Yisong Guo

The Crystal Structure of a High-Spin OxoIron(IV) Complex and Characterization of Its Self-Decay Pathway

[Fe(IV)(O)(TMG(3)tren)](2+) (1; TMG(3)tren = 1,1,1-tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine) is a unique example of an isolable synthetic S = 2 oxoiron(IV) complex, which serves as a model for the high-valent oxoiron(IV) intermediates observed in nonheme iron enzymes. Congruent with DFT calculations predicting a more reactive S = 2 oxoiron(IV) center, 1 has a lifetime significantly shorter than those of related S = 1 oxoiron(IV) complexes. The self-decay of 1 exhibits strictly first-order kinetic behavior and is unaffected by solvent deuteration, suggesting an intramolecular process. This hypothesis was supported by ESI-MS analysis of the iron products and a significant retardation of self-decay upon use of a perdeuteromethyl TMG(3)tren isotopomer, d(36)-1 (KIE = 24 at 25 degrees C). The greatly enhanced thermal stability of d(36)-1 allowed growth of diffraction quality crystals for which a high-resolution crystal structure was obtained. This structure showed an Fe horizontal lineO unit (r = 1.661(2) A) in the intended trigonal bipyramidal geometry enforced by the sterically bulky tetramethylguanidinyl donors of the tetradentate tripodal TMG(3)tren ligand. The close proximity of the methyl substituents to the oxoiron unit yielded three symmetrically oriented short C-D...O nonbonded contacts (2.38-2.49 A), an arrangement that facilitated self-decay by rate-determining intramolecular hydrogen atom abstraction and subsequent formation of a ligand-hydroxylated iron(III) product. EPR and Mossbauer quantification of the various iron products, referenced against those obtained from reaction of 1 with 1,4-cyclohexadiene, allowed formulation of a detailed mechanism for the self-decay process. The solution of this first crystal structure of a high-spin (S = 2) oxoiron(IV) center represents a fundamental step on the path toward a full understanding of these pivotal biological intermediates.

A More Reactive Trigonal Bipyramidal High-Spin Oxoiron(IV) Complex with a cis-Labile Site

The trigonal-bipyramidal high-spin (S = 2) oxoiron(IV) complex [Fe(IV)(O)(TMG(2)dien)(CH(3)CN)](2+) (7) was synthesized and spectroscopically characterized. Substitution of the CH(3)CN ligand by anions, demonstrated here for X = N(3)(-) and Cl(-), yielded additional S = 2 oxoiron(IV) complexes of general formulation [Fe(IV)(O)(TMG(2)dien)(X)](+) (7-X). The reduced steric bulk of 7 relative to the published S = 2 complex [Fe(IV)(O)(TMG(3)tren)](2+) (2) was reflected by enhanced rates of intermolecular substrate oxidation.

Identification of a Catalytic Iron-Hydride at the H-Cluster of [FeFe]-Hydrogenase

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H2 and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (Hhyd) of the [FeFe]-hydrogenase from Chlamydomonas reinhardtii. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]H) with CO and CN- ligands. Mössbauer analysis and density functional theory (DFT) calculations show that Hhyd consists of a reduced [4Fe-4S]H+ coupled to a diferrous [2Fe]H with a terminally bound Fe-hydride. The existence of the Fe-hydride in Hhyd was demonstrated by an unusually low Mössbauer isomer shift of the distal Fe of the [2Fe]H subcluster. A DFT model of Hhyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover.

Mechanism of the C5 stereoinversion reaction in the biosynthesis of carbapenem antibiotics

Carbapenems Through the Looking Glass The carbapenem class of antibiotics is a critical weapon in the ongoing fight against drug-resistant bacteria. Microbial biosynthesis of these compounds, which contain a strained β-lactam ring motif, proceeds via a precursor that has the wrong configuration at one of the ring carbons. Chang et al. (p. 1140) combined x-ray crystallography with multiple spectroscopic probes to map out the mechanism by which the CarC enzyme inverts the precursor configuration to its mirror image. Crystallography and spectroscopy detail a key mechanistic step in the microbial biosynthesis of an important antibiotic class. The bicyclic β-lactam/2-pyrrolidine precursor to all carbapenem antibiotics is biosynthesized by attachment of a carboxymethylene unit to C5 of l-proline followed by β-lactam ring closure. Carbapenem synthase (CarC), an Fe(II) and 2-(oxo)glutarate (Fe/2OG)–dependent oxygenase, then inverts the C5 configuration. Here we report the structure of CarC in complex with its substrate and biophysical dissection of its reaction to reveal the stereoinversion mechanism. An Fe(IV)-oxo intermediate abstracts the hydrogen (H•) from C5, and tyrosine 165, a residue not visualized in the published structures of CarC lacking bound substrate, donates H• to the opposite face of the resultant radical. The reaction oxidizes the Fe(II) cofactor to Fe(III), limiting wild-type CarC to one turnover, but substitution of the H•-donating tyrosine disables stereoinversion and confers to CarC the capacity for catalytic substrate oxidation.

Biophysical Characterization of Iron in Mitochondria Isolated from Respiring and Fermenting Yeast

The distributions of Fe in mitochondria isolated from respiring, respiro-fermenting, and fermenting yeast cells were determined with an integrative biophysical approach involving Mossbauer and electronic absorption spectroscopies, electron paramagnetic resonance, and inductively coupled plasma emission mass spectrometry. Approximately 40% of the Fe in mitochondria from respiring cells was present in respiration-related proteins. The concentration and distribution of Fe in respiro-fermenting mitochondria, where both respiration and fermentation occur concurrently, were similar to those of respiring mitochondria. The concentration of Fe in fermenting mitochondria was also similar, but the distribution differed dramatically. Here, levels of respiration-related Fe-containing proteins were diminished approximately 3-fold, while non-heme HS Fe(II) species, non-heme mononuclear HS Fe(III), and Fe(III) nanoparticles dominated. These changes were rationalized by a model in which the pool of non-heme HS Fe(II) ions serves as feedstock for Fe-S cluster and heme biosynthesis. The integrative approach enabled us to estimate the concentration of respiration-related proteins.

EXAFS and NRVS Reveal a Conformational Distortion of the FeMo-cofactor in the MoFe Nitrogenase Propargyl Alcohol Complex

We have used EXAFS and NRVS spectroscopies to examine the structural changes in the FeMo-cofactor active site of the α-70(Ala) variant of Azotobacter vinelandii nitrogenase on binding and reduction of propargyl alcohol (PA). The Mo K-edge near-edge and EXAFS spectra are very similar in the presence and absence of PA, suggesting PA does not bind at Mo. By contrast, Fe EXAFS spectra show a clear and reproducible change in the long Fe-Fe interaction at ~3.7 Å on PA binding with the apparent appearance of a new Fe-Fe interaction at 3.99 Å. An analogous change in the long Mo-Fe 5.1 Å interaction is not seen. The NRVS spectra exclude the possibility of large-scale structural change of the FeMo-cofactor involving breaking the μ(2) Fe-S-Fe bonds of the Fe(6)S(9)X core. The simplest chemically consistent structural change is that the bound form of PA is coordinated at Fe atoms (Fe6 or Fe7) adjacent to the Mo terminus, with a concomitant movement of the Fe away from the central atom X and along the Fe-X bond by about 0.35 Å. This study comprises the first experimental evidence of the conformational changes of the FeMo-cofactor active site on binding a substrate or product.

Cu(I)-catalyzed aerobic cross-dehydrogenative coupling of terminal alkynes with thiols for the construction of alkynyl sulfides

Highly active and selective aerobic cross-dehydrogenative coupling of terminal alkynes with thiols to construct alkynyl sulfides catalyzed by Cu(I) using molecular oxygen as the oxidant has been demonstrated under mild reaction conditions. The process is applicable to a wide range of alkynes and various thiols and is compatible with a variety of functional groups on both alkyne and thiol coupling partners.

Endoperoxide formation by an α-ketoglutarate-dependent mononuclear non-haem iron enzyme

Many peroxy-containing secondary metabolites have been isolated and shown to provide beneficial effects to human health. Yet, the mechanisms of most endoperoxide biosyntheses are not well understood. Although endoperoxides have been suggested as key reaction intermediates in several cases, the only well-characterized endoperoxide biosynthetic enzyme is prostaglandin H synthase, a haem-containing enzyme. Fumitremorgin B endoperoxidase (FtmOx1) from Aspergillus fumigatus is the first reported α-ketoglutarate-dependent mononuclear non-haem iron enzyme that can catalyse an endoperoxide formation reaction. To elucidate the mechanistic details for this unique chemical transformation, we report the X-ray crystal structures of FtmOx1 and the binary complexes it forms with either the co-substrate (α-ketoglutarate) or the substrate (fumitremorgin B). Uniquely, after α-ketoglutarate has bound to the mononuclear iron centre in a bidentate fashion, the remaining open site for oxygen binding and activation is shielded from the substrate or the solvent by a tyrosine residue (Y224). Upon replacing Y224 with alanine or phenylalanine, the FtmOx1 catalysis diverts from endoperoxide formation to the more commonly observed hydroxylation. Subsequent characterizations by a combination of stopped-flow optical absorption spectroscopy and freeze-quench electron paramagnetic resonance spectroscopy support the presence of transient radical species in FtmOx1 catalysis. Our results help to unravel the novel mechanism for this endoperoxide formation reaction.

Evidence that the Fosfomycin-Producing Epoxidase, HppE, Is a Non–Heme-Iron Peroxidase

Just Add Peroxide The HppE enzyme uses iron to catalyze oxidation of an alcohol to an epoxide ring in the biosynthesis of the antibiotic fosfomycin. Because this process is a two-electron oxidation, it has been unclear how the enzyme reduces its presumed oxidative partner O2 all the way to water. Where do the two extra electrons come from? Wang et al. (p. 991, published 10 October; see the Perspective by Raushel) now show that HppE is actually a peroxidase, and thus reduces H2O2, for which just two electrons are sufficient. The result expands the structural scope of iron-bearing peroxidase enzymes beyond heme motifs. An iron enzyme previously thought to use O2 as an oxidant appears to use peroxide instead. [Also see Perspective by Raushel] The iron-dependent epoxidase HppE converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonate] in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE has been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (H•) from C1 of S-HPP to initiate epoxide ring closure. We show here that the preferred cosubstrate is actually H2O2 and that HppE therefore almost certainly uses an iron(IV)-oxo complex as the H• abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 implies that reduction of O2 and utilization of the resultant H2O2 were actually operant.

Nuclear resonance vibrational spectroscopy and electron paramagnetic resonance spectroscopy of 57Fe-enriched [FeFe] hydrogenase indicate stepwise assembly of the H-cluster.

The [FeFe] hydrogenase from Clostridium pasteurianum (CpI) harbors four Fe-S clusters that facilitate the transfer of an electron to the H-cluster, a ligand-coordinated six-iron prosthetic group that catalyzes the redox interconversion of protons and H(2). Here, we have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study the iron centers in CpI, and we compare our data to that for a [4Fe-4S] ferredoxin as well as a model complex resembling the [2Fe](H) catalytic domain of the H-cluster. To enrich the hydrogenase with (57)Fe nuclei, we used cell-free methods to post-translationally mature the enzyme. Specifically, inactive CpI apoprotein with (56)Fe-labeled Fe-S clusters was activated in vitro using (57)Fe-enriched maturation proteins. This approach enabled us to selectively label the [2Fe](H) subcluster with (57)Fe, which NRVS confirms by detecting (57)Fe-CO and (57)Fe-CN normal modes from the H-cluster nonprotein ligands. The NRVS and iron quantification results also suggest that the hydrogenase contains a second (57)Fe-S cluster. Electron paramagnetic resonance (EPR) spectroscopy indicates that this (57)Fe-enriched metal center is not the [4Fe-4S](H) subcluster of the H-cluster. This finding demonstrates that the CpI hydrogenase retained an (56)Fe-enriched [4Fe-4S](H) cluster during in vitro maturation, providing unambiguous evidence of stepwise assembly of the H-cluster. In addition, this work represents the first NRVS characterization of [FeFe] hydrogenases.