Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

An allosteric redox switch involved in oxygen protection in a CO2 reductase.

Metal-dependent formate dehydrogenases reduce CO2 with high efficiency and selectivity, but are usually very oxygen sensitive. An exception is Desulfovibrio vulgaris W/Sec-FdhAB, which can be handled aerobically, but the basis for this oxygen tolerance was unknown. Here we show that FdhAB activity is controlled by a redox switch based on an allosteric disulfide bond. When this bond is closed, the enzyme is in an oxygen-tolerant resting state presenting almost no catalytic activity and very low formate affinity. Opening this bond triggers large conformational changes that propagate to the active site, resulting in high activity and high formate affinity, but also higher oxygen sensitivity. We present the structure of activated FdhAB and show that activity loss is associated with partial loss of the metal sulfido ligand. The redox switch mechanism is reversible in vivo and prevents enzyme reduction by physiological formate levels, conferring a fitness advantage during O2 exposure.

In-cell investigation of the conformational landscape of the GTPase UreG by SDSL-EPR.

UreG is a cytosolic GTPase involved in the maturation network of urease, an Ni-containing bacterial enzyme. Previous investigations in vitro showed that UreG features a flexible tertiary organization, making this protein the first enzyme discovered to be intrinsically disordered. To determine whether this heterogeneous behavior is maintained in the protein natural environment, UreG structural dynamics was investigated directly in intact bacteria by in-cell EPR. This approach, based on site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy, enables the study of proteins in their native environment. The results show that UreG maintains heterogeneous structural landscape in-cell, existing in a conformational ensemble of two major conformers, showing either random coil-like or compact properties. These data support the physiological relevance of the intrinsically disordered nature of UreG and indicates a role of protein flexibility for this specific enzyme, possibly related to the regulation of promiscuous protein interactions for metal ion delivery.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

Evidence for [2Fe-2S]2+ and Linear [3Fe-4S]1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses.

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 µm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

Probing the Structural Dynamics of a Bacterial Chaperone in Its Native Environment by Nitroxide-Based EPR Spectroscopy.

One of the greatest current challenges in structural biology is to study protein dynamics over a wide range of timescales in complex environments, such as the cell. Among magnetic resonances suitable for this approach, electron paramagnetic resonance spectroscopy coupled to site-directed spin labeling (SDSL-EPR) has emerged as a promising tool to study protein local dynamics and conformational ensembles. In this work, we exploit the sensitivity of nitroxide labels to report protein local dynamics at room temperature. We demonstrate that such studies can be performed while preserving both the integrity of the cells and the activity of the protein under investigation. Using this approach, we studied the structural dynamics of the chaperone NarJ in its natural host, Escherichia coli. We established that spin-labeled NarJ is active inside the cell. We showed that the cellular medium affects NarJ structural dynamics in a site-specific way, while the structural flexibility of the protein is maintained. Finally, we present and discuss data on the time-resolved dynamics of NarJ in cellular context.

Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe-S Cluster Biosynthesis.

Iron-sulfur (Fe-S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe-S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe-S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe-S cluster assembly process and establish that the initiation of Fe-S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Spectroscopic and Structural Characterization of Reduced Desulfovibrio vulgaris Hildenborough W-FdhAB Reveals Stable Metal Coordination during Catalysis.

Metal-dependent formate dehydrogenases are important enzymes due to their activity of CO2 reduction to formate. The tungsten-containing FdhAB formate dehydrogenase from Desulfovibrio vulgaris Hildenborough is a good example displaying high activity, simple composition, and a notable structural and catalytic robustness. Here, we report the first spectroscopic redox characterization of FdhAB metal centers by EPR. Titration with dithionite or formate leads to reduction of three [4Fe-4S]1+ clusters, and full reduction requires Ti(III)-citrate. The redox potentials of the four [4Fe-4S]1+ centers range between -250 and -530 mV. Two distinct WV signals were detected, WDV and WFV, which differ in only the g2-value. This difference can be explained by small variations in the twist angle of the two pyranopterins, as determined through DFT calculations of model compounds. The redox potential of WVI/V was determined to be -370 mV when reduced by dithionite and -340 mV when reduced by formate. The crystal structure of dithionite-reduced FdhAB was determined at high resolution (1.5 Å), revealing the same structural alterations as reported for the formate-reduced structure. These results corroborate a stable six-ligand W coordination in the catalytic intermediate WV state of FdhAB.

Identification and characterization of a noncanonical menaquinone-linked formate dehydrogenase.

The molybdenum/tungsten-bis-pyranopterin guanine dinucleotide family of formate dehydrogenases (FDHs) plays roles in several metabolic pathways ranging from carbon fixation to energy harvesting because of their reaction with a wide variety of redox partners. Indeed, this metabolic plasticity results from the diverse structures, cofactor content, and substrates used by partner subunits interacting with the catalytic hub. Here, we unveiled two noncanonical FDHs in Bacillus subtilis, which are organized into two-subunit complexes with unique features, ForCE1 and ForCE2. We show that the formate oxidoreductase catalytic subunit interacts with an unprecedented partner subunit, formate oxidoreductase essential subunit, and that its amino acid sequence within the active site deviates from the consensus residues typically associated with FDH activity, as a histidine residue is naturally substituted with a glutamine. The formate oxidoreductase essential subunit mediates the utilization of menaquinone as an electron acceptor as shown by the formate:menadione oxidoreductase activity of both enzymes, their copurification with menaquinone, and the distinctive detection of a protein-bound neutral menasemiquinone radical by multifrequency electron paramagnetic resonance (EPR) experiments on the purified enzymes. Moreover, EPR characterization of both FDHs reveals the presence of several [Fe-S] clusters with distinct relaxation properties and a weakly anisotropic Mo(V) EPR signature, consistent with the characteristic molybdenum/bis-pyranopterin guanine dinucleotide cofactor of this enzyme family. Altogether, this work enlarges our knowledge of the FDH family by identifying a noncanonical FDH, which differs in terms of architecture, amino acid conservation around the molybdenum cofactor, and reactivity.

Membrane-Bound Flavocytochrome MsrQ Is a Substrate of the Flavin Reductase Fre in Escherichia coli.

MsrPQ is a new type of methionine sulfoxide reductase (Msr) system found in bacteria. It is specifically involved in the repair of periplasmic methionine residues that are oxidized by hypochlorous acid. MsrP is a periplasmic molybdoenzyme that carries out the Msr activity, whereas MsrQ, an integral membrane-bound hemoprotein, acts as the physiological partner of MsrP to provide electrons for catalysis. Although MsrQ (YedZ) was associated since long with a protein superfamily named FRD (ferric reductase domain), including the eukaryotic NADPH oxidases and STEAP proteins, its biochemical properties are still sparsely documented. Here, we have investigated the cofactor content of the E. coli MsrQ and its mechanism of reduction by the flavin reductase Fre. We showed by electron paramagnetic resonance (EPR) spectroscopy that MsrQ contains a single highly anisotropic low-spin (HALS) b-type heme located on the periplasmic side of the membrane. We further demonstrated that MsrQ holds a flavin mononucleotide (FMN) cofactor that occupies the site where a second heme binds in other members of the FDR superfamily on the cytosolic side of the membrane. EPR spectroscopy indicates that the FMN cofactor can accommodate a radical semiquinone species. The cytosolic flavin reductase Fre was previously shown to reduce the MsrQ heme. Here, we demonstrated that Fre uses the FMN MsrQ cofactor as a substrate to catalyze the electron transfer from cytosolic NADH to the heme. Formation of a specific complex between MsrQ and Fre could favor this unprecedented mechanism, which most likely involves transfer of the reduced FMN cofactor from the Fre active site to MsrQ.

Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs.

Oxidative plant cell-wall processing enzymes are of great importance in biology and biotechnology. Yet, our insight into the functional interplay amongst such oxidative enzymes remains limited. Here, a phylogenetic analysis of the auxiliary activity 7 family (AA7), currently harbouring oligosaccharide flavo-oxidases, reveals a striking abundance of AA7-genes in phytopathogenic fungi and Oomycetes. Expression of five fungal enzymes, including three from unexplored clades, expands the AA7-substrate range and unveils a cellooligosaccharide dehydrogenase activity, previously unknown within AA7. Sequence and structural analyses identify unique signatures distinguishing the strict dehydrogenase clade from canonical AA7 oxidases. The discovered dehydrogenase directly is able to transfer electrons to an AA9 lytic polysaccharide monooxygenase (LPMO) and fuel cellulose degradation by LPMOs without exogenous reductants. The expansion of redox-profiles and substrate range highlights the functional diversity within AA7 and sets the stage for harnessing AA7 dehydrogenases to fine-tune LPMO activity in biotechnological conversion of plant feedstocks.

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility.

UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.

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.

In-Cell EPR: Progress towards Structural Studies Inside Cells.

Exploring the structure and dynamics of biomolecules in the context of their intracellular environment has become the ultimate challenge for structural biology. As the cellular environment is barely reproducible in vitro, investigation of biomolecules directly inside cells has attracted a growing interest. Among magnetic resonance approaches, site-directed spin labeling (SDSL) coupled to electron paramagnetic resonance (EPR) spectroscopy provides competitive and advantageous features to capture protein structure and dynamics inside cells. To date, several in-cell EPR approaches have been successfully applied to both bacterial and eukaryotic cells. In this review, the major advances of in-cell EPR spectroscopy are summarized, as well as the challenges this approach still poses.

Modulating lifetimes and relaxation times of phenoxyl radicals through their incorporation into different hybrid nanostructures.

The development of new open shell systems is essential for advances in spin science. In this work, we report the synthesis and characterization of three nanostructured materials, namely SBA-15 silicas, periodic mesoporous organosilicas (PMOs) and lamellar polysilsesquioxanes, all functionalized with the same diazene-based phenoxyl radical precursor. The impact of the nature of the material, i.e. loading of radical precursor and structure, on half-lifetimes (t1/2) and relaxation times of phenoxyl radicals was investigated. Although phenoxyl radicals are transient in solution, their t1/2 range from hours to years at room temperature (RT) when they are embedded in nanostructured materials. The above mentioned functionalized materials were used to generate the corresponding phenoxyl radicals and their relaxation times were measured (〈T1e〉 and Tm) from 50 K to RT. The results were rationalized in terms of limited mobility of the radical as a result of supramolecular interactions and structure rigidity. All these data show that it is possible to design functionalized nanostructured material with radicals possessing specific electronic relaxation properties which can be of interest in fields like DNP, organic magnetism or spin qubit.

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase.

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties. In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH 8 by 230 mV to 400 mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.

A new mechanistic model for an O2-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans.

The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (Em = -450 mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP+, and that it catalyzes the simultaneous reduction of FdxB and NAD+. Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O2 damage (Hinact). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H2 with a specific activity of 10 µmol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit.

Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges.

Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αß-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αß-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αß-tubulin.

Roles of the F-domain in [FeFe] hydrogenase.

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H+/H2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.

A Bioresistant Nitroxide Spin Label for In-Cell EPR Spectroscopy: In Vitro and In Oocytes Protein Structural Dynamics Studies.

Approaching protein structural dynamics and protein-protein interactions in the cellular environment is a fundamental challenge. Owing to its absolute sensitivity and to its selectivity to paramagnetic species, site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) has the potential to evolve into an efficient method to follow conformational changes in proteins directly inside cells. Until now, the use of nitroxide-based spin labels for in-cell studies has represented a major hurdle because of their short persistence in the cellular context. The design and synthesis of the first maleimido-proxyl-based spin label (M-TETPO) resistant towards reduction and being efficient to probe protein dynamics by continuous wave and pulsed EPR is presented. In particular, the extended lifetime of M-TETPO enabled the study of structural features of a chaperone in the absence and presence of its binding partner at endogenous concentration directly inside cells.