Keywordsbioinorganic chemistry iron-oxo; nonheme iron complexes; high-valent compounds; enzyme models High-valent oxoferryl intermediates have been proposed as the active oxidants in the catalytic cycles of a wide range of mononuclear non-heme oxygen activating enzymes.[1] These high-valent species have now been spectroscopically characterized for four enzymes and were found in all instances to contain high-spin (S = 2) iron(IV) centers.[2] Contemporaneously, the first examples of the existing family of synthetic nonheme oxoiron(IV) complexes were characterized, [3][4][5] which are exclusively octahedral and in all but one case exhibit the S = 1, rather than S = 2, spin-state. Given that DFT suggests higher reactivity for an S = 2 oxoiron(IV) unit, [6,7] it is perhaps not surprising that there is a scarcity of such complexes. Indeed, the only example to date is [Fe IV (O)(H 2 O) 5 ] 2+ (1),

Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer’s disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.

No abstract

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen-and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mö ssbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a ( -1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O 2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein ⌬ 9 -desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.H ypusine is an unusual, but highly conserved, amino acid that is found only in the eukaryotic translational initiation factor 5A (eIF5A), a protein that regulates cell proliferation (1, 2). The biosynthesis of eIF5A involves a posttranslational modification of the eIF5A precursor, where a lysine residue is first modified to deoxyhypusine (Dhp) by deoxyhypusine synthase (DHS) and then the nascent Dhp is hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine (Hpu) (Scheme 1) (1, 2). The importance of hypusine and these 2 enzymes has been shown by several studies where depletion of spermidine (3) or inhibition of either DHS or DOHH (4, 5) leads to a decrease of hypusinecontaining eIF5A [eIF5A(Hpu)] and inhibition of eukaryotic cell growth. Consequently, these results suggest that eIF5A and DOHH could be promising targets for antitumor (6) and anti-HIV-1 therapies (7).The hydroxylase activity of recombinant human DOHH (hDOHH) has been shown to depend on Fe(II) and not on any other physiologically relevant divalent metal ion. An estimated iron-to-holoprotein stoichiometry of 2 is observed (8). Sequence examination, homology modeling, and mutagenesis experiments suggest 2 possible iron binding sites consisting of histidine and carboxylate ligands (8,9). Thus at first glance, hDOHH appears to resemble members of the superfamily of bacterial diiron multicomponent monooxygenases, like methane or toluene monooxygenase, that use nonheme diiron centers to activate dioxygen for the hydroxylat...

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) --> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

No abstract

Methane monooxygenase (MMO) catalyzes the energy dependent oxidation of methane to methanol in methanotrophic bacteria. 1, 2 In these organisms two different methane monooxygenases have been identified, namely a membrane-associated or particulate MMO (pMMO) and a cytoplasmic or soluble MMO (sMMO). In methanotrophs that express both forms of the enzyme, the copper concentration during growth dictates which MMO is expressed. 2-5 In cells cultured under a low copper/biomass ratio, the sMMO is predominately expressed, whereas cells cultured at higher copper/biomass ratios exclusively express the pMMO (sMMO is not transcribed). 6-8 The sMMO is a well characterized three-component enzyme consisting of a hydroxylase, a reductase and a regulatory protein. 9-12 Spectroscopic and X-ray crystallographic studies have established that the hydroxylase contains an oxygen bridged diiron cluster. 13-16 Here we provide evidence that pMMO contains a diiron cluster as well.Owing to the low specific activity and instability of most pMMO preparations, 6, 17-20 comparatively little is known about the molecular properties of this enzyme. As isolated, pMMO is composed of three polypeptides with molecular masses of 45,000, 26,000, and 23,000 Da with a subunit structure of (αβγ) 3 . 6, 17, 18, 20-22 Most researchers agree that each αβγ contains 2 -3 Cu atoms 2, 6, 17-20, 23 although one group has suggested that 15 Cu atoms are arranged into catalytic and electron transfer trinuclear copper clusters. 22, 24, 25 The 2.8 Å resolution crystal structure of pMMO revealed that each αβγ contained a dicopper site, a mononuclear copper site, and a third site occupied by zinc. 21, 23 However, the preparation used for growing the crystal was inactive and did not contain zinc (which was added to the crystallization buffer). 21, 23The involvement of non-heme iron in methane oxidation by the pMMO has been proposed by some laboratories 6, 17, 26-29 and disputed by others. 22, 24, 30 In our laboratory at Iowa State University we have observed that preparations of pMMO showing highest specific activity contain 1-2 iron atoms. 6 We therefore decided to employ Mössbauer spectroscopy to characterize the iron components. This technique is particularly well suited to explore iron environments that are EPR-silent and optically uninformative in the visible region, as is the Author E-mail : emunck@cmu.edu. Table 1 lists analytical and activity data of our purified pMMO sample and of whole cells grown at high copper (80μM) and iron (40μM); the entries are discussed in the Supporting Information. Figure 1 shows 4.2 K Mössbauer spectra of purified pMMO. The central portion of the 45 mT spectrum ( Figure 1A) exhibits two overlapping doublets with ΔE Q (1) = 1.05 mm/ s, δ(1) = 0.50 mm/s (≈20% of total Fe) and ΔE Q (2) = 2.65 mm/s, δ(1) = 1.25 mm/s (≈18% of total Fe); the δ value of doublet 2 is characteristic of a high-spin Fe 2+ with octahedral N/O coordination. The majority of the iron in the spectrum of Figure 1A, perhaps up to 60% of total Fe, belongs to a heter...