Hyperfine Sublevel Correlation Research Articles

Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

Structure and dynamics of catalytically competent but labile paramagnetic metal-hydrides: the Ti(iii)-H in homogeneous olefin polymerization.

Metal hydride complexes find widespread application in catalysis and their properties are often understood on the basis of the available crystal structures. However, some catalytically relevant metal hydrides are only spontaneously formed in situ, cannot be isolated in large quantities or crystallised and their structure is therefore ill defined. One such example is the paramagnetic Ti(iii)-hydride involved in homogeneous Ziegler–Natta catalysis, formed upon activation of CpTi(iv)Cl3 with modified methylalumoxane (MMAO). In this contribution, through a combined use of electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies we identify the nature of the ligands, their bonding interaction and the extent of the spin distribution. From the data, an atomistic and electronic model is proposed, which supports the presence of a self-assembled ion pair between a cationic terminal Ti-hydride and an aluminate anion, with a hydrodynamic radius of ca. 16 Å.

Two-dimensional 67Zn HYSCORE spectroscopy reveals that a Zn-bacteriochlorophyll aP' dimer is the primary donor (P840) in the type-1 reaction centers of Chloracidobacterium thermophilum.

Chloracidobacterium (C.) thermophilum is a microaerophilic, chlorophototrophic species in the phylum Acidobacteria that uses homodimeric type-1 reaction centers (RC) to convert light energy into chemical energy using (bacterio)chlorophyll ((B)Chl) cofactors. Pigment analyses show that these RCs contain BChl aP, Chl aPD, and Zn2+-BChl aP' in the approximate ratio 7.1 : 5.4 : 1. However, the functional roles of these three different Chl species are not yet fully understood. It was recently demonstrated that Chl aPD is the primary electron acceptor. Because Zn2+-(B)Chl aP' is present at low abundance, it was suggested that the primary electron donor might be a dimer of Zn2+-BChl aP' molecules. In this study, we utilize isotopic enrichment and high-resolution two-dimensional (2D) 14N and 67Zn hyperfine sublevel correlation (HYSCORE) spectroscopy to demonstrate that the primary donor cation, P840+, in the C. thermophilum RC is indeed a Zn2+-BChl aP' dimer. Density functional theory (DFT) calculations and the measured electron-nuclear hyperfine parameters of P840+ indicate that the electron spin density on P840+ is distributed nearly symmetrically over two Zn2+-(B)Chl aP' molecules as expected in a homodimeric RC. To our knowledge this is the only example of a photochemical RC in which the Chl molecules of the primary donor are metallated differently than those of the antenna.

Adsorption of Olefins at Cupric Ions in Metal-Organic Framework HKUST-1 Explored by Hyperfine Spectroscopy.

Metal-organic frameworks (MOFs) represent a promising platform for gas storage and separation. In this work, adsorption of olefins in Zn-doped HKUST-1 metal-organic framework is explored with hyperfine spectroscopy. By means of electron-nuclear double resonance and hyperfine sublevel correlation spectroscopy, we detect the interaction between the electron spins of the Cu2+ sites of the MOF and the 1H nuclear spins of adsorbed C2H4 and C4H8. Further analysis of the measured spectra allows us to precisely locate the protons in the vicinity of the Cu2+ ions and thereby establish ensemble-averaged structural models of the olefin molecules adsorbed at the open metal sites of HKUST-1.

Optimizing the transformation of HYSCORE data using the maximum entropy algorithm.

Non-uniform sampling (NUS) in combination with the Maximum Entropy (MaxEnt) algorithm as applied to multi-dimensional NMR data has been thoroughly investigated and the NUS approach shown to provide significant sensitivity improvements as compared to methods using uniformly sampled (US) data and the discrete Fourier transform (DFT). Hyperfine sublevel correlation (HYSCORE) is a standard pulse EPR experiment that can potentially benefit greatly from this approach, but the data present unique challenges as compared to NMR. HYSCORE data typically exhibit a very large range of peak intensities, signals are in the form of irregularly shaped ridges with variable intensities, and time traces are generally truncated to save measurement time. MaxEnt has the advantageous properties that it does not require US data, dampens weak signals (noise) and does not suffer from windowing artifacts due to truncation of the time traces. Critical to the success of the MaxEnt algorithm is the choice of the two input parameters aim and def which describe the data noise and contribution of entropy in the optimization, respectively. In this paper we expand our preliminary study on the application of MaxEnt to the reconstruction of HYSCORE spectra to include a detailed analysis on sensitivity to detect weak peaks, investigate the non-linearity of the transformation and ascertain if it can be characterized by the introduction of synthetic peaks, and define a general range for the choice of aim and def. Furthermore, the ability of the MaxEnt method to remove windowing artefacts in uniformly sampled truncated HYSCORE data is described.

Convolutional Neural Network Analysis of Two-Dimensional Hyperfine Sublevel Correlation Electron Paramagnetic Resonance Spectra.

A machine learning approach is presented for analyzing complex two-dimensional hyperfine sublevel correlation electron paramagnetic resonance (HYSCORE EPR) spectra with the proficiency of an expert spectroscopist. The computer vision algorithm requires no training on experimental data; rather, all of the spin physics required to interpret the spectra are learned from simulations alone. This approach is therefore applicable even when insufficient experimental data exist to train the algorithm. The neural network is demonstrated to be capable of utilizing the full information content of two-dimensional 14N HYSCORE spectra to predict the magnetic coupling parameters and their underlying probability distributions that were previously inaccessible. The predicted hyperfine ( a, T) and 14N quadrupole ( K, η) coupling constants deviate from the previous manual analyses of the experimental spectra on average by 0.11 MHz, 0.09 MHz, 0.19 MHz, and 0.09, respectively.

Elucidating the Role of Zinc-Bacteriochlorophyll A' in the Primary Photochemistry of Chloroacidobacterium thermophilum Reaction Centers

Chloracidobacterium (Cab.) thermophilum is a microaerophilic, chlorophototrophic species in the phylum of Acidobacteria that contains homodimeric Type I reaction centers (RC). Photosynthetic RCs in bacteria convert light energy into chemical energy using (bacterio)chlorophyll ((B)Chl) cofactors. In addition to participating in energy transfer in the light-harvesting complexes, the (B)Chl molecules also act as the primary electron donor and primary electron acceptor in Type I RCs. Previously, reversed-phase high-performance liquid chromatography of Cab. thermophilum RCs revealed the presence of (B)Chl a, Chl a, and Zn2+-(B)Chl a' in a ratio of about 12.8:8.0:2.0. Moreover, it was shown that Cab. thermophilum uses chlorosomes and FMO for light harvesting, and synthesizes (B)Chl c in addition to (B)Chl a, Chl a, and Zn2+-(B)Chl a'. It has been demonstrated that Chl a is the primary electron acceptor in Cab. thermophilum RC and since Zn2+-(B)Chl a' is only present in the RC, it was suggested that it could function as the primary electron donor. In this study, we utilize high-resolution, two-dimensional (2D) 14N and 67Zn hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic and geometric structure of the primary donor cation, P840+, in the Cab. thermophilum RC. Additionally, we perform density functional theory (DFT) calculations to determine the electron spin-density distribution and electron-nuclear hyperfine coupling parameters of P840+ for comparison with the parameters that are obtained from the experimental 2D HYSCORE measurements. Our studies indicate that that the primary donor, P840, of the Cab. RC is comprised of Zn2+-sBChl a' molecules.

EPR study of luminescence mechanism of Nd3+-doped borate aluminate glass

The luminescence mechanism of Nd3+: B2O3-Al2O3-CaO glasses with different amounts of B2O3 were investigated based on their electron paramagnetic resonance spectra and luminescence properties. The results of the echo-detection field sweep exhibited two different maxima indicating that Nd3+ ions occupy two different sites. 27Al and 11B signals in two-dimensional hyperfine sublevel correlation spectra were observed in the (+, +) quadrant, indicating that both 27Al and 11B are located in the second or higher order Nd3+ coordination shell. In addition, the fraction of the 11B nucleus present in the Nd3+ coordination sphere increased with an increase in the amount of B2O3. The continuous changes in the hypersensitive transition intensity and luminescence properties are strong indicators for the change in the Nd3+ coordination environment.

Polypyridyl-Based Copper Phenanthrene Complexes: A New Type of Stabilized Artificial Chemical Nuclease.

The building of robust and versatile inorganic scaffolds with artificial metallo-nuclease (AMN) activity is an important goal for bioinorganic, biotechnology, and metallodrug research fields. Here, a new type of AMN combining a tris-(2-pyridylmethyl)amine (TPMA) scaffold with the copper(II) N,N'-phenanthrene chemical nuclease core is reported. In designing these complexes, the stabilization and flexibility of TPMA together with the prominent chemical nuclease activity of copper 1,10-phenanthroline (Phen) were targeted. A second aspect was the opportunity to introduce designer phenazine DNA intercalators (e.g., dipyridophenazine; DPPZ) for improved DNA recognition. Five compounds of formula [Cu(TPMA)(N,N')]2+ (where N,N' is 2,2-bipyridine (Bipy), Phen, 1,10-phenanthroline-5,6-dione (PD), dipyridoquinoxaline (DPQ), or dipyridophenazine (DPPZ)) were developed and characterized by X-ray crystallography. Solution stabilities were studied by continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE), and Davies electron-nuclear double resonance (ENDOR) spectroscopies, which demonstrated preferred geometries in which phenanthrene ligands were coordinated to the copper(II) TPMA core. Complexes with Phen, DPQ, and DPPZ ligands possessed enhanced DNA binding activity, with DPQ and DPPZ compounds showing excellent intercalative effects. These complexes are effective AMNs and analysis with spin-trapping scavengers of reactive oxygen species and DNA repair enzymes with glycosylase/endonuclease activity demonstrated a distinctive DNA oxidation activity compared to classical Sigman- and Fenton-type reagents.

Investigation of luminescence mechanism of Nd3+-doped calcium aluminate glasses: Effect of glass-formers

The relationship between the luminescence properties and the glass structure of Nd3+-doped CaO-Al2O3-(B2O3, SiO2, or Ga2O3) glasses are investigated. The luminescence properties of CaO-Al2O3-B2O3 system are twice as much as that of the CaO-Al2O3-(SiO2 or Ga2O3) system. The glass structure and the coordination environment of the Nd3+ ions were investigated using nuclear magnetic resonance, Raman and electron paramagnetic resonance spectroscopy. All Al, Ga, and Si atoms are four coordinated. The B atoms have three- and four-coordinated boron units, however, most of them are three coordinated. The hyperfine sublevel correlation spectra reveal that the Al and B nuclei are in the coordination sphere of Nd3+ ions for aluminoborate glasses; however, only 27Al signals could be observed for aluminosilicate and aluminogallate glasses. Owing to the increased number of AlO4-BO3 connections and 11B nuclei around the Nd3+ ions, B has a significant effect on the luminescence properties of the Nd3+ ions. The increase in GaO4 or SiO4 does not change the three dimensional-tetrahedral network, and the coordination environment of Nd3+ ions varies slightly. Thus, the effect of Ga2O3 and SiO2 on the luminescence properties of the Nd3+ ions is not as great as that of B2O3.

Exploring the Interaction of Ammonia with Supported Vanadia Catalysts by Continuous Wave and Pulsed Electron Paramagnetic Resonance Spectroscopy

Continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) spectroscopy are used to investigate the nature of vanadium species on V2O5/TiO2 catalysts prepared via wet impregnation and gas-phase grafting routes. CW EPR experiments show in both cases the formation of VO2+ vanadyl ions upon reaction with ammonia; moreover, phase memory time filtered echo-detected EPR experiments at X- and Q-band frequencies provide direct experimental evidence for the reduction of the TiO2 substrate and the formation of Ti3+ ions. X-band hyperfine sublevel correlation (HYSCORE) experiments are used to investigate the local environment of VO2+ ions. The direct nitrogen coordination of residual ammonia fragments in the equatorial plane of the vanadyl ion is demonstrated.

Nitric Oxide Modulates Endonuclease III Redox Activity by a 800 mV Negative Shift upon [Fe4S4] Cluster Nitrosylation.

Here we characterize the [Fe4S4] cluster nitrosylation of a DNA repair enzyme, endonuclease III (EndoIII), using DNA-modified gold electrochemistry and protein film voltammetry, electrophoretic mobility shift assays, mass spectrometry of whole and trypsin-digested protein, and a variety of spectroscopies. Exposure of EndoIII to nitric oxide under anaerobic conditions transforms the [Fe4S4] cluster into a dinitrosyl iron complex, [(Cys)2Fe(NO)2]-, and Roussin's red ester, [(μ-Cys)2Fe2(NO)4], in a 1:1 ratio with an average retention of 3.05 ± 0.01 Fe per nitrosylated cluster. The formation of the dinitrosyl iron complex is consistent with previous reports, but the Roussin's red ester is an unreported product of EndoIII nitrosylation. Hyperfine sublevel correlation (HYSCORE) pulse EPR spectroscopy detects two distinct classes of NO with 14N hyperfine couplings consistent with the dinitrosyl iron complex and reduced Roussin's red ester. Whole-protein mass spectrometry of EndoIII nitrosylated with 14NO and 15NO support the assignment of a protein-bound [(μ-Cys)2Fe2(NO)4] Roussin's red ester. The [Fe4S4]2+/3+ redox couple of DNA-bound EndoIII is observable using DNA-modified gold electrochemistry, but nitrosylated EndoIII does not display observable redox activity using DNA electrochemistry on gold despite having a similar DNA-binding affinity as the native protein. However, direct electrochemistry of protein films on graphite reveals the reduction potential of native and nitrosylated EndoIII to be 127 ± 6 and -674 ± 8 mV vs NHE, respectively, corresponding to a shift of approximately -800 mV with cluster nitrosylation. Collectively, these data demonstrate that DNA-bound redox activity, and by extension DNA-mediated charge transport, is modulated by [Fe4S4] cluster nitrosylation.

Cu(II) EPR Reveals Two Distinct Binding Sites and Oligomerization of Innate Immune Protein Calgranulin C

S100A12 or Calgranulin C is a homodimeric antimicrobial protein of the S100 family of EF-hand calcium-modulated proteins. S100A12 is involved in many diseases such as inflammation, tumor invasion, cancer and neurological disorders such as Alzheimer’s disease. The binding of transition metal ions to the protein is important as the sequestering of the metal ion induces conformational changes in the protein, inhibiting the growth of various pathogenic microorganisms. In this work, we probe the Cu2+ binding properties of Calgranulin C. We demonstrate that the two Cu2+ binding sites in Calgranulin C show different coordination environments in solution. Continuous wave-electron spin resonance (CW-ESR) spectra of Cu2+-bound protein clearly show two distinct components at higher Cu2+:protein ratios, which is indicative of the two different binding environments for the Cu2+ ions. The g|| and A|| values are also different for the two components, indicating that the number of directly coordinated nitrogen in each site differs. Furthermore, we perform CW-ESR titrations to obtain the binding affinity of the Ca2+-loaded protein to Cu2+ ions. We observe a positive cooperativity in binding of the two Cu2+ ions. To further probe the Cu2+ coordination, we also perform electron spin echo envelope modulation (ESEEM) experiment. We perform ESEEM at two different fields where one Cu2+ binding site dominates the other. At both sites we see distinct signatures of Cu2+–histidine coordination. However, we clearly see that the ESEEM spectra corresponding to the two Cu2+ binding sites are significantly different. There is clear change in the intensity of the double quantum peak with respect to the nuclear quadrupole interaction peak at the two different fields. Furthermore, ESEEM along with hyperfine sublevel correlation show that only one of the two Cu2+ binding sites has backbone coordination, confirming our previous observation. Finally, we perform double electron–electron resonance spectroscopy to probe if the difference in binding environment is due to the Cu2+ binding to different sites in the protein. We obtain a distance distribution with a sharp peak at ~ 3 nm and a broad peak at ~ 4 nm. The shorter distance agrees with the Cu2+–Cu2+ distance expected for a dimer from the crystal structure. The longer distance is consistent with the Cu2+–Cu2+ distance when oligomerization occurs.

Relationship investigation of structure and properties of Nd3+: Ga2O3-Al2O3-PbO-CaO via Raman, infrared, NMR and EPR spectroscopy

A detailed study structure and luminescence properties of Nd3+: Ga2O3-Al2O3-PbO-CaO laser glasses was conducted. The network structure was mainly dominated by Al (IV) or Ga (IV). With the increase of the Al2O3 content, the Al (IV) content increases, whereas the Ga (IV) content decreases. Hyperfine sublevel correlation (HYSCORE) spectra reveal 27Al and 207Pb weak hyperfine coupling, which indicates that these nuclei are in the second- or higher-order Nd3+ coordination sphere. No 69Ga or 71Ga signals can be detected in the three-pulse electron spin echo envelope modulation (3P-ESEEM) and HYSCORE spectra. For germanate glass, the local environment of the Nd3+ ions is characterized by the Pb ligand, for aluminum gallium glasses, the Nd3+ ions are surrounded by Al3+ and Pb2+ ions, and for aluminum glass, Nd3+ ions are mainly surrounded by 27Al cations. The change in the structure and in the local environment of Nd3+ ions results in a change in the luminescence properties of glasses.

Low-Coordinated Titanium(III) Alkyl-Molecular and Surface-Complexes: Detailed Structure from Advanced EPR Spectroscopy.

The structure of paramagnetic surface species is notoriously difficult to determine. For TiIII centers related to Ziegler-Natta catalysis, we demonstrate here that detailed structural information can be obtained by advanced EPR spectroscopy and DFT computations, benchmarked on molecular analogs. The hyperfine sublevel correlation (HYSCORE) spectra obtained after reaction with 13 C-labeled ethylene provides information about the coupling with a proton in the first coordination sphere of TiIII as well as significant 13 C hyperfine coupling and thereby allows structural assignment of the surface species.

Low‐Coordinated Titanium(III) Alkyl—Molecular and Surface—Complexes: Detailed Structure from Advanced EPR Spectroscopy

AbstractThe structure of paramagnetic surface species is notoriously difficult to determine. For TiIII centers related to Ziegler–Natta catalysis, we demonstrate here that detailed structural information can be obtained by advanced EPR spectroscopy and DFT computations, benchmarked on molecular analogs. The hyperfine sublevel correlation (HYSCORE) spectra obtained after reaction with 13C‐labeled ethylene provides information about the coupling with a proton in the first coordination sphere of TiIII as well as significant 13C hyperfine coupling and thereby allows structural assignment of the surface species.

Salen Complexes as Fire Protective Agents for Thermoplastic Polyurethane: Deep Electron Paramagnetic Resonance Spectroscopy Investigation.

The contribution of copper complexes of salen-based Schiff bases N, N'-bis(salicylidene)ethylenediamine (C1), N, N'-bis(4-hydroxysalicylidene)ethylenediamine (C2), and N, N'-bis(5-hydroxysalicylidene)ethylenediamine (C3) to the flame retardancy of thermoplastic polyurethane (TPU) is investigated in the context of minimizing the inherent flammability of TPU. Thermal and fire properties of TPU are evaluated. It is observed that fire performances vary depending upon the substitution of the salen framework. Cone calorimetry [mass loss calorimetry (MLC)] results show that, in TPU at 10 wt % loading, C2 and C3 reduce the peak of heat release rate by 46 and 50%, respectively. At high temperature, these copper complexes undergo polycondensation leading to resorcinol-type resin in the condensed phase and thus acting as intumescence reinforcing agents. C3 in TPU is particularly interesting because it delays significantly the time to ignition (MLC experiment). In addition, pyrolysis combustion flow calorimetry shows reduction in the heat release rate curve, suggesting its involvement in gas-phase action. Structural changes of copper complexes and radical formation during thermal treatment as well as their influence on fire retardancy of TPU in the condensed phase are investigated by spectroscopic studies supported by microscopic and powder diffraction studies. Electron paramagnetic resonance (EPR) spectroscopy was fully used to follow the redox changes of Cu(II) ions as well as radical formation of copper complexes/TPU formulations in their degradation pathways. Pulsed EPR technique of hyperfine sublevel correlation spectroscopy reveals evolution of the local surrounding of copper and radicals with a strong contribution of nitrogen fragments in the degradation products. Further, the spin state of radicals was investigated by the two-dimensional technique of phase-inverted echo-amplitude detected nutation experiment. Two different radicals were detected, that is, one monocarbon radical and an oxygen biradical. Thus, the EPR study permits to deeply investigate the mode of action of copper salen complexes in TPU.

Low-temperature binding of NO adsorbed on MIL-100(Al)-A case study for the application of high resolution pulsed EPR methods and DFT calculations.

The low-temperature binding of nitric oxide (NO) in the metal-organic framework MIL-100(Al) has been investigated by pulsed electron nuclear double resonance and hyperfine sublevel correlation spectroscopy. Three NO adsorption species have been identified. Among them, one species has been verified experimentally to bind directly to an 27Al atom and all its relevant 14N and 27Al hyperfine interaction parameters have been determined spectroscopically. Those parameters fit well to the calculated ones of a theoretical cluster model, which was derived by density functional theory (DFT) in the present work and describes the low temperature binding of NO to the regular coordinatively unsaturated Al3+ site of the MIL-100(Al) structure. As a result, the Lewis acidity of that site has been characterized using the NO molecule as an electron paramagnetic resonance active probe. The DFT derived wave function analysis revealed a bent end-on coordination of the NO molecule adsorbed at that site which is almost purely ionic and has a weak binding energy. The calculated flat potential energy surface of this species indicates the ability of the NO molecule to freely rotate at intermediate temperatures while it is still binding to the Al3+ site. For the other two NO adsorption species, no structural models could be derived, but one of them is indicated to be adsorbed at the organic part of the metal-organic framework. Hyperfine interactions with protons, weakly coupled to the observed NO adsorption species, have also been measured by pulsed electron paramagnetic resonance and found to be consistent with their attribution to protons of the MIL-100(Al) benzenetricarboxylate ligand molecules.

Pulse EPR Study of Gas Adsorption in Cu2+-Doped Metal–Organic Framework [Zn2(1,4-bdc)2(dabco)

Gas separation and storage are the hot topics for addressing current challenges in energy and environmental science, including the air pollution problems and alternative fuels, and metal–organic frameworks (MOFs) have a great potential in these fields. Herewith, we present the electron paramagnetic resonance (EPR) study of the adsorption of several gases (hydrogen D2, methane 13CH4 and CD4, and carbon dioxide 13CO2) in Cu2+-doped MOF [Zn2(1,4-bdc)2(dabco)]. The obtained compound of composition [Zn1.993Cu0.007(1,4-bdc)2(dabco)] is suitable for studying adsorption geometries at Cu2+ ions and in their closest environments using pulse EPR. In attempt to characterize D2, 13CH4, CD4, and 13CO2 adsorption sites, we applied echo-detected EPR along with hyperfine sublevel correlation spectroscopy and pulse electron-nuclear double resonance spectroscopy. Altogether, these methods demonstrated the preferred location of gas molecules in the framework being at least 6 A away from the copper ions. In addition, EPR spectroscopy allowed determination of the proton environment of copper and confirmed its incorporation into the MOF lattice, which is hard to establish using other techniques.

Using Hyperfine Electron Paramagnetic Resonance Spectroscopy to Define the Proton-Coupled Electron Transfer Reaction at Fe-S Cluster N2 in Respiratory Complex I.

Energy-transducing respiratory complex I (NADH:ubiquinone oxidoreductase) is one of the largest and most complicated enzymes in mammalian cells. Here, we used hyperfine electron paramagnetic resonance (EPR) spectroscopic methods, combined with site-directed mutagenesis, to determine the mechanism of a single proton-coupled electron transfer reaction at one of eight iron-sulfur clusters in complex I, [4Fe-4S] cluster N2. N2 is the terminal cluster of the enzyme's intramolecular electron-transfer chain and the electron donor to ubiquinone. Because of its position and pH-dependent reduction potential, N2 has long been considered a candidate for the elusive "energy-coupling" site in complex I at which energy generated by the redox reaction is used to initiate proton translocation. Here, we used hyperfine sublevel correlation (HYSCORE) spectroscopy, including relaxation-filtered hyperfine and single-matched resonance transfer (SMART) HYSCORE, to detect two weakly coupled exchangeable protons near N2. We assign the larger coupling with A(1H) = [-3.0, -3.0, 8.7] MHz to the exchangeable proton of a conserved histidine and conclude that the histidine is hydrogen-bonded to N2, tuning its reduction potential. The histidine protonation state responds to the cluster oxidation state, but the two are not coupled sufficiently strongly to catalyze a stoichiometric and efficient energy transduction reaction. We thus exclude cluster N2, despite its proton-coupled electron transfer chemistry, as the energy-coupling site in complex I. Our work demonstrates the capability of pulse EPR methods for providing detailed information on the properties of individual protons in even the most challenging of energy-converting enzymes.