Diiron Center Research Articles

Synthesis and molecular structures of the 52-electron triiron telluride clusters [Fe3(CO)8(μ3-Te)2(κ2-diphosphine)] - Electrochemical properties and activity as proton reduction catalysts

Abstract Heating the 50-electron cluster [Fe3(CO)9 (μ3-Te)2] (1) with the diphosphines Ph2P-R-PPh2 [R = -CH2CH2- (dppe), Z-CH=CH- (dppv), 1,2-C6H4 (dppb), -CH2CH2CH2- (dpp), ferrocenyl (dppf), naphthalenyl (dppbn)] in benzene affords the 52-electron diphosphine-containing tellurium-capped triiron clusters [Fe3(CO)8 (μ3-Te)2 (κ2-diphosphine)] (diphosphine = dppe, dppv, dppb, dpp, dppf, dppnd) (2–7) in moderate yields, resulting from both phosphine addition and carbonyl loss. With 1,2-bis(diphenylphosphino)benzene (dppb) a second product is the cubane cluster [Fe4(CO)10(μ3-Te)4 (κ2-dppb)] (8). Cyclic voltammetry measurements on 2–7 reveal that all clusters show irreversible reductive behaviour at ca. −1.85 V with a series of associated small back oxidation waves, suggesting that reduction leads to significant structural change but that this can be reversed chemically. Oxidation occurs at relatively low potentials and is diphosphine-dependent. The first oxidation appears at ca. +0.35 V for 2–6 with a small degree of reversibility but is as low as +0.14 V for the bis(diphenylphosphino)naphthalene derivative 7 and in some cases is followed by further closely-spaced oxidation. Addition of [Cp2Fe][PF6] to 2–7 results in the formation of new clusters formulated as [Fe3(CO)8(μ3-Te)2(κ2-diphosphine)]+, with their IR spectra suggesting oxidation at the diiron centre. This is supported by computational studies (DFT) of the bis(diphenylphosphino)propane cluster 5 showing that the HOMO is the Fe Fe σ-bonding orbital, while the LUMO is centered on the diphosphine-substituted iron atom and has significant Fe Te σ∗-anti-bonding character consistent with the irreversible nature of the reduction. Complexes 2–7 have been examined as proton reduction catalysts in the presence of para-toluenesulfonic acid (TsOH). All are active at their first reduction potential, with a second catalytic process being observed at slightly higher potentials. While their overall electrocatalytic behaviour is similar to that noted for [Fe2(CO)6{μ-E(CH2)3E}] (E = S, Se, Te), the DFT results suggest that as the added electron is localised on the unique iron atom. The mechanistic aspects of hydrogen formation are likely to be quite different from the more widely studied diiron models.

Structure and Function of Mammalian Stearoyl-COA Desaturase

Membrane bound stearoyl-CoA desaturase (SCD) is an iron-containing metalloenzyme. It catalyzes the rate-limiting step in the conversion of saturated fatty acids to monounsaturated fatty acids. The formation of a double bond is catalyzed by a diiron center in the protein, and is coupled to NAD(P)H oxidation via cytochrome b5 and cytochrome b5 reductase. In mammals, SCD1-deficiency leads to resistance against diet-induced obesity and diabetes because of elevated levels of lipid metabolism and insulin sensitivity. Many efforts have been made to develop effective SCD1 inhibitors for the treatment of obesity, ectopic fat deposition, diabetes and other metabolic syndromes. SCD1 is also a potential drug target for cancers because its activity is upregulated and required for propagating cancer cells. Crystal structures of mouse and human SCD1 have been reported recently, however, in both structures the presumed diiron center is occupied by zinc ions. The mis-incorporation of zinc ions during heterologous overexpression in insect cells renders the purified SCD1 protein biochemically inactive, and raises the question of whether the structures faithfully represent the iron-containing SCD1. The inactive SCD1 also hinders our ability to unravel the mechanism of catalysis and identification of inhibitors. Therefore, we developed a procedure to remove zinc ions and reconstitute ferrous ions into the mouse SCD1 protein. After confirming the presence of irons in SCD1, we then crystallized and solved the structure of the iron-loaded SCD1 to 3.5 Å. Initial analysis showed that the iron containing SCD1 structure is similar to that of the zinc containing SCD1, indicating that the previous structure was not altered significantly by the presence of zinc ions. The iron containing SCD1 also enabled us to conduct further experiments to dissect the redox reaction mechanism.

Diversity and complexity of flavodiiron NO/O2 reductases.

Flavodiiron proteins (FDPs) are a family of enzymes endowed with nitric oxide (NO) or oxygen reductase activities, forming the innocuous nitrous oxide (N2O) or water molecules, respectively. FDPs are widespread in the three life kingdoms, and have a modular nature, being each monomer minimally constituted by a metallo-β-lactamase-like domain containing a catalytic diiron centre, followed by a flavodoxin one, with a flavin mononucleotide. Since their discovery, additional domains have been found in FDPs, attached to the C-terminus, and containing either extra metal (iron) centers or extra flavin binding modules. Following an extensive analysis of genomic databases, we identified novel domain compositions, and proposed a new classification of FDPs in eight classes based on the nature and number of extra domains.

Substrate specificity and membrane topologies of the iron-containing ω3 and ω6 desaturases from Mortierella alpina.

Polyunsaturated fatty acids (PUFAs) are essential lipids for cell function, normal growth, and development, serving as key structural components of biological membranes and modulating critical signal transduction events. Omega-3 (n-3) long chain PUFAs (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect against inflammatory diseases and enhance brain development and function. This had led to a marked increase in demand for fish and fish oils in human diets, supplements, and aquaculture and created a need for new, sustainable n-3 LC-PUFA sources. We have studied for the first time homogenous preparations of the membrane-type ω6 and ω3 fatty acid desaturases from the fungus Mortierella alpina, as a model system to produce PUFAs. These desaturases possess a di-iron metal center and are selective for 18:1 n-9 and 18:2 n-6 acyl-CoA substrates, respectively. Sequence alignments and membrane topology predictions support that these enzymes have unique cap regions that may include the rearrangement and repositioning of the active site, especially when compared to the mammalian stearoyl-coenzyme A desaturase-1 (SCD1) and the related sphingolipid α-hydroxylase (Scs7p) that act upon different substrates.

Alternative oxidase (AOX) constitutes a small family of proteins in Citrus clementina and Citrus sinensis L. Osb.

The alternative oxidase (AOX) protein is present in plants, fungi, protozoa and some invertebrates. It is involved in the mitochondrial respiratory chain, providing an alternative route for the transport of electrons, leading to the reduction of oxygen to form water. The present study aimed to characterize the family of AOX genes in mandarin (Citrus clementina) and sweet orange (Citrus sinensis) at nucleotide and protein levels, including promoter analysis, phylogenetic analysis and C. sinensis gene expression. This study also aimed to do the homology modeling of one AOX isoform (CcAOXd). Moreover, the molecular docking of the CcAOXd protein with the ubiquinone (UQ) was performed. Four AOX genes were identified in each citrus species. These genes have an open reading frame (ORF) ranging from 852 bp to 1150 bp and a number of exons ranging from 4 to 9. The 1500 bp-upstream region of each AOX gene contained regulatory cis-elements related to internal and external response factors. CsAOX genes showed a differential expression in citrus tissues. All AOX proteins were predicted to be located in mitochondria. They contained the conserved motifs LET, NERMHL, LEEEA and RADE-H as well as several putative post-translational modification sites. The CcAOXd protein was modeled by homology to the AOX of Trypanosona brucei (45% of identity). The 3-D structure of CcAOXd showed the presence of two hydrophobic helices that could be involved in the anchoring of the protein in the inner mitochondrial membrane. The active site of the protein is located in a hydrophobic environment deep inside the AOX structure and contains a diiron center. The molecular docking of CcAOXd with UQ showed that the binding site is a recessed pocket formed by the helices and submerged in the membrane. These data are important for future functional studies of citrus AOX genes and/or proteins, as well as for biotechnological approaches leading to AOX inhibition using UQ homologs.

In-crystal reaction cycle of a toluene-bound diiron hydroxylase.

Electrophilic aromatic substitution is one of the most important and recognizable classes of organic chemical transformation. Enzymes create the strong electrophiles that are needed for these highly energetic reactions by using O2, electrons, and metals or other cofactors. Although the nature of the oxidants that carry out electrophilic aromatic substitution has been deduced from many approaches, it has been difficult to determine their structures. Here we show the structure of a diiron hydroxylase intermediate formed during a reaction with toluene. Density functional theory geometry optimizations of an active site model reveal that the intermediate is an arylperoxo Fe2+/Fe3+ species with delocalized aryl radical character. The structure suggests that a carboxylate ligand of the diiron centre may trigger homolytic cleavage of the O-O bond by transferring a proton from a metal-bound water. Our work provides the spatial and electronic constraints needed to propose a comprehensive mechanism for diiron enzyme arene hydroxylation that accounts for many prior experimental results.

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship

BackgroundAldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the kcat value below 1 min−1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.ResultsThe amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The kcat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%.ConclusionsSome important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the kcat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.

Redox sensing molecular mechanism of an iron metabolism regulatory protein FBXL5

FBXL5 is a subunit of the SCFFBXL5 ubiquitin ligase complex that targets the proteasomal degradation of iron regulatory protein IRP2, which is an important regulator in iron metabolism. The degradation of FBXL5 itself is regulated in an iron- and oxygen-responsive manner through its diiron center containing Hr-like domain. Although the crystal structure of the Hr-like domain of FBXL5 and its degradation based on iron/oxygen sensing has been reported, the redox sensing molecular mechanism is still not clear. Herein the redox properties of FBXL5 were investigated via EPR, direct electrochemistry, SRCD, fluorescence emission spectroscopy, and redox kinetics. The results indicated that the conformation and function of FBXL5 are tuned by the redox states of the diiron center. The redox reactions of the diiron center are accompanied with conformational changes and iron release, which are associated with FBXL5 stability and degradation. These results provide insights into the redox sensing mechanism by which FBXL5 can serve as an iron metabolism regulator within mammalian cells.

Structure of Escherichia coli Flavodiiron Nitric Oxide Reductase

Flavodiiron proteins (FDPs) are present in organisms from all domains of life and have been described so far to be involved in the detoxification of oxygen or nitric oxide (NO), acting as O2 and/or NO reductases. The Escherichia coli FDP, named flavorubredoxin (FlRd), is the most extensively studied FDP. Biochemical and in vivo studies revealed that FlRd is involved in NO detoxification as part of the bacterial defense mechanisms against reactive nitrogen species. E. coli FlRd has a clear preference for NO as a substrate in vitro, exhibiting a very low reactivity toward O2. To contribute to the understanding of the structural features defining this substrate selectivity, we determined the crystallographic structure of E. coli FlRd, both in the isolated and reduced states. The overall tetrameric structure revealed a highly conserved flavodiiron core domain, with a metallo-β-lactamase-like domain containing a diiron center, and a flavodoxin domain with a flavin mononucleotide cofactor. The metal center in the oxidized state has a μ-hydroxo bridge coordinating the two irons, while in the reduced state, this moiety is not detected. Since only the flavodiiron domain was observed in these crystal structures, the structure of the rubredoxin domain was determined by NMR. Tunnels for the substrates were identified, and through molecular dynamics simulations, no differences for O2 or NO permeation were found. The present data represent the first structure for a NO-selective FDP.

Using Biosynthetic Models of Heme-Copper Oxidase and Nitric Oxide Reductase in Myoglobin to Elucidate Structural Features Responsible for Enzymatic Activities.

In biology, a heme-Cu center in heme-copper oxidases (HCOs) is used to catalyze the four-electron reduction of oxygen to water, while a heme-nonheme diiron center in nitric oxide reductases (NORs) is employed to catalyze the two-electron reduction of nitric oxide to nitrous oxide. Although much progress has been made in biochemical and biophysical studies of HCOs and NORs, structural features responsible for similarities and differences within the two enzymatic systems remain to be understood. Here, we discuss the progress made in the design and characterization of myoglobin-based enzyme models of HCOs and NORs. In particular, we focus on use of these models to understand the structure-function relations between HCOs and NORs, including the role of nonheme metals, conserved amino acids in the active site, heme types and hydrogen-bonding network in tuning enzymatic activities and total turnovers. Insights gained from these studies are summarized and future directions are proposed.

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO.

Molecular mechanisms underlying the repair of nitrosylated [Fe-S] clusters by the microbial protein YtfE remain poorly understood. The X-ray crystal structure of YtfE, in combination with EPR, magnetic circular dichroism (MCD), UV, and (17) O-labeling electron spin echo envelope modulation measurements, show that each iron of the oxo-bridged Fe(II) -Fe(III) diiron core is coordinatively unsaturated with each iron bound to two bridging carboxylates and two terminal histidines in addition to an oxo-bridge. Structural analysis reveals that there are two solvent-accessible tunnels, both of which converge to the diiron center and are critical for capturing substrates. The reactivity of the reduced-form Fe(II) -Fe(II) YtfE toward nitric oxide demonstrates that the prerequisite for N2 O production requires the two iron sites to be nitrosylated simultaneously. Specifically, the nitrosylation of the two iron sites prior to their reductive coupling to produce N2 O is cooperative. This result suggests that, in addition to any repair of iron centers (RIC) activity, YtfE acts as an NO-trapping scavenger to promote the NO to N2 O transformation under low NO flux, which precedes nitrosative stress.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C.farreri (denoted as CfFER). The full length of CfFER is 848bp and contains a 5'-UTR of 113bp, a 3'-UTR of 219bp, and a complete open reading frame (ORF) of 516bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12h post-infection (hpi) and then normalized at 48hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C.farreri and protection against pathogen challenge.

Trichomonas vaginalis Repair of Iron Centres Proteins: The Different Role of Two Paralogs

Trichomonas vaginalis, the causative parasite of one of the most prevalent sexually transmitted diseases is, so far, the only protozoan encoding two putative Repair of Iron Centres (RIC) proteins. Homologs of these proteins have been shown to protect bacteria from the chemical stress imposed by mammalian immunity. In this work, the biochemical and functional characterisation of the T. vaginalis RICs revealed that the two proteins have different properties. Expression of ric1 is induced by nitrosative stress but not by hydrogen peroxide, while ric2 transcription remained unaltered under similar conditions. T. vaginalis RIC1 contains a di-iron centre, but RIC2 apparently does not. Only RIC1 resembles bacterial RICs on spectroscopic profiling and repairing ability of oxidatively-damaged iron-sulfur clusters. Unexpectedly, RIC2 was found to bind DNA plasmid and T. vaginalis genomic DNA, a function proposed to be related with its leucine zipper domain. The two proteins also differ in their cellular localization: RIC1 is expressed in the cytoplasm only, and RIC2 occurs both in the nucleus and cytoplasm. Therefore, we concluded that the two RIC paralogs have different roles in T. vaginalis, with RIC2 showing an unprecedented DNA binding ability when compared with all other until now studied RICs.

The Dual Role of the Plastid Terminal Oxidase PTOX: Between a Protective and a Pro-oxidant Function.

The Dual Role of the Plastid Terminal Oxidase PTOX: Between a Protective and a Pro-oxidant Function.

Diiron centre mutations in Ciona intestinalis alternative oxidase abolish enzymatic activity and prevent rescue of cytochrome oxidase deficiency in flies.

The mitochondrial alternative oxidase, AOX, carries out the non proton-motive re-oxidation of ubiquinol by oxygen in lower eukaryotes, plants and some animals. Here we created a modified version of AOX from Ciona instestinalis, carrying mutations at conserved residues predicted to be required for chelation of the diiron prosthetic group. The modified protein was stably expressed in mammalian cells or flies, but lacked enzymatic activity and was unable to rescue the phenotypes of flies knocked down for a subunit of cytochrome oxidase. The mutated AOX transgene is thus a potentially useful tool in studies of the physiological effects of AOX expression.

Special Delivery: The Role of Iron Chaperones in the Distribution of Iron in Developing Red Cells

Abstract Iron is an essential nutrient for every cell in the human body, yet it can also be a potent cellular toxin. Iron is essential because enzymes that require iron co-factors (namely, heme, iron-sulfur clusters, mononuclear and diiron centers) are involved in virtually every major metabolic process in the cell. Hundreds of iron, zinc, copper, and manganese proteins are expressed in human cells, yet little is known about the mechanisms by which these metalloproteins acquire their native metal ligands and avoid mis-metallation. Significant advances have been made in understanding the delivery of iron to iron-dependent enzymes in the cytosol. Poly(rC)-binding proteins (PCBPs) are multifunctional adaptors that mediate interactions between single-stranded nucleic acids, iron cofactors, and other proteins, affecting the fate and activity of the components of these interactions. PCBP1 is an iron-binding protein that delivers iron to ferritin in human cells via a direct protein-protein interaction and can be described as an iron chaperone. PCBP2, a human paralog of PCBP1, is also involved in the delivery of iron to ferritin, both in yeast cells and in human cells, suggesting that PCBP1 and PCBP2 work together in iron delivery. PCBP1 and PCBP2 can also deliver iron to the two major families of non-heme iron enzymes: the mononuclear and dinuclear iron-dependent oxygenases. The prolyl hydroxylases (PHDs) are mononuclear iron enzymes that regulate the degradation of hypoxia inducible factor 1 (HIF1). Misregulation of the HIF transcription factors leads to the development of a variety of cancers in humans. Cells depend on the iron chaperones PCBP1 and PCBP2 to maintain iron in the enzymatic active site of PHDs and the related enzyme, asparagyl hydroxylase and to maintain proper regulation of HIF1a, especially under conditions of iron limitation. Deoxyhypusine hydroxylase (DOHH) is a dinuclear iron enzyme that is required for the posttranslational modification of a single lysine residue on eukaryotic initiation factor 5A (eIF5A). EIF5A and the conversion of this conserved lysine to hypusine are essential in all eukaryotes, as it enables the translation of peptides containing polyproline sequences. We found that cells depleted of PCBP1 or PCBP2 exhibited reduced activity of DOHH, which was due to a loss of iron in the active site of the enzyme. Thus, PCBPs are basic components of a cytosolic iron delivery system that serves both of the major classes of non-heme iron enzymes in the cytosol. Recent work has indicated that a second type of iron delivery system in the cytosol is mediated by a monothiol glutaredoxin, Glrx3, which, in vitro, can bind and transfer iron-sulfur clusters to recipient apo-iron-sulfur proteins. We have determined that PCBP1 directly interacts with Glrx3-containing complexes and can affect the coordination of iron-sulfur clusters by Glrx3. The huge flux of iron through the developing erythroid cell represents unique challenges for the utilization of cellular iron. We have examined the role of PCBPs as iron chaperones in terminal erythroid differentiation. The role of ferritin in erythroid cell maturation is controversial, but our data indicate that ferritin, PCBPs and NCOA4 are critical factors in erythrocyte development. The flux of iron through ferritin via the lysosome appears to be critical for the transfer of iron to mitochondria for heme synthesis. Disclosures No relevant conflicts of interest to declare.

A Diatom Ferritin Optimized for Iron Oxidation but Not Iron Storage

Ferritin from the marine pennate diatom Pseudo-nitzschia multiseries (PmFTN) plays a key role in sustaining growth in iron-limited ocean environments. The di-iron catalytic ferroxidase center of PmFTN (sites A and B) has a nearby third iron site (site C) in an arrangement typically observed in prokaryotic ferritins. Here we demonstrate that Glu-44, a site C ligand, and Glu-130, a residue that bridges iron bound at sites B and C, limit the rate of post-oxidation reorganization of iron coordination and the rate at which Fe(3+) exits the ferroxidase center for storage within the mineral core. The latter, in particular, severely limits the overall rate of iron mineralization. Thus, the diatom ferritin is optimized for initial Fe(2+) oxidation but not for mineralization, pointing to a role for this protein in buffering iron availability and facilitating iron-sparing rather than only long-term iron storage.

Linking chlorophyll biosynthesis to a dynamic plastoquinone pool

Chlorophylls are essential cofactors in photosynthesis. All steps in the chlorophyll pathway are well characterized except for the cyclase reaction in which the fifth ring of the chlorophyll molecule is formed during conversion of Mg-protoporphyrin IX monomethyl ester into Protochlorophyllide. The only subunit of the cyclase identified so far, is AcsF (Xantha-l in barley and Chl27 in Arabidopsis). This subunit contains a typical consensus di-iron-binding sequence and belongs to a subgroup of di-iron proteins, such as the plastid terminal oxidase (PTOX) in the chloroplast and the alternative oxidase (AOX) found in mitochondria. In order to complete the catalytic cycle, the irons of these proteins need to be reduced from Fe(3+) to Fe(2+) and either a reductase or another form of reductant is required. It has been reported that the alternative oxidase (AOX) and the plastid terminal oxidase (PTOX) utilize the di-iron center to oxidise ubiquinol and plastoquinol, respectively. In this paper, we have used a specific inhibitor of di-iron proteins as well as Arabidopsis and barley mutants affected in regulation of photosynthetic electron flow, to show that the cyclase step indeed is directly coupled to the plastoquinone pool. Thus, plastoquinol might act as an electron donor for the cyclase reaction and thereby fulfil the role of a cyclase reductase. That would provide a functional connection between the redox status of the thylakoids and the biosynthesis of chlorophyll.

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

AbstractReview: 56 refs.

Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of "Fe2O2" intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.