Ricardo Garcia-Serres

A mononuclear nonheme iron(IV)-oxo complex which is more reactive than cytochrome P450 model compound I

A highly reactive mononuclear nonheme iron(IV)-oxo complex with a low-spin (S = 1) triplet ground state in both C–H bond activation and oxo transfer reactions is reported; this nonheme iron(IV)-oxo complex is more reactive than an iron(IV)-oxo porphyrin π-cation radical (i.e., a model of cytochrome P450 compound I) and is the most reactive species in kinetic studies among nonheme iron(IV)-oxo complexes reported so far. DFT calculations support the experimental results with extremely low activation barriers in the C–H bond activation of cyclohexane and 1,4-cyclohexadiene. The DFT calculations reveal that the S = 1 state is set up to easily lead to the highly reactive S = 2 high-spin iron(IV)-oxo species.

Iron-Sulfur (Fe-S) Cluster Assembly THE SufBCD COMPLEX IS A NEW TYPE OF Fe-S SCAFFOLD WITH A FLAVIN REDOX COFACTOR

Assembly of iron-sulfur (Fe-S) clusters and maturation of Fe-S proteins in vivo require complex machineries. In Escherichia coli, under adverse stress conditions, this process is achieved by the SUF system that contains six proteins as follows: SufA, SufB, SufC, SufD, SufS, and SufE. Here, we provide a detailed characterization of the SufBCD complex whose function was so far unknown. Using biochemical and spectroscopic analyses, we demonstrate the following: (i) the complex as isolated exists mainly in a 1:2:1 (B:C:D) stoichiometry; (ii) the complex can assemble a [4Fe-4S] cluster in vitro and transfer it to target proteins; and (iii) the complex binds one molecule of flavin adenine nucleotide per SufBC2D complex, only in its reduced form (FADH2), which has the ability to reduce ferric iron. These results suggest that the SufBC2D complex functions as a novel type of scaffold protein that assembles an Fe-S cluster through the mobilization of sulfur from the SufSE cysteine desulfurase and the FADH2-dependent reductive mobilization of iron.

[Fe(IV)═O(TBC)(CH3CN)]2+: comparative reactivity of iron(IV)-oxo species with constrained equatorial cyclam ligation.

[Fe(IV)═O(TBC)(CH(3)CN)](2+) (TBC = 1,4,8,11-tetrabenzyl-1,4,8,11-tetraazacyclotetradecane) is characterized, and its reactivity differences relative to [Fe(IV)═O(TMC)(CH(3)CN)](2+) (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) are evaluated in hydrogen atom (H-atom) abstraction and oxo-transfer reactions. Structural differences are defined using X-ray absorption spectroscopy and correlated to reactivities using density functional theory. The S = 1 ground states are highly similar and result in large activation barriers (~25 kcal/mol) due to steric interactions between the cyclam chelate and the substrate (e.g., ethylbenzene) associated with the equatorial π-attack required by this spin state. Conversely, H-atom abstraction reactivity on an S = 2 surface allows for a σ-attack with an axial substrate approach. This results in decreased steric interactions with the cyclam and a lower barrier (~9 kcal/mol). For [Fe(IV)═O(TBC)(CH(3)CN)](2+), the S = 2 excited state in the reactant is lower in energy and therefore more accessible at the transition state due to a weaker ligand field associated with the steric interactions of the benzyl substituents with the trans-axial ligand. This study is further extended to the oxo-transfer reaction, which is a two-electron process requiring both σ- and π-electron transfer and thus a nonlinear transition state. In oxo-transfer, the S = 2 has a lower barrier due to sequential vs concerted (S = 1) two electron transfer which gives a high-spin ferric intermediate at the transition state. The [Fe(IV)═O(TBC)(CH(3)CN)](2+) complex is more distorted at the transition state, with the iron farther out of the equatorial plane due to the steric interaction of the benzyl groups with the trans-axial ligand. This allows for better orbital overlap with the substrate, a lower barrier, and an increased rate of oxo-transfer.

Dinitrogen Activation Upon Reduction of a Triiron(II) Complex

Reaction of a trinuclear iron(II) complex, Fe3 Br3 L (1), with KC8 under N2 leads to dinitrogen activation products (2) from which Fe3 (NH)3 L (2-1; L is a cyclophane bridged by three β-diketiminate arms) was characterized by X-ray crystallography. (1) H NMR spectra of the protonolysis product of 2 synthesized under (14) N2 and (15) N2 confirm atmospheric N2 reduction, and ammonia is detected by the indophenol assay (yield ∼30 %). IR and Mössbauer spectroscopy, and elemental analysis on 2 and 2-1 as well as the tri(amido)triiron(II) 3 and tri(methoxo)triiron 4 congeners support our assignment of the reduction product as containing protonated N-atom bridges.

Post-translational Modification of Ribosomal Proteins STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF RimO FROM THERMOTOGA MARITIMA, A RADICAL S-ADENOSYLMETHIONINE METHYLTHIOTRANSFERASE

Post-translational modifications of ribosomal proteins are important for the accuracy of the decoding machinery. A recent in vivo study has shown that the rimO gene is involved in generation of the 3-methylthio derivative of residue Asp-89 in ribosomal protein S12 (Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 1826–1831). This reaction is formally identical to that catalyzed by MiaB on the C2 of adenosine 37 near the anticodon of several tRNAs. We present spectroscopic evidence that Thermotoga maritima RimO, like MiaB, contains two [4Fe-4S] centers, one presumably bound to three invariant cysteines in the central radical S-adenosylmethionine (AdoMet) domain and the other to three invariant cysteines in the N-terminal UPF0004 domain. We demonstrate that holo-RimO can specifically methylthiolate the aspartate residue of a 20-mer peptide derived from S12, yielding a mixture of mono- and bismethylthio derivatives. Finally, we present the 2.0 Å crystal structure of the central radical AdoMet and the C-terminal TRAM (tRNA methyltransferase 2 and MiaB) domains in apo-RimO. Although the core of the open triose-phosphate isomerase (TIM) barrel of the radical AdoMet domain was conserved, RimO showed differences in domain organization compared with other radical AdoMet enzymes. The unusually acidic TRAM domain, likely to bind the basic S12 protein, is located at the distal edge of the radical AdoMet domain. The basic S12 protein substrate is likely to bind RimO through interactions with both the TRAM domain and the concave surface of the incomplete TIM barrel. These biophysical results provide a foundation for understanding the mechanism of methylthioation by radical AdoMet enzymes in the MiaB/RimO family.

The role of CyaY in iron sulfur cluster assembly on the E. coli IscU scaffold protein.

Progress in understanding the mechanism underlying the enzymatic formation of iron-sulfur clusters is difficult since it involves a complex reaction and a multi-component system. By exploiting different spectroscopies, we characterize the effect on the enzymatic kinetics of cluster formation of CyaY, the bacterial ortholog of frataxin, on cluster formation on the scaffold protein IscU. Frataxin/CyaY is a highly conserved protein implicated in an incurable ataxia in humans. Previous studies had suggested a role of CyaY as an inhibitor of iron sulfur cluster formation. Similar studies on the eukaryotic proteins have however suggested for frataxin a role as an activator. Our studies independently confirm that CyaY slows down the reaction and shed new light onto the mechanism by which CyaY works. We observe that the presence of CyaY does not alter the relative ratio between [2Fe2S]2+ and [4Fe4S]2+ but directly affects enzymatic activity.

Heme Binding Induces Dimerization and Nitration of Truncated β-Amyloid Peptide Aβ16 Under Oxidative Stress†

Several observations suggest a link between (ferric) heme and Alzheimer s disease (AD), for example, an increase in heme oxygenase 1, 2] a loss of complex IV, and abnormal iron metabolism. These observations suggest that heme metabolism is altered in age-related disorders. As a possible explanation, hemin has been proposed to interact with b-amyloid peptides (Ab), and it is now well established that Ab peptides bind ferric heme. The adducts of hemin–Ab peptides also exhibit increased peroxidase activity with respect to free hemin, but these complexes have not been characterized. Herein we provide thermodynamic and spectroscopic data showing that hemin b can coordinate two Ab16 molecules to form the low-spin, six-coordinated complex [hemin(Ab16)2], and this is in equilibrium with the high-spin [hemin(Ab16)] species, the relative amounts of which strongly depend on peptide concentration and temperature. In the presence of H2O2, hemin-Ab16 complexes produce dimerization of the peptide through dityrosine cross-linking, and in addition the peptide undergoes endogenous nitration at Tyr10 when nitrite is also present in solution. This result is important because both cross-linking and nitration at Tyr10 critically enhance Ab aggregation and plaque formation, thus showing that the binding of heme to Ab can indeed be a factor contributing to neuroinflammation and Ab aggregation. The endogenous peroxidative (H2O2) and nitrative (H2O2/NO2 ) activities exhibited by the hemin–Ab16 peptide add to, and are likely more deleterious than, the oxidation and nitration of external substrates which have been previously reported. Upon adding Ab16 to a 50 mm aqueous phosphate buffer solution of hemin b at pH 7.4 and 300 K, the broad Soret band of the complex near l = 385 nm progressively changes to give a sharp, red-shifted band at l = 414 nm, with additional visible bands at l = 535 and 566 nm (Figure 1C, and see the Supporting Information). The conversion from the high-spin to the low-spin hemin species is complete after addition of 20 equivalents of Ab16, and spectrophotometric data analysis (see the Supporting Information) confirms a 2:1 Ab16/hemin stoichiometry, thus indicating the formation of the [hemin(Ab16)2] complex. The Mçssbauer and H NMR spectra are consistent with a low-spin (S = 1/2) iron(III) center coordinated by two axial imidazole ligands. However, below saturating amounts of Ab16, the Mçssbauer spectrum clearly indicates the presence of a minor high-spin species (Figure 2). Figure 1. UV/vis spectra recorded in 50 mm phosphate buffer solution at pH 7.4. A) Hemin b (1.2 10 5 m). B) Hemin b in the presence of an excess (20 equiv) of Ab16 peptide at 319 K. C) The same as (B) but at 300 K, the spectral changes are fully reversible. The D symbol indicates an increase in temperature.

Highly reactive nonheme iron(III) iodosylarene complexes in alkane hydroxylation and sulfoxidation reactions.

High-spin iron(III) iodosylarene complexes bearing an N-methylated cyclam ligand are synthesized and characterized using various spectroscopic methods. The nonheme high-spin iron(III) iodosylarene intermediates are highly reactive oxidants capable of activating strong C-H bonds of alkanes; the reactivity of the iron(III) iodosylarene intermediates is much greater than that of the corresponding iron(IV) oxo complex. The electrophilic character of the iron(III) iodosylarene complexes is demonstrated in sulfoxidation reactions.

4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

Background: 4-Demethylwyosine synthase (TYW1) is a tRNA-modifying metalloenzyme involved in the biosynthesis of wyosine. Results: TYW1 enzyme belongs to the Radical-SAM superfamily with two Fe-S clusters involved in catalysis. Conclusion: The canonical Radical-SAM cluster binds and activates SAM co-factor, whereas the additional [4Fe-4S] cluster is shown to interact with the pyruvate co-substrate. Significance: This study helps to understand how radical-SAM enzymes with two Fe-S centers can synergistically achieve challenging radical insertion reactions. Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

4-demethylwyosine synthase from Pyrococcus abyssi is a Radical-SAM enzyme with an additional [4Fe-4S]+2 cluster which interacts with the pyruvate co-substrate

Background: 4-Demethylwyosine synthase (TYW1) is a tRNA-modifying metalloenzyme involved in the biosynthesis of wyosine. Results: TYW1 enzyme belongs to the Radical-SAM superfamily with two Fe-S clusters involved in catalysis. Conclusion: The canonical Radical-SAM cluster binds and activates SAM co-factor, whereas the additional [4Fe-4S] cluster is shown to interact with the pyruvate co-substrate. Significance: This study helps to understand how radical-SAM enzymes with two Fe-S centers can synergistically achieve challenging radical insertion reactions. Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called “Radical-SAM.” These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX3CX2C motif, and S-adenosyl-l-methionine (SAM) to generate a 5′-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.