Kiyoung Park

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.

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.

An engineering model of the masking for the noise-robust speech recognition

Abstract The masking effect of human hearing is modeled by lateral and unilateral inhibition, and tested for isolated word recognition tasks. Frequency masking suppresses unwanted signals close to the dominant signal of interest in frequency domain, and the weak signals following dominant ones in the time are suppressed by temporal forward masking. The masking effect filters out unimportant signals, which may improve the performance of speech recognition systems. With the parameters derived from the psychological observations, proposed model shows good analogy to psychoacoustic masking effects as well as superior recognition performance.

Out-of-Vocabulary Rejection based on Selective Attention Model

Based on a psychological selective attention theory a new algorithm is developed to provide reliable out-of-vocabulary (OOV) rejection for speech recognition systems in noisy environments. The developed attention model is based on Broadbents ‘early filtering’ theory, and the attention adaptation process utilizes a gradient-descent error minimization algorithm with error backpropagation rule. The developed model is applied to isolated-word recognition tasks, and much higher in-vocabulary recognition rates are achieved with the same out-of-vocabulary rejection rates.

Combined spectroscopic and computational analysis of the vibrational properties of vitamin B12 in its Co3+, Co2+, and Co1+ oxidation states.

While the geometric and electronic structures of vitamin B12 (cyanocobalamin, CNCbl) and its reduced derivatives Co(2+)cobalamin (Co(2+)Cbl) and Co(1+)cobalamin (Co(1+)Cbl(-)) are now reasonably well established, their vibrational properties, in particular their resonance Raman (rR) spectra, have remained quite poorly understood. The goal of this study was to establish definitive assignments of the corrin-based vibrational modes that dominate the rR spectra of vitamin B12 in its Co(3+), Co(2+), and Co(1+) oxidation states. rR spectra were collected for all three species with laser excitation in resonance with the most intense corrin-based π → π* transitions. These experimental data were used to validate the computed vibrational frequencies, eigenvector compositions, and relative rR intensities of the normal modes of interest as obtained by density functional theory (DFT) calculations. Importantly, the computational methodology employed in this study successfully reproduces the experimental observation that the frequencies and rR excitation profiles of the corrin-based vibrational modes vary significantly as a function of the cobalt oxidation state. Our DFT results suggest that this variation reflects large differences in the degree of mixing between the occupied Co 3d orbitals and empty corrin π* orbitals in CNCbl, Co(2+)Cbl, and Co(1+)Cbl(-). As a result, vibrations mainly involving stretching of conjugated C-C and C-N bonds oriented along one axis of the corrin ring may, in fact, couple to a perpendicularly polarized electronic transition. This unusual coupling between electronic transitions and vibrational motions of corrinoids greatly complicates an assignment of the corrin-based normal modes of vibrations on the basis of their rR excitation profiles.

Structure/function correlations over binuclear non-heme iron active sites

Binuclear non-heme iron enzymes activate O2 to perform diverse chemistries. Three different structural mechanisms of O2 binding to a coupled binuclear iron site have been identified utilizing variable-temperature, variable-field magnetic circular dichroism spectroscopy (VTVH MCD). For the μ-OH-bridged Fe(II)2 site in hemerythrin, O2 binds terminally to a five-coordinate Fe(II) center as hydroperoxide with the proton deriving from the μ-OH bridge and the second electron transferring through the resulting μ-oxo superexchange pathway from the second coordinatively saturated Fe(II) center in a proton-coupled electron transfer process. For carboxylate-only-bridged Fe(II)2 sites, O2 binding as a bridged peroxide requires both Fe(II) centers to be coordinatively unsaturated and has good frontier orbital overlap with the two orthogonal O2 π* orbitals to form peroxo-bridged Fe(III)2 intermediates. Alternatively, carboxylate-only-bridged Fe(II)2 sites with only a single open coordination position on an Fe(II) enable the one-electron formation of Fe(III)–O2− or Fe(III)–NO− species. Finally, for the peroxo-bridged Fe(III)2 intermediates, further activation is necessary for their reactivities in one-electron reduction and electrophilic aromatic substitution, and a strategy consistent with existing spectral data is discussed.

Properties of independent components of self-motion optical flow

In this paper we describe the properties of independent components of optical flow of moving objects. Video sequences of objects seen by an observer moving at various angles, directions and distances are used to produce optical flow maps. These maps are then, recessed using independent component analysis, which yields filters that resemble the receptive fields of dorsal medial superior temporal cells of the primate brain. Contraction, expansion, rotation and translation receptive fields have been identified. Our results support Barlows sensory coding theory and are in-line with other work on independent components of image and video intensities.

Mechanistic Insights into Tunable Metal-Mediated Hydrolysis of Amyloid-β Peptides

An amyloidogenic peptide, amyloid-β (Aβ), has been implicated as a contributor to the neurotoxicity of Alzheimers disease (AD) that continues to present a major socioeconomic burden for our society. Recently, the use of metal complexes capable of cleaving peptides has arisen as an efficient tactic for amyloid management; unfortunately, little has been reported to pursue this strategy. Herein, we report a novel approach to validate the hydrolytic cleavage of divalent metal complexes toward two major isoforms of Aβ (Aβ40 and Aβ42) and tune their proteolytic activity based on the choice of metal centers (M = Co, Ni, Cu, and Zn) which could be correlated to their anti-amyloidogenic properties. Such metal-dependent tunability was facilitated employing a tetra-N-methylated cyclam (TMC) ligand that imparts unique geometric and stereochemical control, which has not been available in previous systems. Co(II)(TMC) was identified to noticeably cleave Aβ peptides and control their aggregation, reporting the first Co(II) complex for such reactivities to the best of our knowledge. Through detailed mechanistic investigations by biochemical, spectroscopic, mass spectrometric, and computational studies, the critical importance of the coordination environment and acidity of the aqua-bound complexes in promoting amide hydrolysis was verified. The biological applicability of Co(II)(TMC) was also illustrated via its potential blood-brain barrier permeability, relatively low cytotoxicity, regulatory capability against toxicity induced by both Aβ40 and Aβ42 in living cells, proteolytic activity with Aβ peptides under biologically relevant conditions, and inertness toward cleavage of structured proteins. Overall, our approaches and findings on reactivities of divalent metal complexes toward Aβ, along with the mechanistic insights, demonstrate the feasibility of utilizing such metal complexes for amyloid control.

Nuclear resonance vibrational spectroscopic and computational study of high-valent diiron complexes relevant to enzyme intermediates

High-valent intermediates of binuclear nonheme iron enzymes are structurally unknown despite their importance for understanding enzyme reactivity. Nuclear resonance vibrational spectroscopy combined with density functional theory calculations has been applied to structurally well-characterized high-valent mono- and di-oxo bridged binuclear Fe model complexes. Low-frequency vibrational modes of these high-valent diiron complexes involving Fe motion have been observed and assigned. These are independent of Fe oxidation state and show a strong dependence on spin state. It is important to note that they are sensitive to the nature of the Fe2 core bridges and provide the basis for interpreting parallel nuclear resonance vibrational spectroscopy data on the high-valent oxo intermediates in the binuclear nonheme iron enzymes.

Nuclear Resonance Vibrational Spectroscopy and DFT study of Peroxo-Bridged Biferric Complexes: Structural Insight into Peroxo Intermediates of Binuclear Non-heme Iron Enzymes†

Binuclear non-heme iron enzymes utilize O2 to catalyze a variety of reactions, including hydrogen atom abstraction, desaturation, electrophilic aromatic substitution, and so on. In most cases, their catalytic cycles begin with the reductive binding of O2 by biferrous centers to form high-spin antiferromagnetically coupled (AFC) peroxo-bridged biferric intermediates. These peroxo intermediates can either react with substrate or convert to more reactive high-valent species. Because of their transient nature, structural information must be deduced from spectroscopic data, which are rich for some peroxo intermediates, while for others too limited for geometric and electronic structural insight. The peroxo intermediate of W48F/D84E ribonucleotide reductase (RR), referred to as P, does exhibit distinct spectral features. These include electronic absorption (Abs) and resonance Raman (rR) spectra that are equivalent to those of cis m-1,2 end-on peroxo-bridged Fe2 model complexes, thus providing a basis for the computational model of P as a cis m-1,2 peroxo-bridged Fe2 species (with the (Glu)4(His)2 ligand set of this protein active site). However, P is not reactive and must convert to a second-peroxo-level intermediate P’ that does not have Abs spectral features for rR-based structural elucidation. For systems that do not have chromophores or are photoactive, nuclear resonance vibrational spectroscopy (NRVS) is an alternative to rR spectroscopy. NRVS is a synchrotron-based technique that probes vibrational side bands of Fe nuclear transitions. Its spectral intensity is determined by the amount of Fe displacement in each normal mode, thus allowing the specific investigation of the Fe active site with high sensitivity and without the limitation of the selection rules of rR spectroscopy. In this study, we establish the basis for the NRVS analysis of peroxo-bridged Fe2 intermediates, based on structurally well-characterized synthetic model complexes. We have measured the NRVS spectra of [Fe2(mOH)(mO2)(6Me2-BPP)2] + (1) and [Fe2(mO)(mO2)(6Me2-BPP)2] (2 ; Figure 1; 6Me2-BPP = N,N-bis(6methyl-2-pyridylmethyl)-3-aminopropionate). These complexes are cis m-1,2 peroxo-bridged species, the former with an additional hydroxo bridge and the latter with an oxo