Film-electrochemical EPR spectroscopy to investigate electron transfer in membrane proteins in their native environment.

Film-electrochemical electron paramagnetic resonance spectroscopy (FE-EPR) enables simultaneous electrochemical and spectroscopic characterisation of paramagnetic electron-transfer centres, including in soluble proteins. We now report a modified set-up FE-EPR with tuneable macroporous working electrodes and demonstrate the feasibility to investigate electron transfer in membrane proteins in their native membrane environment.

The Effect of Reactive Oxygen Species on Respiratory Complex I Activity in Liposomes.

Respiratory complex I (R-CI) is an essential enzyme in the mitochondrial electron transport chain but also a major source of reactive oxygen species (ROS), which are implicated in neurodegenerative diseases and ageing. While the mechanism of ROS production by R-CI is well-established, the feedback of ROS on R-CI activity is poorly understood. Here, we perform EPR spectroscopy on R-CI incorporated in artificial membrane vesicles to reveal that ROS (particularly hydroxyl radicals) reduce R-CI activity by making the membrane more polar and by increasing its hydrogen bonding capability. Moreover, the mechanism that we have uncovered reveals that the feedback of ROS on R-CI activity via the membrane is transient and not permanent; lipid peroxidation is negligible for the levels of ROS generated under these conditions. Our successful use of modular proteoliposome systems in conjunction with EPR spectroscopy and other biophysical techniques is a powerful approach for investigating ROS effects on other membrane proteins.

Mixed Valence {Ni2+Ni1+} Clusters as Models of Acetyl Coenzyme A Synthase Intermediates.

Acetyl coenzyme A synthase (ACS) catalyzes the formation and deconstruction of the key biological metabolite, acetyl coenzyme A (acetyl-CoA). The active site of ACS features a {NiNi} cluster bridged to a [Fe4S4]n+ cubane known as the A-cluster. The mechanism by which the A-cluster functions is debated, with few model complexes able to replicate the oxidation states, coordination features, or reactivity proposed in the catalytic cycle. In this work, we isolate the first bimetallic models of two hypothesized intermediates on the paramagnetic pathway of the ACS function. The heteroligated {Ni2+Ni1+} cluster, [K(12-crown-4)2][1], effectively replicates the coordination number and oxidation state of the proposed "Ared" state of the A-cluster. Addition of carbon monoxide to [1]- allows for isolation of a dinuclear {Ni2+Ni1+(CO)} complex, [K(12-crown-2)n][2] (n = 1-2), which bears similarity to the "ANiFeC" enzyme intermediate. Structural and electronic properties of each cluster are elucidated by X-ray diffraction, nuclear magnetic resonance, cyclic voltammetry, and UV/vis and electron paramagnetic resonance spectroscopies, which are supplemented by density functional theory (DFT) calculations. Calculations indicate that the pseudo-T-shaped geometry of the three-coordinate nickel in [1]- is more stable than the Y-conformation by 22 kcal mol-1, and that binding of CO to Ni1+ is barrierless and exergonic by 6 kcal mol-1. UV/vis absorption spectroscopy on [2]- in conjunction with time-dependent DFT calculations indicates that the square-planar nickel site is involved in electron transfer to the CO π*-orbital. Further, we demonstrate that [2]- promotes thioester synthesis in a reaction analogous to the production of acetyl coenzyme A by ACS.

A Paramagnetic Nickel-Zinc Hydride Complex.

Reaction of a molecular zinc-hydride [{(ArNCMe)2CH}ZnH] (Ar = 2,6-di-isopropylphenyl) with 0.5 equiv. of [Ni(CO)Cp]2 led to the isolation of a nickel-zinc hydride complex containing a bridging 3-centre,2-electron Ni-H-Zn interaction. This species has been characterized in the solid-state by single crystal X-ray diffraction. DFT calculations are consistent with its formulation as a σ-complex derived from coordination of the zinc-hydride to a paramagnetic nickel(I) fragment. Continuous wave and pulse EPR experiments suggest that this species is not stable in solution and suggest that this species is labile in solution. Further experiments show that in the presence of catalytic quantities of nickel(I) precursors, zinc-hydride bonds can undergo either H/D-exchange with D2 or dehydrocoupling to form Zn-Zn bonds. In combination, the data support the activation and functionalisation of zinc-hydride bonds at nickel(I) centres.

Heterobimetallic 3d-4f complexes supported by a Schiff-base tripodal ligand.

Complexes featuring multiple metal centres are of growing interest regarding metal-metal cooperation and its tuneability. Here the synthesis and characterisation of heterobimetallic complexes of a 3d metal (4: Mn, 5: Co) and lanthanum supported by a (1,1,1-tris[(3-methoxysalicylideneamino)methyl]ethane) ligand is reported, as well as discussion of their electronic structure via electron paramagnetic resonance (EPR) spectroscopy, electrochemical experiments and computational studies. Competitive binding experiments of the ligand and various metal salts unequivocally demonstrate that in these heterobimetallic complexes the 3d metal (Mn, Co) selectively occupies the κ6-N3O3 binding site of the ligand, whilst La occupies the κ6-O6 metal binding site in line with their relative oxophilicities. EPR spectroscopy supported by density functional theory analysis indicates that the 3d metal is high spin in both cases (S = 5/2 (Mn), 3/2 (Co)). Cyclic voltammetry studies on the Mn/La and Co/La bimetallic complexes revealed a quasi-reversible Mn2+/3+ redox process and poorly-defined irreversible oxidation events respectively.

Understanding the effects of targeted modifications on the 1 : 2 Choline And GEranate structure.

1 : 2 Choline-and-geranate (CAGE) is an ionic liquid (IL) widely studied for its biomedical applications. However, both its industrial-scale preparation and its long-term storage are problematic so finding more suitable candidates which retain its advantageous properties is crucial. As a first step towards this we have conducted a targeted modification study to understand the effects of specific functional groups on the properties of CAGE. 1 : 2 Choline-and-octanoate and 1 : 2 butyltrimethylammonium-and-octanoate were synthesised and their thermal and rheological properties examined in comparison to those of CAGE. Using differential scanning calorimetry and polarising microscopy, the model compound was found to be an isotropic liquid, while the analogues were room-temperature liquid-crystals which transition to isotropic liquids upon heating. Dynamic mechanical analysis showed that the thermal behaviour of the studied systems was even more complex, with the ILs also undergoing a thermally-activated relaxation process. Furthermore, we have used electron paramagnetic resonance (EPR) spectroscopy, along with a variety of spin probes with different functional groups, in order to understand the chemical environment experienced by solutes in each system. The EPR spectra indicate that the radicals experience two distinct environments (polar and nonpolar) in the liquid-crystalline phase, but only one average environment in the isotropic phase. The liquid-crystalline phase experiments also showed that the relative populations of the two domains depend on the nature of the solutes, with polar or strongly hydrogen-bonding solutes preferring the polar domain. For charged solutes, the EPR spectra showed line-broadening, suggesting that their ionic nature leads to complex, unresolved interactions.

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.

Using light scattering to assess how phospholipid-protein interactions affect complex I functionality in liposomes.

Complex I is an essential membrane protein in respiration, oxidising NADH and reducing ubiquinone to contribute to the proton-motive force that powers ATP synthesis. Liposomes provide an attractive platform to investigate complex I in a phospholipid membrane with the native hydrophobic ubiquinone substrate and proton transport across the membrane, but without convoluting contributions from other proteins present in the native mitochondrial inner membrane. Here, we use dynamic and electrophoretic light scattering techniques (DLS and ELS) to show how physical parameters, in particular the zeta potential (ζ-potential), correlate strongly with the biochemical functionality of complex I-containing proteoliposomes. We find that cardiolipin plays a crucial role in the reconstitution and functioning of complex I and that, as a highly charged lipid, it acts as a sensitive reporter on the biochemical competence of proteoliposomes in ELS measurements. We show that the change in ζ-potential between liposomes and proteoliposomes correlates linearly with protein retention and catalytic oxidoreduction activity of complex I. These correlations are dependent on the presence of cardiolipin, but are otherwise independent of the liposome lipid composition. Moreover, changes in the ζ-potential are sensitive to the proton motive force established upon proton pumping by complex I, thereby constituting a complementary technique to established biochemical assays. ELS measurements may thus serve as a more widely useful tool to investigate membrane proteins in lipid systems, especially those that contain charged lipids.

Bio-Electrocatalytic Conversion of Food Waste to Ethylene via Succinic Acid as the Central Intermediate.

Ethylene is an important feedstock in the chemical industry, but currently requires production from fossil resources. The electrocatalytic oxidative decarboxylation of succinic acid offers in principle an environmentally friendly route to generate ethylene. Here, a detailed investigation of the role of different carbon electrode materials and characteristics revealed that a flat electrode surface and high ordering of the carbon material are conducive for the reaction. A range of electrochemical and spectroscopic approaches such as Koutecky-Levich analysis, rotating ring-disk electrode (RRDE) studies, and Tafel analysis as well as quantum chemical calculations, electron paramagnetic resonance (EPR), and in situ infrared (IR) spectroscopy generated further insights into the mechanism of the overall process. A distinct reaction intermediate was detected, and the decarboxylation onset potential was determined to be 2.2-2.3 V versus the reversible hydrogen electrode (RHE). Following the mechanistic studies and electrode optimization, a two-step bio-electrochemical process was established for ethylene production using succinic acid sourced from food waste. The initial step of this integrated process involves microbial hydrolysis/fermentation of food waste into aqueous solutions containing succinic acid (0.3 M; 3.75 mmol per g bakery waste). The second step is the electro-oxidation of the obtained intermediate succinic acid to ethylene using a flow setup at room temperature, with a productivity of 0.4-1 µmol ethylene cmelectrode -2 h-1. This approach provides an alternative strategy to produce ethylene from food waste under ambient conditions using renewable energy.

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.

Functional basis of electron transport within photosynthetic complex I.

Photosynthesis and respiration rely upon a proton gradient to produce ATP. In photosynthesis, the Respiratory Complex I homologue, Photosynthetic Complex I (PS-CI) is proposed to couple ferredoxin oxidation and plastoquinone reduction to proton pumping across thylakoid membranes. However, little is known about the PS-CI molecular mechanism and attempts to understand its function have previously been frustrated by its large size and high lability. Here, we overcome these challenges by pushing the limits in sample size and spectroscopic sensitivity, to determine arguably the most important property of any electron transport enzyme - the reduction potentials of its cofactors, in this case the iron-sulphur clusters of PS-CI (N0, N1 and N2), and unambiguously assign them to the structure using double electron-electron resonance. We have thus determined the bioenergetics of the electron transfer relay and provide insight into the mechanism of PS-CI, laying the foundations for understanding of how this important bioenergetic complex functions.

Rotaxane CoII Complexes as Field-Induced Single-Ion Magnets.

Mechanically chelating ligands have untapped potential for the engineering of metal ion properties. Here we demonstrate this principle in the context of CoII -based single-ion magnets. Using multi-frequency EPR, susceptibility and magnetization measurements we found that these complexes show some of the highest zero field splittings reported for five-coordinate CoII complexes to date. The predictable coordination behaviour of the interlocked ligands allowed the magnetic properties of their CoII complexes to be evaluated computationally a priori and our combined experimental and theoretical approach enabled us to rationalize the observed trends. The predictable magnetic behaviour of the rotaxane CoII complexes demonstrates that interlocked ligands offer a new strategy to design metal complexes with interesting functionality.

A conserved arginine residue is critical for stabilizing the N2 FeS cluster in mitochondrial complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase), the first enzyme of the electron-transport chain, captures the free energy released by NADH oxidation and ubiquinone reduction to translocate protons across an energy-transducing membrane and drive ATP synthesis during oxidative phosphorylation. The cofactor that transfers the electrons directly to ubiquinone is an iron-sulfur cluster (N2) located in the NDUFS2/NUCM subunit. A nearby arginine residue (R121), which forms part of the second coordination sphere of the N2 cluster, is known to be posttranslationally dimethylated but its functional and structural significance are not known. Here, we show that mutations of this arginine residue (R121M/K) abolish the quinone-reductase activity, concomitant with disappearance of the N2 signature from the electron paramagnetic resonance (EPR) spectrum. Analysis of the cryo-EM structure of NDUFS2-R121M complex I at 3.7 Å resolution identified the absence of the cubane N2 cluster as the cause of the dysfunction, within an otherwise intact enzyme. The mutation further induced localized disorder in nearby elements of the quinone-binding site, consistent with the close connections between the cluster and substrate-binding regions. Our results demonstrate that R121 is required for the formation and/or stability of the N2 cluster and highlight the importance of structural analyses for mechanistic interpretation of biochemical and spectroscopic data on complex I variants.

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.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Using a chimeric respiratory chain and EPR spectroscopy to determine the origin of semiquinone species previously assigned to mitochondrial complex I.

BACKGROUND: For decades, semiquinone intermediates have been suggested to play an essential role in catalysis by one of the most enigmatic proton-pumping enzymes, respiratory complex I, and different mechanisms have been proposed on their basis. However, the difficulty in investigating complex I semiquinones, due to the many different enzymes embedded in the inner mitochondrial membrane, has resulted in an ambiguous picture and no consensus. RESULTS: In this paper, we re-examine the highly debated origin of semiquinone species in mitochondrial membranes using a novel approach. Our combination of a semi-artificial chimeric respiratory chain with pulse EPR spectroscopy (HYSCORE) has enabled us to conclude, unambiguously and for the first time, that the majority of the semiquinones observed in mitochondrial membranes originate from complex III. We also identify a minor contribution from complex II. CONCLUSIONS: We are unable to attribute any semiquinone signals unambiguously to complex I and, reconciling our observations with much of the previous literature, conclude that they are likely to have been misattributed to it. We note that, for this earlier work, the tools we have relied on here to deconvolute overlapping EPR signals were not available. Proposals for the mechanism of complex I based on the EPR signals of semiquinone species observed in mitochondrial membranes should thus be treated with caution until future work has succeeded in isolating any complex I semiquinone EPR spectroscopic signatures present.

A Precious-Metal-Free Hybrid Electrolyzer for Alcohol Oxidation Coupled to CO2 -to-Syngas Conversion.

Electrolyzers combining CO2 reduction (CO2 R) with organic substrate oxidation can produce fuel and chemical feedstocks with a relatively low energy requirement when compared to systems that source electrons from water oxidation. Here, we report an anodic hybrid assembly based on a (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) electrocatalyst modified with a silatrane-anchor (STEMPO), which is covalently immobilized on a mesoporous indium tin oxide (mesoITO) scaffold for efficient alcohol oxidation (AlcOx). This molecular anode was subsequently combined with a cathode consisting of a polymeric cobalt phthalocyanine on carbon nanotubes to construct a hybrid, precious-metal-free coupled AlcOx-CO2 R electrolyzer. After three-hour electrolysis, glycerol is selectively oxidized to glyceraldehyde with a turnover number (TON) of ≈1000 and Faradaic efficiency (FE) of 83 %. The cathode generated a stoichiometric amount of syngas with a CO:H2 ratio of 1.25±0.25 and an overall cobalt-based TON of 894 with a FE of 82 %. This prototype device inspires the design and implementation of nonconventional strategies for coupling CO2 R to less energy demanding, and value-added, oxidative chemistry.