Magnetic resonance imaging of human neural stem cells in rodent and primate brain.

Stem cell transplantation therapies are currently under investigation for central nervous system disorders. Although preclinical models show benefit, clinical translation is somewhat limited by the absence of reliable noninvasive methods to confirm targeting and monitor transplanted cells in vivo. Here, we assess a novel magnetic resonance imaging (MRI) contrast agent derived from magnetotactic bacteria, magneto-endosymbionts (MEs), as a translatable methodology for in vivo tracking of stem cells after intracranial transplantation.

Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location, Fate, and Host Cell Interactions.

Purpose: The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.

Characterization of Magneto-Endosymbionts as MRI Cell Labeling and Tracking Agents.

Purpose: Magneto-endosymbionts (MEs) show promise as living magnetic resonance imaging (MRI) contrast agents for in vivo cell tracking. Here we characterize the biomedical imaging properties of ME contrast agents, in vitro and in vivo.

Procedures: By adapting and engineering magnetotactic bacteria to the intracellular niche, we are creating magneto-endosymbionts (MEs) that offer advantages relative to passive iron-based contrast agents (superparamagnetic iron oxides, SPIOs) for cell tracking.

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF.

Binuclear non-heme iron enzymes activate O for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally.

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).

Circular dichroism, magnetic circular dichroism, and variable temperature variable field magnetic circular dichroism studies of biferrous and mixed-valent myo-inositol oxygenase: insights into substrate activation of O2 reactivity.

myo-Inositol oxygenase (MIOX) catalyzes the 4e(-) oxidation of myo-inositol (MI) to D-glucuronate using a substrate activated Fe(II)Fe(III) site. The biferrous and Fe(II)Fe(III) forms of MIOX were studied with circular dichroism (CD), magnetic circular dichroism (MCD), and variable temperature variable field (VTVH) MCD spectroscopies. The MCD spectrum of biferrous MIOX shows two ligand field (LF) transitions near 10000 cm(-1), split by ~2000 cm(-1), characteristic of six coordinate (6C) Fe(II) sites, indicating that the modest reactivity of the biferrous form toward O2 can be attributed to the saturated coordination of both irons.

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

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).

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.

Spectroscopic studies of the iron and manganese reconstituted tyrosyl radical in Bacillus cereus ribonucleotide reductase R2 protein.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in DNA synthesis where ribonucleotides are reduced to the corresponding deoxyribonucleotides. Class Ib RNRs consist of two homodimeric subunits: R1E, which houses the active site; and R2F, which contains a metallo cofactor and a tyrosyl radical that initiates the ribonucleotide reduction reaction. We studied the R2F subunit of B.

Hybrid genetic algorithm with an adaptive penalty function for fitting multimodal experimental data: application to exchange-coupled non-Kramers binuclear iron active sites.

A Genetic Algorithm (GA) is a stochastic optimization technique based on the mechanisms of biological evolution. These algorithms have been successfully applied in many fields to solve a variety of complex nonlinear problems. While they have been used with some success in chemical problems such as fitting spectroscopic and kinetic data, many have avoided their use due to the unconstrained nature of the fitting process.

Definition of the intermediates and mechanism of the anticancer drug bleomycin using nuclear resonance vibrational spectroscopy and related methods.

Bleomycin (BLM) is a glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage. The last detectable intermediate prior to DNA cleavage is a low spin Fe(III) peroxy level species, termed activated bleomycin (ABLM). DNA strand scission is initiated through the abstraction of the C-4' hydrogen atom of the deoxyribose sugar unit.

Peroxo-type intermediates in class I ribonucleotide reductase and related binuclear non-heme iron enzymes.

We have performed a systematic study of chemically possible peroxo-type intermediates occurring in the non-heme di-iron enzyme class Ia ribonucleotide reductase, using spectroscopically calibrated computational chemistry. Density functional computations of equilibrium structures, Fe-O and O-O stretch frequencies, Mossbauer isomer shifts, absorption spectra, J-coupling constants, electron affinities, and free energies of O(2) and proton or water binding are presented for a series of possible intermediates. The results enable structure-property correlations and a new rationale for the changes in carboxylate conformations occurring during the O(2) reaction of this class of non-heme iron enzymes.

Spectroscopic definition of the biferrous and biferric sites in de novo designed four-helix bundle DFsc peptides: implications for O2 reactivity of binuclear non-heme iron enzymes.

DFsc is a single chain de novo designed four-helix bundle peptide that mimics the core protein fold and primary ligand set of various binuclear non-heme iron enzymes. DFsc and the E11D, Y51L, and Y18F single amino acid variants have been studied using a combination of near-IR circular dichroism (CD), magnetic circular dichroism (MCD), variable temperature variable field MCD (VTVH MCD), and X-ray absorption (XAS) spectroscopies. The biferrous sites are all weakly antiferromagnetically coupled with mu-1,3 carboxylate bridges and one 4-coordinate and one 5-coordinate Fe, very similar to the active site of class I ribonucleotide reductase (R2) providing open coordination positions on both irons for dioxygen to bridge.

A combined NRVS and DFT study of Fe(IV)=O model complexes: a diagnostic method for the elucidation of non-heme iron enzyme intermediates.

Fe=O biomimetic model complexes have been characterized using nuclear vibrational resonance spectroscopy (NRVS). Features and systematic trends in the low energy region reflect equatorial and axial bonding differences that relate to differences in reactivity. These trends have been computationally extended to predict the spectra of putative Fe=O intermediates in non-heme iron enzymes and show the utility of the NRVS method for structural insight.

Circular dichroism and magnetic circular dichroism studies of the biferrous site of the class Ib ribonucleotide reductase from Bacillus cereus: comparison to the class Ia enzymes.

The rate limiting step in DNA biosynthesis is the reduction of ribonucleotides to form the corresponding deoxyribonucleotides. This reaction is catalyzed by ribonucleotide reductases (RNRs) and is an attractive target against rapidly proliferating pathogens. Class I RNRs are binuclear non-heme iron enzymes and can be further divided into subclasses.

Oxygen reactivity of the biferrous site in the de novo designed four helix bundle peptide DFsc: nature of the "intermediate" and reaction mechanism.

The DFsc and DFscE11D de novo designed protein scaffolds support biomimetic diiron cofactor sites that react with dioxygen forming a 520 nm "intermediate" species with an apparent pseudo-first-order formation rate constant of 2.2 and 4.8 s-1, respectively.

A role for iron in an ancient carbonic anhydrase.

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (alpha class) and plants (beta class); however, two additional classes (gamma and delta) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the gamma class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All of the enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc.