Operando film-electrochemical EPR spectroscopy tracks radical intermediates in surface-immobilized catalysts.

The development of surface-immobilized molecular redox catalysts is an emerging research field with promising applications in sustainable chemistry. In electrocatalysis, paramagnetic species are often key intermediates in the mechanistic cycle but are inherently difficult to detect and follow by conventional in situ techniques. We report a new method, operando film-electrochemical electron paramagnetic resonance spectroscopy (FE-EPR), which enables mechanistic studies of surface-immobilized electrocatalysts. This technique enables radicals formed during redox reactions to be followed in real time under flow conditions, at room temperature and in aqueous solution. Detailed insight into surface-immobilized catalysts, as exemplified here through alcohol oxidation catalysis by a surface-immobilized nitroxide, is possible by detecting active-site paramagnetic species sensitively and quantitatively operando, thereby enabling resolution of the reaction kinetics. Our finding that the surface electron-transfer rate, which is of the same order of magnitude as the rate of catalysis (accessible from operando FE-EPR), limits catalytic efficiency has implications for the future design of better surface-immobilized catalysts.

Controlling and exploiting intrinsic unpaired electrons in metalloproteins.

Electron paramagnetic resonance spectroscopy encompasses a versatile set of techniques that allow detailed insight into intrinsically occurring paramagnetic centers in metalloproteins and enzymes that undergo oxidation-reduction reactions. In this chapter, we discuss the process from isolating the protein to acquiring and analyzing pulse EPR spectra, adopting a practical perspective. We start with considerations when preparing the protein sample, explain techniques and procedures available for determining the reduction potential of the redox-active center of interest and provide details on methodologies to trap a given paramagnetic state for detailed pulse EPR studies, with an emphasis on biochemical and spectroscopic tools available when multiple EPR-active species are present. We elaborate on some of the most commonly used pulse EPR techniques and the choices the user has to make, considering advantages and disadvantages and how to avoid pitfalls. Examples are provided throughout.

Functional group introduction and aromatic unit variation in a set of π-conjugated macrocycles: revealing the central role of local and global aromaticity.

π-Conjugated macrocycles are molecules with unique properties that are increasingly exploited for applications and the question of whether they can sustain global aromatic or antiaromatic ring currents is particularly intriguing. However, there are only a small number of experimental studies that investigate how the properties of π-conjugated macrocycles evolve with systematic structural changes. Here, we present such a systematic experimental study of a set of [2.2.2.2]cyclophanetetraenes, all with formally Hückel antiaromatic ground states, and combine it with an in-depth computational analysis. The study reveals the central role of local and global aromaticity for rationalizing the observed optoelectronic properties, ranging from extremely large Stokes shifts of up to 1.6 eV to reversible fourfold reduction, a highly useful feature for charge storage/accumulation applications. A recently developed method for the visualization of chemical shielding tensors (VIST) is applied to provide unique insight into local and global ring currents occurring in different planes along the macrocycle. Conformational changes as a result of the structural variations can further explain some of the observations. The study contributes to the development of structure-property relationships and molecular design guidelines and will help to understand, rationalize, and predict the properties of other π-conjugated macrocycles. It will also assist in the design of macrocycle-based supramolecular elements with defined properties.

Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy.

Metal ions play an important role in diverse biological processes, and much of the basic knowledge derived from studying native bioinorganic systems are applied in the synthesis of new molecules with the aim of diagnosing and treating diseases. At first glance, metalloproteins and metallodrugs are very different systems, but metal ion coordination, redox chemistry and substrate binding play essential roles in advancing both of these research fields. In this article, we discuss recent metalloprotein and metallodrug studies where electron paramagnetic resonance spectroscopy served as a major tool to gain a better understanding of metal-based structures and their function.

A sensitivity leap for X-band EPR using a probehead with a cryogenic preamplifier.

Inspired by the considerable success of cryogenically cooled NMR cryoprobes, we present an upgraded X-band EPR probehead, equipped with a cryogenic low-noise preamplifier. Our setup suppresses source noise, can handle the high microwave powers typical in X-band pulsed EPR, and is compatible with the convenient resonator coupling and sample access found on commercially available spectrometers. Our approach allows standard pulsed and continuous-wave EPR experiments to be performed at X-band frequency with significantly increased sensitivity compared to the unmodified setup. The probehead demonstrates a voltage signal-to-noise ratio (SNR) enhancement by a factor close to 8× at a temperature of 6 K, and remains close to 2× at room temperature. By further suppressing room-temperature noise at the expense of reduced microwave power (and thus minimum π-pulse length), the factor of SNR improvement approaches 15 at 6 K, corresponding to an impressive 200-fold reduction in EPR measurement time. We reveal the full potential of this probehead by demonstrating such SNR improvements using a suite of typical hyperfine and dipolar spectroscopy experiments on exemplary samples.

1,2H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI).

The quinol oxidation site QD in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI QD quinol oxidation site is analyzed in detail using 1,2H hyperfine (hf) spectroscopy in combination with H2O/D2O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the QD site (MSKD) previously studied by us. DMSKD and MSKD are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSKD field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two ß-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (Aiso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSKD and (ii) a near in-plane orientation of its isoprenyl chain at Cß relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.

Probing the Menasemiquinone Binding Mode to Nitrate Reductase†A by Selective 2 H and 15 N Labeling, HYSCORE Spectroscopy, and DFT Modeling.

In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15 N selective labeling of His15 Nδ or Lys15 Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15 Nδ as the direct hydrogen-bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15 Nζ consistent with a through-space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2 H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2 H-and hence of the 1 H-methyl isotropic hyperfine coupling by 2 H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen-bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one-sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.