Yoshitaka Yoda

Elucidation of the Fe(iv)=O intermediate in the catalytic cycle of the halogenase SyrB2

SUMMARY Mononuclear non-haem iron (NHFe) enzymes catalyse a wide variety of oxidative reactions including halogenation, hydroxylation, ring closure, desaturation, and aromatic ring cleavage. These are highly important for mammalian somatic processes such as phenylalanine metabolism, production of neurotransmitters, hypoxic response, and the biosynthesis of natural products.1–3 The key reactive intermediate in the catalytic cycles of these enzymes is an S = 2 FeIV=O species, which has been trapped for a number of NHFe enzymes4–8 including the halogenase SyrB2, the subject of this study. Computational studies to understand the reactivity of the enzymatic NHFe FeIV=O intermediate9–13 are limited in applicability due to the paucity of experimental knowledge regarding its geometric and electronic structures, which determine its reactivity. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes of Fe on the nature of the FeIV=O active site.14–16 Here we present the first NRVS structural characterisation of the reactive FeIV=O intermediate of a NHFe enzyme. This FeIV=O intermediate reacts via an initial H-atom abstraction step, with its subsquent halogenation (native) or hydroxylation (non-native) rebound reactivity being dependent on the substrate.17 A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate is able to direct the orientation of the FeIV=O intermediate, presenting specific frontier molecular orbitals (FMOs) which can activate the selective halogenation versus hydroxylation reactivity.Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neurotransmitters, the hypoxic response and the biosynthesis of secondary metabolites. The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe(iv)=O species, which has been trapped for a number of NHFe enzymes, including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(iv)=O intermediate are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe(iv)=O active site. Here we present NRVS structural characterization of the reactive Fe(iv)=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae. This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates. A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe(iv)=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.

Characterization of Iron Dinitrosyl Species Formed in the Reaction of Nitric Oxide with a Biological Rieske Center

Reactions of nitric oxide with cysteine-ligated iron-sulfur cluster proteins typically result in disassembly of the iron-sulfur core and formation of dinitrosyl iron complexes (DNICs). Here we report the first evidence that DNICs also form in the reaction of NO with Rieske-type [2Fe-2S] clusters. Upon treatment of a Rieske protein, component C of toluene/o-xylene monooxygenase from Pseudomonas sp. OX1, with an excess of NO(g) or NO-generators S-nitroso-N-acetyl-D,L-pencillamine and diethylamine NONOate, the absorbance bands of the [2Fe-2S] cluster are extinguished and replaced by a new feature that slowly grows in at 367 nm. Analysis of the reaction products by electron paramagnetic resonance, Mössbauer, and nuclear resonance vibrational spectroscopy reveals that the primary product of the reaction is a thiolate-bridged diiron tetranitrosyl species, [Fe(2)(μ-SCys)(2)(NO)(4)], having a Roussins red ester (RRE) formula, and that mononuclear DNICs account for only a minor fraction of nitrosylated iron. Reduction of this RRE reaction product with sodium dithionite produces the one-electron-reduced RRE, having absorptions at 640 and 960 nm. These results demonstrate that NO reacts readily with a Rieske center in a protein and suggest that dinuclear RRE species, not mononuclear DNICs, may be the primary iron dinitrosyl species responsible for the pathological and physiological effects of nitric oxide in such systems in biology.

Construction of a precision diffractometer for nuclear Bragg scattering at the Photon Factory

A versatile precision diffractometer for nuclear Bragg scattering was designed and constructed at the Photon Factory. Standardized component goniometers can be arranged to address various requirements of incident beam conditioning for nuclear Bragg scattering, including a high‐energy resolution monochromator with wide angular acceptance as well as a circularly polarized incident beam.

Electronic and magnetic phase diagram of superconductors, SmFeAsO1?xFx

A crystallographic and magnetic phase diagram of SmFeAsO1−xFx is determined as a function of x in terms of temperature based on electrical transport and magnetization, synchrotron powder x-ray diffraction, 57Fe Mossbauer spectra (MS), and 149Sm nuclear resonant forward scattering (NRFS) measurements. MS revealed that the magnetic moments of Fe were aligned antiferromagnetically at ~144 K (TN(Fe)). The magnetic moment of Fe (MFe) is estimated to be 0.34 μB/Fe at 4.2 K for undoped SmFeAsO; MFe is quenched in superconducting F-doped SmFeAsO. 149Sm NRFS spectra revealed that the magnetic moments of Sm start to order antiferromagnetically at 5.6 K (undoped) and 4.4 K (TN(Sm)) (x=0.069). Results clearly indicate that the antiferromagnetic (AF) Sm sublattice coexists with the superconducting phase in SmFeAsO1−xFx below TN(Sm), while the AF Fe sublattice does not coexist with the superconducting phase.

A Possible Novel Magnetic Ordering in SmRu4P12

We have carried out X-ray absorption spectroscopy, 149 Sm nuclear resonant forward scattering, and 99 Ru Mossbauer spectroscopy of SmRu 4 P 12 . The X-ray absorption spectra indicate that the Sm ions are trivalent up to 14 K. The quantum beats due to magnetic dipolar ordering are observed at 4.5 K in 149 Sm nuclear resonant forward scattering. The temperature dependence of time-integrated intensity in 149 Sm nuclear forward scattering suggests that the magnetic dipolar ordering occurs just below the metal–insulator transition temperature. No significant changes in the parameters are observed between 77 and 5 K in 99 Ru Mossbauer spectroscopy. The present result suggests a magnetic ordering accompanied by a magnetic dipole moment at the metal–insulator transition temperature in SmRu 4 P 12 in spite of the proposal of the magnetic ordering accompanied by a T β -type octupole.

Observation of the Fe—CN and Fe—CO Vibrations in the Active Site of [NiFe] Hydrogenase by Nuclear Resonance Vibrational Spectroscopy

Nuclear inelastic scattering of (57)Fe labeled [NiFe] hydrogenase is shown to give information on different states of the enzyme. It was thus possible to detect and assign Fe-CO and Fe-CN bending and stretching vibrations of the active site outside the spectral range of the Fe-S cluster normal modes.

Subnanosecond time-resolved x-ray measurements using an organic-inorganic perovskite scintillator

We have developed a fast x-ray detector using an organic-inorganic perovskite scintillator of phenethylamine lead bromide (PhE-PbBr4). The scintillator had a dominant light emission with a fast decay time of 9.9 ns. An x-ray detector equipped with a 0.9-mm-thick PhE-PbBr4 crystal was used to detect nuclear resonant scattering in N61i (the first excited level: 67.41 keV; lifetime: 7.6 ns) by using synchrotron radiation. With this detector, we could successfully record the decaying gamma rays emitted from N61i with a time resolution of 0.7 ns (full width at half maximum) and a relatively high detection efficiency of 24%.

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.

Polarizer/analyzer filter for nuclear resonant scattering of synchrotron radiation

A silicon (840) channel‐cut crystal with a large asymmetry is used to linearly polarize synchrotron radiation generated by an undulator source at the 14.413 keV nuclear resonance of 57Fe. The resulting σ‐polarized radiation is then scattered from an 57Fe foil placed in a magnetic field that effects σ→π polarization conversion within the resonant bandwidth. A second crystal of the same type is placed in a crossed position to suppress the nonresonant radiation while transmitting the π‐polarized resonant radiation. A polarization suppression factor of 6×10−7 has been obtained. The suppression of the nonresonant radiation allowed monitoring the decay of the nuclear levels after 1 ns of their excitation.

Geometric and Electronic Structure of the Mn(IV)Fe(III) Cofactor in Class Ic Ribonucleotide Reductase: Correlation to the Class Ia Binuclear Non-Heme Iron Enzyme

The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the β (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable Mn(IV)Fe(III) cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn(IV)Fe(III) cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe(III), whereas MCD reflects the spin-allowed transitions mostly on the Mn(IV). We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn(IV) at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH(-) to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn(IV)Fe(III) cofactor as having a μ-oxo, μ-hydroxo core and a terminal hydroxo ligand on the Mn(IV). From DFT calculations, the Mn(IV) at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH(-) terminal ligand on this Mn(IV) provides a high proton affinity that could gate radical translocation to the α (R1) subunit.