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Nonheme diiron enzymes harness the chemical potential of oxygen to catalyze challenging reactions in biology. In their resting state, these enzymes have a diferrous cofactor that is coordinated by histidine and carboxylate ligands. Upon exposure to oxygen, the cofactor oxidizes to its diferric state forming a peroxo- adduct, capable of catalyzing a wide range of oxidative chemistries such as desaturation and heteroatom oxidation. Despite their versatility and prowess, an emerging subset of nonheme diiron enzymes has inherent cofactor instability making them resistant to structural characterization. This feature is widespread among members of the heme-oxygenase-like diiron oxidase/oxygenase (HDO) superfamily. HDOs have a flexible core structure that remodels upon metal binding. Although ~9600 HDOs have been unearthed, few have undergone functional characterization to date. In this chapter, we describe the methods that have been used to characterize the HDO N-oxygenase, SznF. We demonstrate the overexpression and purification of apo-SznF and methodology specifically designed to aid in obtaining an X-ray structure of holo-SznF. We also describe the characterization of the transient SznF-peroxo-Fe(III)2 complex by stopped-flow absorption and Mössbauer spectroscopies. These studies provide the framework for the characterization of new members of the HDO superfamily.

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Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes the deformylation of aldehydes to generate alkanes/alkenes. In this study, we report for the first time that under anaerobic or limited oxygen conditions, Prochlorococcus marinus (PmADO) can generate full-length fatty alcohols from fatty aldehydes without eliminating a carbon unit. In contrast to ADO's native activity, which requires electrons from the Fd/FNR electron transfer complex, ADO's aldehyde reduction activity requires only NAD(P)H. Our results demonstrated that the yield of alcohol products could be affected by oxygen concentration and the type of aldehyde. Under strictly anaerobic conditions, yields of octanol were up to 31%. Moreover, metal cofactors are not involved in the aldehyde reductase activity of PmADO because the yields of alcohols obtained from apoenzyme and holoenzyme treated with various metals were similar under anaerobic conditions. In addition, PmADO prefers medium-chain aldehydes, specifically octanal (kcat/Km around 15 × 10-3 µM-1min-1). The findings herein highlight a new activity of PmADO, which may be applied as a biocatalyst for the industrial synthesis of fatty alcohols.

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Ferritin-like carboxylate-bridged non-heme diiron enzymes activate O2 for a variety of difficult reactions throughout nature. These reactions often begin by abstraction of hydrogen from strong CH bonds. The enzymes activate O2 at their diferrous cofactors to form canonical diferric peroxo intermediates, with a range of possible coordination modes. Herein, we explore the ability of high-energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD XAS) to provide insight into the nature of peroxo level intermediates in non-heme diiron proteins. Freeze quenched (FQ) peroxo intermediates from p-aminobenzoate N-oxygenase (AurF), aldehyde-deformylating oxygenase (ADO), and the ß subunit of class Ia ribonucleotide reductase from Escherichia coli (Ecß) are investigated. All three intermediates are proposed to adopt different peroxo binding modes, and each exhibit different Fe Kα HERFD XAS pre-edge features and intensities. As these FQ-trapped samples consist of multiple species, deconvolution of HERFD XAS spectra based on speciation, as determined by Mössbauer spectroscopy, is also necessitated - yielding 'pure' diferric peroxo HERFD XAS spectra from dilute protein samples. Finally, the impact of a given peroxo coordination mode on the HERFD XAS pre-edge energy and intensity is evaluated through time-dependent density functional theory (TDDFT) calculations of the XAS spectra on a series of hypothetical model complexes, which span a full range of possible peroxo coordination modes to a diferric core. The utility of HERFD XAS for future studies of enzymatic intermediates is discussed.

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Iron and oxygen chemistry is mediated by iron proteins for many biological functions. Carboxylate-bridged diiron enzymes including ferritin have the common mechanism of oxygen activation via peroxodiferric intermediates. However, the route for iron uptake and the structural identification of intermediates still remain incomplete. The 4-fold symmetry channel of Helicobacter pylori ferritin was previously proposed as the iron-uptake route in eubacteria, but the amino acid residues at the 4-fold channel are not highly conserved. Here, we show evidence for a short path for iron uptake from His93 on the surface to the ferroxidase center in H. pylori ferritin and Escherichia coli ferritin. The amino acid residues along this path are highly conserved in Gram-negative bacteria and some archaea, and the mutants containing S20A and H93L showed significantly decreased iron oxidation. Surprisingly, the E. coli ferritin S20A crystal structure showed oxygen binding and side-on, symmetric µ-η2:η2 peroxodiferric and oxodiferric intermediates. The results provide the structural basis for understanding the chemical nature of intermediates in iron oxidation in bacteria and some of archaea.

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Although cells express hundreds of metalloenzymes, the mechanisms by which apoenzymes receive their metal cofactors are largely unknown. Poly(rC)-binding proteins PCBP1 and PCBP2 are multifunctional adaptor proteins that bind iron and deliver it to ferritin for storage or to prolyl and asparagyl hydroxylases to metallate the mononuclear iron center. Here, we show that PCBP1 and PCBP2 also deliver iron to deoxyhypusine hydroxylase (DOHH), the dinuclear iron enzyme required for hypusine modification of the translation factor eukaryotic initiation factor 5A. Cells depleted of PCBP1 or PCBP2 exhibited loss of DOHH activity and loss of the holo form of the enzyme in cells, particularly when cells were made mildly iron-deficient. Lysates containing PCBP1 and PCBP2 converted apo-DOHH to holo-DOHH in vitro with greater efficiency than lysates lacking PCBP1 or PCBP2. PCBP1 bound to DOHH in iron-treated cells but not in control or iron-deficient cells. Depletion of PCBP1 or PCBP2 had no effect on the cytosolic Fe-S cluster enzyme xanthine oxidase but led to loss of cytosolic aconitase activity. Loss of aconitase activity was not accompanied by gain of RNA-binding activity, a pattern suggesting the incomplete disassembly of the [4Fe-4S] cluster. PCBP depletions had minimal effects on total cellular iron, mitochondrial iron levels, and heme synthesis. Thus, PCBP1 and PCBP2 may serve as iron chaperones to multiple classes of cytosolic nonheme iron enzymes and may have a particular role in restoring metal cofactors that are spontaneously lost in iron deficient cells.

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Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for any biological system. The nature of the diiron cluster and the newly recognized products from ADO catalysis hold implications for the mechanism of C-C bond cleavage.