position B08), and a 1,2,5,-oxadiazole MMV665805 (plate A, position C03) were selected and subsequently evaluated in silico for their potential to complex iron ions. As a proof of principle, a bioanalytical assay was performed and the inhibition of hypusine biosynthesis was determined by GC-MS. All tested compounds proved to be active in this assay and MMV665805 exhibited the strongest inhibitory effect. Notably, the results were in accordance with the preliminary quantum-mechanical calculations suggesting the strongest iron complexation capacity for MMV665805. This compound might be a useful tool as well as a novel lead structure for inhibitors of P. falciparum DOHH.

Insights into the structure of the diiron site of RIC from Escherichia coli

Repair of Iron Centres (RICs) are a widely-spread family of diiron proteins involved in the protection of iron–sulphur-containing enzymes from nitrosative and oxidative stress. Here, homology-based modelling was used to predict putative ligands of the RIC diiron centre in E. coli. Site-directed mutagenesis studies showed that several conserved residues modulate the spectroscopic properties of the diiron centre, and mutations in H129, E133 and E208 abrogated RIC ability to protect aconitase. Taken together, these data led to a structural model of a diiron centre inserted in a four-helix bundle fold and coordinated by H84, H129, H160, H204, E133 and E208. Moreover, two μ-carboxylate bridges involving E133 and E208 were found to be required for assembly of a stable diiron centre.

X-Ray Structure of a Mammalian Stearoyl-Coa Desaturase-1

Stearoyl-CoA desaturase (SCD) is conserved in all eukaryotes and introduces the first double bond into saturated fatty acyl CoAs. Since the monounsaturated products of SCD are key precursors of membrane phospholipids, cholesterol esters, and triglycerides, SCD is pivotal in fatty acid metabolism. Humans have two SCD homologs (SCD1 and SCD5), and mice have four (SCD1-SCD4). SCD1-deficient mice do not become obese or diabetic when fed a high-fat diet because of improved lipid metabolic profiles and insulin sensitivity. Thus, SCD1 is a promising pharmacological target in the treatment of obesity, diabetes, and other metabolic diseases. SCD1 is an integral membrane protein located in the endoplasmic reticulum, and catalyzes the formation of a cis-double bond between the 9th and 10th carbons of stearoyl- or palmitoyl-CoA. The reaction requires molecular oxygen, which is activated by a diiron center, and cytochrome b5, which reduces the diiron center. To better understand the structural basis of these characteristics of SCD function, we crystallized and solved the structure of mouse SCD1 bound to a stearoyl-CoA molecule at 2.6 Å resolution. The structure shows a novel fold comprising four transmembrane helices capped by a cytosolic domain. The acyl chain of the bound stearoyl- CoA is enclosed in a tunnel buried in the cytosolic domain, and the geometry of the tunnel suggests the structural basis for the regioselectivity and stereospecificity of the desaturation reaction. The structure reveals a dimetal center coordinated by a unique configuration of nine conserved histidine residues that implies a potentially novel mechanism for oxygen activation. The structure also illustrates a potential pathway for substrate access and product egress, and a possible route for electron transfer from cytochrome b5 to the diiron center.

(19)F NMR study of ligand dynamics in carboxylate-bridged diiron(II) complexes supported by a macrocyclic ligand.

A series of asymmetrically carboxylate-bridged diiron(ii) complexes featuring fluorine atoms as NMR spectroscopic probes, [Fe2(PIM)(Ar(4F-Ph)CO2)2] (10), [Fe2(F2PIM)(Ar(Tol)CO2)2] (11), and [Fe2(F2PIM)(Ar(4F-Ph)CO2)2] (12), were prepared and characterized by X-ray crystallography, Mössbauer spectroscopy, and VT (19)F NMR spectroscopy. These complexes are part of a rare family of syn N-donor diiron(ii) compounds, [Fe2(X2PIM)(RCO2)2], that are structurally very similar to the active site of the hydroxylase enzyme component of reduced methane monooxygenase (MMOHred). Solution characterization of these complexes demonstrates that they undergo intramolecular carboxylate rearrangements, or carboxylate shifts, a dynamic feature relevant to the reactivity of the diiron centers in bacterial multicomponent monooxygenases.

Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases.

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

Component interactions and electron transfer in toluene/o-xylene monooxygenase.

The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases.

Structural basis for biomolecular recognition in overlapping binding sites in a diiron enzyme system.

Productive biomolecular recognition requires exquisite control of affinity and specificity. Accordingly, nature has devised many strategies to achieve proper binding interactions. Bacterial multicomponent monooxygenases provide a fascinating example, where a diiron hydroxylase must reversibly interact with both ferredoxin and catalytic effector in order to achieve electron transfer and O2 activation during catalysis. Because these two accessory proteins have distinct structures, and because the hydroxylase-effector complex covers the entire surface closest to the hydroxylase diiron centre, how ferredoxin binds to the hydroxylase has been unclear. Here we present high-resolution structures of toluene 4-monooxygenase hydroxylase complexed with its electron transfer ferredoxin and compare them with the hydroxylase-effector structure. These structures reveal that ferredoxin or effector protein binding produce different arrangements of conserved residues and customized interfaces on the hydroxylase in order to achieve different aspects of catalysis.

The crystal structure of ferritin from Chlorobium tepidum reveals a new conformation of the 4-fold channel for this protein family.

Ferritins are ubiquitous iron-storage proteins found in all kingdoms of life. They share a common architecture made of 24 subunits of five α-helices. The recombinant Chlorobium tepidum ferritin (rCtFtn) is a structurally interesting protein since sequence alignments with other ferritins show that this protein has a significantly extended C-terminus, which possesses 12 histidine residues as well as several aspartate and glutamic acid residues that are potential metal ion binding residues. We show that the macromolecular assembly of rCtFtn exhibits a cage-like hollow shell consisting of 24 monomers that are related by 4-3-2 symmetry; similar to the assembly of other ferritins. In all ferritins of known structure the short fifth α-helix adopts an acute angle with respect to the four-helix bundle. However, the crystal structure of the rCtFtn presented here shows that this helix adopts a new conformation defining a new assembly of the 4-fold channel of rCtFtn. This conformation allows the arrangement of the C-terminal region into the inner cavity of the protein shell. Furthermore, two Fe(III) ions were found in each ferroxidase center of rCtFtn, with an average FeA-FeB distance of 3Å; corresponding to a diferric μ-oxo/hydroxo species. This is the first ferritin crystal structure with an isolated di-iron center in an iron-storage ferritin. The crystal structure of rCtFtn and the biochemical results presented here, suggests that rCtFtn presents similar biochemical properties reported for other members of this protein family albeit with distinct structural plasticity.

Mutations in SACPD-C result in a range of elevated stearic acid concentration in soybean seed.

Soybean oil has a wide variety of uses, and stearic acid, which is a relatively minor component of soybean oil is increasingly desired for both industrial and food applications. New soybean mutants containing high levels of the saturated fatty acid stearate in seeds were recently identified from a chemically mutagenized population. Six mutants ranged in stearate content from 6–14% stearic acid, which is 1.5 to 3 times the levels contained in wild-type seed of the Williams 82 cultivar. Candidate gene sequencing revealed that all of these lines carried amino acid substitutions in the gene encoding the delta-9-stearoyl-acyl-carrier protein desaturase enzyme (SACPD-C) required for the conversion of stearic acid to oleic acid. Five of these missense mutations were in highly conserved residues clustered around the predicted di-iron center of the SACPD-C enzyme. Co-segregation analysis demonstrated a positive association of the elevated stearate trait with the SACPD-C mutation for three populations. These missense mutations may provide additional alleles that may be used in the development of new soybean cultivars with increased levels of stearic acid.

Trace-gas metabolic versatility of the facultative methanotroph Methylocella silvestris

The climate-active gas methane is generated both by biological processes and by thermogenic decomposition of fossil organic material, which forms methane and short-chain alkanes, principally ethane, propane and butane. In addition to natural sources, environments are exposed to anthropogenic inputs of all these gases from oil and gas extraction and distribution. The gases provide carbon and/or energy for a diverse range of microorganisms that can metabolize them in both anoxic and oxic zones. Aerobic methanotrophs, which can assimilate methane, have been considered to be entirely distinct from utilizers of short-chain alkanes, and studies of environments exposed to mixtures of methane and multi-carbon alkanes have assumed that disparate groups of microorganisms are responsible for the metabolism of these gases. Here we describe the mechanism by which a single bacterial strain, Methylocella silvestris, can use methane or propane as a carbon and energy source, documenting a methanotroph that can utilize a short-chain alkane as an alternative to methane. Furthermore, during growth on a mixture of these gases, efficient consumption of both gases occurred at the same time. Two soluble di-iron centre monooxygenase (SDIMO) gene clusters were identified and were found to be differentially expressed during bacterial growth on these gases, although both were required for efficient propane utilization. This report of a methanotroph expressing an additional SDIMO that seems to be uniquely involved in short-chain alkane metabolism suggests that such metabolic flexibility may be important in many environments where methane and short-chain alkanes co-occur.

Bioinspired Hydrogenase Models: The Mixed-Valence Triiron Complex [Fe3(CO)7(μ-edt)2] and Phosphine Derivatives [Fe3(CO)7-x (PPh3) x (μ-edt)2] (x = 1, 2) and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2] as Proton Reduction Catalysts.

The mixed-valence triiron complexes [Fe3(CO)7–x(PPh3)x(μ-edt)2] (x = 0–2; edt = SCH2CH2S) and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2] (diphosphine = dppv, dppe, dppb, dppn) have been prepared and structurally characterized. All adopt an anti arrangement of the dithiolate bridges, and PPh3 substitution occurs at the apical positions of the outer iron atoms, while the diphosphine complexes exist only in the dibasal form in both the solid state and solution. The carbonyl on the central iron atom is semibridging, and this leads to a rotated structure between the bridged diiron center. IR studies reveal that all complexes are inert to protonation by HBF4·Et2O, but addition of acid to the pentacarbonyl complexes results in one-electron oxidation to yield the moderately stable cations [Fe3(CO)5(PPh3)2(μ-edt)2]+ and [Fe3(CO)5(κ2-diphosphine)(μ-edt)2]+, species which also result upon oxidation by [Cp2Fe][PF6]. The electrochemistry of the formally Fe(I)–Fe(II)–Fe(I) complexes has been investigated. Each undergoes a quasi-reversible oxidation, the potential of which is sensitive to phosphine substitution, generally occurring between 0.15 and 0.50 V, although [Fe3(CO)5(PPh3)2(μ-edt)2] is oxidized at −0.05 V. Reduction of all complexes is irreversible and is again sensitive to phosphine substitution, varying between −1.47 V for [Fe3(CO)7(μ-edt)2] and around −1.7 V for phosphine-substituted complexes. In their one-electron-reduced states, all complexes are catalysts for the reduction of protons to hydrogen, the catalytic overpotential being increased upon successive phosphine substitution. In comparison to the diiron complex [Fe2(CO)6(μ-edt)], [Fe3(CO)7(μ-edt)2] catalyzes proton reduction at 0.36 V less negative potentials. Electronic structure calculations have been carried out in order to fully elucidate the nature of the oxidation and reduction processes. In all complexes, the HOMO comprises an iron–iron bonding orbital localized between the two iron atoms not ligated by the semibridging carbonyl, while the LUMO is highly delocalized in nature and is antibonding between both pairs of iron atoms but also contains an antibonding dithiolate interaction.

The Production of Nitrous Oxide by the Heme/Nonheme Diiron Center of Engineered Myoglobins (FeBMbs) Proceeds through a trans-Iron-Nitrosyl Dimer

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N2O. Engineering a nonheme FeB site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N2O in solution and to show that the presence of a distal FeBII is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}7 species with ν(FeNO)heme and ν(NO)heme at 522 and 1660 cm–1, and a second NO molecule is bound to the nonheme FeB site with a ν(NO)FeB at 1755 cm–1. Stopped-flow UV–vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to FeB is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex with characteristic ν(FeNO)heme at 570 ± 2 cm–1 and ν(NO)FeB at 1755 cm–1. Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in FeBMb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex to decay with production of N2O at a rate of 0.7 s–1 at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs.