Electron Paramagnetic Resonance Spectroscopy as a Probe of Hydrogen Bonding in Heme-Thiolate Proteins

Abstract

Despite utilizing a common cofactor binding motif, hemoproteins bearing a cysteine-derived thiolate ligand (heme-thiolate proteins) are involved in a diverse array of biological processes ranging from drug metabolism to transcriptional regulation. Though the origin of heme-thiolate functional divergence is not well understood, growing evidence suggests that the hydrogen bonding (H-bonding) environment surrounding the Fe-coordinating thiolate influences protein function. Outside of X-ray crystallography, few methods exist to characterize these critical H-bonding interactions. Electron paramagnetic resonance (EPR) spectra of heme-thiolate proteins bearing a six-coordinate, Fe­(III) heme exhibit uniquely narrow low-spin (S = 1/2), rhombic signals, which are sensitive to changes in the heme-thiolate H-bonding environment. To establish a well-defined relationship between the magnitude of g-value dispersion in this unique EPR signal and the strength of the heme-thiolate H-bonding environment, we synthesized and characterized of a series of six-coordinate, aryl-thiolate-ligated Fe­(III) porphyrin complexes bearing a tunable intramolecular H-bond. Spectroscopic investigation of these complexes revealed a direct correlation between H-bond strength and g-value dispersion in the rhombic EPR signal. Using density functional theory (DFT), we elucidated the electronic origins of the narrow, rhombic EPR signal in heme-thiolates, which arises from an Fe–S pπ–dπ bonding interaction. Computational analysis of the intramolecularly H-bonded heme-thiolate models revealed that H-bond donation to the coordinating thiolate reduces thiolate donor strength and weakens this Fe–S interaction, giving rise to larger g-value dispersion. By defining the relationship between heme-thiolate electronic structure and rhombic EPR signal, it is possible to compare thiolate donor strengths among heme-thiolate proteins through analysis of low-spin, Fe­(III) EPR spectra. Thus, this study establishes EPR spectroscopy as a valuable tool for exploring how second coordination sphere effects influence heme-thiolate protein function.

Heme-Thiolate Perturbation in Cystathionine β-Synthase by Mercury Compounds

“…Although the Hg–S Cys52 distances were larger than in the isolated model systems, we still observed an elongation of the Fe–S Cys52 bond (Table ). Of note, the Fe–S Cys52 distance computed for low-spin Fe­(III)-CBS in the absence of mercury compounds was consistent with previous experimental and theoretical determinations . For p -CMB and p -MB, two conformations were studied, one with the mercury compound partially inside the protein (“in”) and the other one with the mercury compound in the solvent (“out”).…”

“…Of note, the Fe−S Cys52 distance computed for low-spin Fe(III)-CBS in the absence of mercury compounds was consistent with previous experimental and theoretical determinations. 90 For p-CMB and p-MB, two conformations were studied, one with the mercury compound partially inside the protein ("in") and the other one with the mercury compound in the solvent ("out"). The "in" conformation was more effective in weakening the Fe−S Cys52 bond, as evidenced in the larger Fe−S Cys52 distances and the smaller Hg−S Cys52 distances observed in the optimized structure, compared to the "out" conformation.…”

The Biologically Relevant Coordination Chemistry of Iron and Nitric Oxide: Electronic Structure and Reactivity

“…These complexes were obtained by the reaction of the corresponding 5C ferric heme-thiolate precursors (all with TPP 2– as the porphyrin ligand) with NO at −80 °C, and subsequently characterized using low-temperature UV–vis, IR, and rRaman spectroscopy ( Figure ). In these model systems, thiophenolate ligands were used that either contain (a) various electron-withdrawing substituents (i.e., the “electron poor” thiolate series), or (b) one intramolecular hydrogen bond to the thiolate sulfur (ligands – SPh-NHPh- p R, where R is a functional group allowing for tunability of the hydrogen-bond strength), , as shown in Scheme . These model systems allowed for the experimental demonstration that in heme-thiolate ls-{FeNO} 6 complexes: Both electron-withdrawing groups and hydrogen bonds can modulate the thiolate donor strength in a similar way. ,, The thiolate donor strength directly modulates the Fe–NO and N–O bond strengths via a thermodynamic σ- trans effect (more precisely, a σ- trans interaction, since it is a thermodynamic effect) that can be experimentally quantified by spectroscopic determination of the Fe–NO and N–O stretching frequencies. Hydrogen bonds are able to provide additional protection for the thiolate ligand against S -nitrosylation and potentially other side reactions as well. ,, The cumulative strength of the proximal hydrogen bonds to the thiolate ligand in proteins and model systems can in turn be gauged by determination of the Fe–NO and N–O stretching frequencies of the corresponding ls-{FeNO} 6 adducts, by comparison of these data to the vibrational correlation plot shown in Figure . …”

“…(B) The thiophenolate ligand series that contains one intramolecular hydrogen bond (indicated by a dashed line). Here, the R group can be varied to tune the strength of this hydrogen bond. , .…”

Design Considerations of a Dual Mode X-Band EPR Resonator for Rapid In-Situ Microwave Heating

“…The Zeeman splitting of electronic spin states via an applied DC magnetic field is essential for detecting Electron Paramagnetic Resonance (EPR) spectra of paramagnetic substances [1]. The interactions between an unpaired electron with neighboring magnetic nuclei and/or other unpaired electrons impart spectral features from which information on molecular structure, bonding, dynamics in solution, concentration and reactivity can be ascertained [2][3][4][5]. EPR plays a major role in investigating industrially relevant catalytic reactions involving paramagnetic species and free radicals [6][7][8][9].…”