Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolution of all of the principal components of the g tensors of the two radicals and revealed significant differences in the g tensor anisotropies. The spectra together with 2H ENDOR spectra and supporting DFT calculations show that the g tensor anisotropy is directly diagnostic of the presence or absence as well as the strength of a hydrogen bond to the indole nitrogen. The approach is a powerful one for identifying and characterizing hydrogen bonds that are critical in the regulation of tryptophan-assisted electron transfer and tryptophan-mediated redox chemistry in proteins.

Manganese-oxide minerals (MnOx) are widely distributed over the Earth’s surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnOx minerals by oxidizing Mn2+(aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as “Mnx” that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE3F3G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E3F3 hexamer. This isolated MnxG shows activity for Mn2+(aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g∥ = 2.320 and A∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g∥ = 2.210 and A∥ = 615 MHz and resides in the putative hexameric MnxE3F3 subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.

Fluorinated tyrosines (FnY’s, n = 2 and 3) have been site-specifically incorporated into E. coli class Ia ribonucleotide reductase (RNR) using the recently evolved M. jannaschii Y-tRNA synthetase/tRNA pair. Class Ia RNRs require four redox active Y’s, a stable Y radical (Y·) in the β subunit (position 122 in E. coli), and three transiently oxidized Y’s (356 in β and 731 and 730 in α) to initiate the radical-dependent nucleotide reduction process. FnY (3,5; 2,3; 2,3,5; and 2,3,6) incorporation in place of Y122-β and the X-ray structures of each resulting β with a diferric cluster are reported and compared with wt-β2 crystallized under the same conditions. The essential diferric-FnY· cofactor is self-assembled from apo FnY-β2, Fe2+, and O2 to produce ∼1 Y·/β2 and ∼3 Fe3+/β2. The FnY· are stable and active in nucleotide reduction with activities that vary from 5% to 85% that of wt-β2. Each FnY·-β2 has been characterized by 9 and 130 GHz electron paramagnetic resonance and high-field electron nuclear double resonance spectroscopies. The hyperfine interactions associated with the 19F nucleus provide unique signatures of each FnY· that are readily distinguishable from unlabeled Y·’s. The variability of the abiotic FnY pKa’s (6.4 to 7.8) and reduction potentials (−30 to +130 mV relative to Y at pH 7.5) provide probes of enzymatic reactions proposed to involve Y·’s in catalysis and to investigate the importance and identity of hopping Y·’s within redox active proteins proposed to protect them from uncoupled radical chemistry.

NifB utilizes two equivalents of S-adenosyl methionine (SAM) to insert a carbide atom and fuse two substrate [Fe–S] clusters forming the NifB cofactor (NifB-co), which is then passed to NifEN for further modification to form the iron–molybdenum cofactor (FeMo-co) of nitrogenase. Here, we demonstrate that NifB from the methanogen Methanocaldococcus infernus is a radical SAM enzyme able to reductively cleave SAM to 5′-deoxyadenosine radical and is competent in FeMo-co maturation. Using electron paramagnetic resonance spectroscopy we have characterized three [4Fe–4S] clusters, one SAM binding cluster, and two auxiliary clusters probably acting as substrates for NifB-co formation. Nitrogen coordination to one or more of the auxiliary clusters in NifB was observed, and its mechanistic implications for NifB-co dissociation from the maturase are discussed.

Three maturase enzymes—HydE, HydF, and HydG—synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)]− species is a key intermediate in H-cluster maturation.

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe–4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes—HydE, HydF, and HydG—in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe2+, l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)]− intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)]− species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe2+, Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN–, and the identification of the final organometallic products of the reaction.

A variety of metal oxides can catalyze the oxidation of water to molecular oxygen when polarized by a sufficiently high electrochemical potential. Minimizing the overpotential and increasing the rate of the oxygen-evolving reaction (OER) are key goals in making such materials a component of viable energy storage devices. However, the structural factors that imbue the metal oxides with their catalytic power are difficult to assess as these solids contain many distinct metal-ion sites, have a varying amount of defect sites within the lattice, and can be composed of multiple phases. In the present study, we determined the magnetic properties for a series of dimeric cobalt complexes in which the two metal centers are bridged by a dioxygen moiety. Our spectroscopically validated electronic structure description indicates that each species is best described as two Co(III) ions that are bound to a μ–η1η1 superoxide ligand. Intriguingly, we found evidence that the two compounds that possess oxygen-evolving activity coordinate the superoxide ion in an unusual, nonplanar fashion. It appears as if the intermediately long Co···Co distance of 3.9 Å is responsible for the unusual superoxide binding geometry. This structural factor may be an important element in the design of solid-state OER catalysts.

Oxalate decarboxylase (OxDC) catalyzes the disproportionation of oxalic acid monoanion into CO2 and formate. The enzyme has long been hypothesized to utilize dioxygen to form mononuclear Mn(III) or Mn(IV) in the catalytic site during turnover. Recombinant OxDC, however, contains only tightly bound Mn(II), and direct spectroscopic detection of the metal in higher oxidation states under optimal catalytic conditions (pH 4.2) has not yet been reported. Using parallel mode electron paramagnetic resonance spectroscopy, we now show that substantial amounts of Mn(III) are indeed formed in OxDC, but only in the presence of oxalate and dioxygen under acidic conditions. These observations provide the first direct support for proposals in which Mn(III) removes an electron from the substrate to yield a radical intermediate in which the barrier to C–C bond cleavage is significantly decreased. Thus, OxDC joins a small list of enzymes capable of stabilizing and controlling the reactivity of the powerful oxidizing species Mn(III).

The antimicrobial protein calprotectin (CP), a hetero-oligomer of the S100 family members S100A8 and S100A9, is the only identified mammalian Mn(II)-sequestering protein. Human CP uses Ca(II) ions to tune its Mn(II) affinity at a biologically unprecedented hexahistidine site that forms at the S100A8/S100A9 interface, and the molecular basis for this phenomenon requires elucidation. Herein, we investigate the remarkable Mn(II) coordination chemistry of human CP using X-ray crystallography as well as continuous-wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopies. An X-ray crystallographic structure of Mn(II)-CP containing one Mn(II), two Ca(II), and two Na(I) ions per CP heterodimer is reported. The CW EPR spectrum of Ca(II)- and Mn(II)-bound CP prepared with a 10:0.9:1 Ca(II):Mn(II):CP ratio is characterized by an unusually low zero-field splitting of 485 MHz (E/D = 0.30) for the S = 5/2 Mn(II) ion, consistent with the high symmetry of the His6 binding site observed crystallographically. Results from electron spin–echo envelope modulation and electron–nuclear double resonance experiments reveal that the six Mn(II)-coordinating histidine residues of Ca(II)- and Mn(II)-bound CP are spectroscopically equivalent. The observed 15N (I = 1/2) hyperfine couplings (A) arise from two distinct classes of nitrogen atoms: the coordinating ε-nitrogen of the imidazole ring of each histidine ligand (A = [3.45, 3.71, 5.91] MHz) and the distal δ-nitrogen (A = [0.11, 0.18, 0.42] MHz). In the absence of Ca(II), the binding affinity of CP for Mn(II) drops by two to three orders of magnitude and coincides with Mn(II) binding at the His6 site as well as other sites. This study demonstrates the role of Ca(II) in enabling high-affinity and specific binding of Mn(II) to the His6 site of human calprotectin.

The dynamics of manganese solid formation (as MnOx) by the multicopper oxidase (MCO)-containing Mnx protein complex were examined by electron paramagnetic resonance (EPR) spectroscopy. Continuous-wave (CW) EPR spectra of samples of Mnx, prepared in atmosphere and then reacted with Mn(II) for times ranging from 7 to 600 s, indicate rapid oxidation of the substrate manganese (with two-phase pseudo-first-order kinetics modeled using rate coefficients of: k1obs = 0.205 ± 0.001 s–1 and k2obs = 0.019 ± 0.001 s–1). This process occurs on approximately the same time scale as in vitro solid MnOx formation when there is a large excess of Mn(II). We also found CW and pulse EPR spectroscopic evidence for at least three classes of Mn(II)-containing species in the reaction mixtures: (i) aqueous Mn(II), (ii) a specifically bound mononuclear Mn(II) ion coordinated to the Mnx complex by one nitrogenous ligand, and (iii) a weakly exchange-coupled dimeric Mn(II) species. These findings provide new insights into the molecular mechanism of manganese mineralization.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN–, and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe–4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe2+ binding and synthon release.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe–4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN–, and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the “dangler” Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe–4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe–4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe2+ binding and synthon release.

Proton reduction and H2 oxidation are key elementary reactions for solar fuel production. Hydrogenases interconvert H+ and H2 with remarkable efficiency and have therefore received much attention in this context. For [FeFe]-hydrogenases, catalysis occurs at a unique cofactor called the H-cluster. In this article, we discuss ways in which EPR spectroscopy has elucidated aspects of the bioassembly of the H-cluster, with a focus on four case studies: EPR spectroscopic identification of a radical en route to the CO and CN– ligands of the H-cluster, tracing 57Fe from the maturase HydG into the H-cluster, characterization of the auxiliary Fe–S cluster in HydG, and isotopic labeling of the CN– ligands of HydA for electronic structure studies of its Hox state. Advances in cell-free maturation protocols have enabled several of these mechanistic studies, and understanding H-cluster maturation may in turn provide insights leading to improvements in hydrogenase production for biotechnological applications.

In exchange-coupled mixed-valence spin systems, the magnitude and sign of the effective ligand hyperfine interaction (HFI) can be useful in determining the formal oxidation state of the coordinating metal ion, as well as provide information about the coordination geometry. This is due to the fact that the observed ligand HFI is a function of the projection factor (Clebsch-Gordon coefficient) that maps the site spin value Si of the local paramagnetic center onto the total spin of the exchange-coupled system, ST. Recently, this relationship has been successfully exploited in identifying the oxidation state of the Mn ion coordinated by the sole nitrogenous ligand to the oxygen-evolving complex in certain states of photosystem II. The origin and evolution of these efforts is described.

These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.

The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous l-tyrosine. Using selectively labeled tyrosine substrates, the cyanides were isotopically labeled via a recently developed in vitro maturation procedure allowing advanced electron paramagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN– ligands—a reporter of spin density on their respective coordinating iron ions—collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.

The binding of the substrate analogue methanol to the catalytic Mn4CaO5 cluster of the water-oxidizing enzyme photosystem II is known to alter the electronic structure properties of the oxygen-evolving complex without retarding O2-evolution under steady-state illumination conditions. We report the binding mode of 13C-labeled methanol determined using 9.4 GHz (X-band) hyperfine sublevel-correlation (HYSCORE) and 34 GHz (Q-band) electron spin–echo electron nuclear double resonance (ESE-ENDOR) spectroscopies. These results are compared to analogous experiments on a mixed-valence Mn(III)Mn(IV) complex (2-OH-3,5-Cl2-salpn)2Mn(III)Mn(IV) (salpn = N,N′-bis(3,5-dichlorosalicylidene)-1,3-diamino-2-hydroxypropane) in which methanol ligates to the Mn(III) ion (Larson et al. (1992) J. Am. Chem. Soc., 114, 6263). In the mixed-valence Mn(III,IV) complex, the hyperfine coupling to the 13C of the bound methanol (Aiso = 0.65 MHz, T = 1.25 MHz) is appreciably larger than that observed for 13C methanol associated with the Mn4CaO5 cluster poised in the S2 state, where only a weak dipolar hyperfine interaction (Aiso = 0.05 MHz, T = 0.27 MHz) is observed. An evaluation of the 13C hyperfine interaction using the X-ray structure coordinates of the Mn4CaO5 cluster indicates that methanol does not bind as a terminal ligand to any of the manganese ions in the oxygen-evolving complex. We favor methanol binding in place of a water ligand to the Ca2+ in the Mn4CaO5 cluster or in place of one of the waters that form hydrogen bonds with the oxygen bridges of the cluster.

The ruthenium “blue dimer” [(bpy)2Ru(OH2)]2O4+—the first well-defined molecular complex able to catalyze water oxidation at low overpotentials—has been the subject of numerous experimental and computational studies. However, elements of the reaction mechanism remain controversial. Of particular interest is the nature of the O–O bond-forming step. Herein, we report the first advanced electron paramagnetic resonance (EPR) spectroscopic studies of a high-valent intermediate that appears under conditions in which the catalyst is actively turning over. Results from previous studies have suggested that this intermediate is derived from [(bpy)2RuV(O)]2O4+, denoted {5,5}. Under photooxidizing conditions, the corresponding EPR signal disappears at a rate comparable to the turnover rate of the catalyst once the illumination source is removed. In the present work, the electronic and geometric structures of this species were explored using a variety of EPR techniques. Continuous wave (CW) EPR spectroscopy was used to probe the hyperfine coupling of the Ru ions, while corresponding ligand 14N hyperfine couplings were characterized with electron spin echo envelope modulation (ESEEM) and hyperfine sublevel correlation spectroscopy (HYSCORE) methods. Finally, 1H/2H ENDOR was performed to monitor any exchangeable protons. Our studies strongly suggest that the accumulating transient is an S = 1/2 species. This spin state formulation of the so-called {5,5} species is consistent with only a limited number of electronic structures, each of which is discussed. Notably, the observed large metal hyperfine coupling indicates that the orbital carrying the unpaired spin has significant ruthenyl-oxyl character, contrary to an earlier electronic structure description that had tentatively assigned the signal to formation of a bipyridine ligand radical.

Recoverin, a member of the neuronal calcium sensor (NCS) branch of the calmodulin superfamily, is expressed in retinal photoreceptor cells and serves as a calcium sensor in vision. Ca2+-induced conformational changes in recoverin cause extrusion of its covalently attached myristate (termed Ca2+-myristoyl switch) that promotes translocation of recoverin to disk membranes during phototransduction in retinal rod cells. Here we report double electron–electron resonance (DEER) experiments on recoverin that probe Ca2+-induced changes in distance as measured by the dipolar coupling between spin-labels strategically positioned at engineered cysteine residues on the protein surface. The DEER distance between nitroxide spin-labels attached at C39 and N120C is 2.5 ± 0.1 nm for Ca2+-free recoverin and 3.7 ± 0.1 nm for Ca2+-bound recoverin. An additional DEER distance (5–6 nm) observed for Ca2+-bound recoverin may represent an intermolecular distance between C39 and N120. 15N NMR relaxation analysis and CW-EPR experiments both confirm that Ca2+-bound recoverin forms a dimer at protein concentrations above 100 μM, whereas Ca2+-free recoverin is monomeric. We propose that Ca2+-induced dimerization of recoverin at the disk membrane surface may play a role in regulating Ca2+-dependent phosphorylation of dimeric rhodopsin. The DEER approach will be useful for elucidating dimeric structures of NCS proteins in general for which Ca2+-induced dimerization is functionally important but not well understood.

We report high-frequency (up to 219 GHz) and correspondingly high-field electron paramagnetic resonance spectra and X-ray crystal structure of [Co4O4(pyridine)4(acetate)4]+ ([Co4O4(py)4(OAc)4]+) that serves as a structural and spectroscopic model of cobalt-oxide films that are capable of oxidizing water. These results are, in large part, consistent with those from our earlier, lower-frequency study and serve to benchmark future high-field studies on paramagnetic states of the catalyst film.High-frequency and -field electron paramagnetic resonance spectra and X-ray crystal structure studies of [Co4O4(pyridine)4(acetate)4]+ confirm earlier electronic structure descriptions of this spectroscopic and functional model of cobalt-oxide films that are capable of oxidizing water.

Recently, Nocera and coworkers claim that an “artificial leaf” – able to harvest up to 4.7% of incident photonic energy to split water and produce fuels using only Earth abundant elements – has been produced with much of its design inspired by photosynthetic elements [1]. In this short review, we compare the structure and function of the cobalt-based water oxidation center of the artificial leaf with what is known about the manganese-based oxygen-evolving complex (OEC) of photosystem II (PSII). The geometric structure, proposed oxidation state topology, and putative mechanism of water oxidation for the respective catalytic sites are discussed.Highlights► The base unit of the Co-OEC structural motif mimics the OEC in PSII. ► Spectroscopic findings evince that the resting state is of a Co(III)/Co(IV) nature. ► Interlayer buffers facilitate water splitting through a charge balance mechanism.

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5′-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the μ-sulfide of the nearby [2Fe–2S]2+ cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe–2S]+ cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe–2S]+ cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe–2S]+ cluster.

We report the generation and characterization of a new high-spin iron(IV)–oxo complex supported by a trigonal nonheme pyrrolide platform. Oxygen-atom transfer to [(tpaMes)FeII]− (tpaAr = tris(5-arylpyrrol-2-ylmethyl)amine) in acetonitrile solution affords the Fe(III)–alkoxide product [(tpaMes2MesO)FeIII]− resulting from intramolecular C–H oxidation with no observable ferryl intermediates. In contrast, treatment of the phenyl derivative [(tpaPh)FeII]− with trimethylamine N-oxide in acetonitrile solution produces the iron(IV)–oxo complex [(tpaPh)FeIV(O)]− that has been characterized by a suite of techniques, including mass spectrometry as well as UV–vis, FTIR, Mössbauer, XAS, and parallel-mode EPR spectroscopies. Mass spectral, FTIR, and optical absorption studies provide signatures for the iron–oxo chromophore, and Mössbauer and XAS measurements establish the presence of an Fe(IV) center. Moreover, the Fe(IV)–oxo species gives parallel-mode EPR features indicative of a high-spin, S = 2 system. Preliminary reactivity studies show that the high-spin ferryl tpaPh complex is capable of mediating intermolecular C–H oxidation as well as oxygen-atom transfer chemistry.

Soluble guanylate cyclase (sGC) is a heme-containing enzyme that senses nitric oxide (NO). Formation of a heme Fe–NO complex is essential to sGC activation, and several spectroscopic techniques, including electron paramagnetic resonance (EPR) spectroscopy, have been aimed at elucidating the active enzyme conformation. Of these, only EPR spectra (X-band ∼9.6 GHz) have shown differences between low- and high-activity Fe–NO states, and these states are modeled in two different heme domain truncations of sGC, β1(1–194) and β2(1–217), respectively (Derbyshire et al., Biochemistry 2008, 47, 3892–3899). The EPR signal of the low-activity sGC Fe–NO complex exhibits a broad lineshape that has been interpreted as resulting from site-to-site inhomogeneity, and simulated using g strain, a continuous distribution about the principal values of a given g tensor. This approach, however, fails to account for visible features in the X-band EPR spectra as well as the g anisotropy observed at higher microwave frequencies. Herein we analyze X-, Q-, and D-band EPR spectra and show that both the broad lineshape and the spectral structure of the sGC EPR signal at multiple microwave frequencies can be simulated successfully with a superposition of only two distinct g tensors. These tensors represent different populations that likely differ in Fe–NO bond angle, hydrogen bonding, or the geometry of the amino acid residues. One of these conformations can be linked to a form of the enzyme with higher activity.

Although cytochrome P450cam from Pseudomonas putida, the archetype for all heme monooxygenases, has long been known to have a closed active site, recent reports show that the enzyme can also be crystallized in at least two clusters of open conformations. This suggests that the enzyme may undergo significant conformational changes during substrate binding and catalytic turnover. However, these conformations were observed in the crystalline state, and information is needed about the conformations that are populated in solution. In this study, double electron–electron resonance experiments were performed to observe substrate-induced changes in distance as measured by the dipolar coupling between spin labels introduced onto the surface of the enzyme on opposite sides of the substrate access channel. The double electron–electron resonance data show a decrease of 0.8 nm in the distance between spin labels placed at S48C and S190C upon binding the substrate camphor. A rotamer distribution model based on the crystal structures adequately describes the observed distance distributions. These results demonstrate conclusively that, in the physiologically relevant solution state, the substrate-free enzyme exists in the open P450cam-O conformation and that camphor binding results in conversion to the closed P450cam-C form. This approach should be useful for investigating many other P450s, including mammalian forms, in which the role of conformational change is of central importance but not well understood.

Building upon recent study of cobalt-oxide electrocatalysts in fluoride-buffered electrolyte at pH 3.4, we have undertaken a mechanistic investigation of cobalt-catalyzed water oxidation in aqueous buffering electrolytes from pH 0–14. This work includes electrokinetic studies, cyclic voltammetric analysis, and electron paramagnetic resonance (EPR) spectroscopic studies. The results illuminate a set of interrelated mechanisms for electrochemical water oxidation in alkaline, neutral, and acidic media with electrodeposited Co-oxide catalyst films (CoOxcfs) as well as for a homogeneous Co-catalyzed electrochemical water oxidation reaction. Analysis of the pH dependence of quasi-reversible features in cyclic voltammograms of the CoOxcfs provides the basis for a Pourbaix diagram that closely resembles a Pourbaix diagram derived from thermodynamic free energies of formation for a family of Co-based layered materials. Below pH 3, a shift from heterogeneous catalysis producing O2 to homogeneous catalysis yielding H2O2 is observed. Collectively, the results reported here provide a foundation for understanding the structure, stability, and catalytic activity of aqueous cobalt electrocatalysts for water oxidation.

Multifrequency electron paramagnetic resonace (EPR) spectroscopy and electronic structure calculations were performed on [Co4O4(C5H5N)4(CH3CO2)4]+ (1+), a cobalt tetramer with total electron spin S = 1/2 and formal cobalt oxidation states III, III, III, and IV. The cuboidal arrangement of its cobalt and oxygen atoms is similar to that of proposed structures for the molecular cobaltate clusters of the cobalt–phosphate (Co–Pi) water-oxidizing catalyst. The Davies electron–nuclear double resonance (ENDOR) spectrum is well-modeled using a single class of hyperfine-coupled 59Co nuclei with a modestly strong interaction (principal elements of the hyperfine tensor are equal to [−20(±2), 77(±1), −5(±15)] MHz). Mims 1H ENDOR spectra of 1+ with selectively deuterated pyridine ligands confirm that the amount of unpaired spin on the cobalt-bonding partner is significantly reduced from unity. Multifrequency 14N ESEEM spectra (acquired at 9.5 and 34.0 GHz) indicate that four nearly equivalent nitrogen nuclei are coupled to the electron spin. Cumulatively, our EPR spectroscopic findings indicate that the unpaired spin is delocalized almost equally across the eight core atoms, a finding corroborated by results from DFT calculations. Each octahedrally coordinated cobalt ion is forced into a low-spin electron configuration by the anionic oxo and carboxylato ligands, and a fractional electron hole is localized on each metal center in a Co 3dxz,yz-based molecular orbital for this essentially [Co+3.1254O4] system. Comparing the EPR spectrum of 1+ with that of the catalyst film allows us to draw conclusions about the electronic structure of this water-oxidation catalyst.

Multifrequency electron spin-echo envelope modulation (ESEEM) spectroscopy is used to ascertain the nature of the bonding interactions of various active site amino acids with the Mn ions that compose the oxygen-evolving cluster (OEC) in photosystem II (PSII) from the cyanobacterium Synechocystis sp. PCC 6803 poised in the S2 state. Spectra of natural isotopic abundance PSII (14N-PSII), uniformly 15N-labeled PSII (15N-PSII), and 15N-PSII containing 14N-histidine (14N-His/15N-PSII) are compared. These complementary data sets allow for a precise determination of the spin Hamiltonian parameters of the postulated histidine nitrogen interaction with the Mn ions of the OEC. These results are compared to those from a similar study on PSII isolated from spinach. Upon mutation of His332 of the D1 polypeptide to a glutamate residue, all isotopically sensitive spectral features vanish. Additional Ka- and Q-band ESEEM experiments on the D1-D170H site-directed mutant give no indication of new 14N-based interactions.

Human mitoNEET is a homodimeric iron−sulfur protein located in the outer mitochondrial membrane with unknown function, but which is known to interact with thiazolidinedione diabetes drugs. Each monomer houses a [2Fe−2S] cluster with an unusual (Cys)3(His)1 ligation. The His ligand is important for enabling cluster release and for tuning the redox potential. We use multifrequency (X-, Ka-, and Q-band) and multitechnique (continuous-wave, electron spin-echo envelope modulation (ESEEM), pulsed electron−nuclear double resonance (ENDOR), and hyperfine sublevel correlation (HYSCORE)) electron paramagnetic resonance spectroscopy to investigate the cluster in its paramagnetic reduced [Fe2+Fe3+] (S = 1/2) state. It has a rhombic g tensor (2.007, 1.937, 1.897) with an average g value of 1.947 that falls between those of Rieske-type and ferredoxin-type [2Fe−2S] clusters. Simulation and least-squares fitting of orientation-selective Ka- and Q-band ENDOR, 1D ESEEM, and HYSCORE spectra of 14N and 15N-labeled mitoNEET yield the principal values and orientations of both the hyperfine tensor (14N, Aiso = −6.25 MHz, T = −0.94 MHz) and the quadrupolar tensor (e2Qq/h = −2.47 MHz, η = 0.38) of the ligating histidine nitrogen Nδ. From these, we can infer the absolute g tensor orientation with respect to the cluster: The g2 axis is close to perpendicular to the [2Fe−2S] plane, and g1 and g3 are in-plane, but skewed from the Fe−Fe and S−S axes. In X-band ENDOR and ESEEM spectra, a weakly coupled nitrogen is visible, most likely the Nε of the histidine in the protonated state. We find that the cluster is in a valence-localized state, where Fe2+ is His-bound. The field-sweep spectra show evidence of intercluster dipolar coupling that can be simulated using an uncoupled spin model for each cluster (SFe2+ = 2, SFe3+ = 5/2). The parameters determined in this work can function as reporters on how the cluster structure is altered upon pH changes and drug binding.

Thin-film water oxidation catalysts (Co−Pi) prepared by electrodeposition from phosphate electrolyte and Co(NO3)2 have been characterized by electron paramagnetic resonance (EPR) spectroscopy. Co−Pi catalyst films exhibit EPR signals corresponding to populations of both Co(II) and Co(IV). As the deposition voltage is increased into the region where water oxidation prevails, the population of Co(IV) rises and the population of Co(II) decreases. The changes in the redox speciation of the film can also be induced, in part, by prolonged water oxidation catalysis in the absence of additional catalyst deposition. These results provide spectroscopic evidence for the formation of Co(IV) species during water oxidation catalysis at neutral pH.

Nitric oxide synthase (NOS), a homodimeric enzyme with a flavin reductase domain and a P450-type heme-containing oxygenase domain, catalyzes the formation of NO from l-arginine, NADPH, and O2 in a two-step reaction sequence. In the first step, a tetrahydrobiopterin (H4B) cofactor bound near one of the heme propionate groups acts as an electron donor to the P450-type heme active site, yielding a one-electron oxidized radical that is subsequently re-reduced. In solution, H4B undergoes two-electron oxidation, showing that the enzyme significantly alters the proton- and electron-transfer properties of the cofactor. Multifrequency EPR and ENDOR spectroscopy were used to determine magnetic parameters, and from them the (de)protonation state of the H4B radical in the oxygenase domain dimer of inducible NO synthase that was trapped by rapid freeze quench. From 9.5 and 330−416 GHz EPR and from 34 GHz 1H ENDOR spectroscopy, the g tensor of the radical and the hyperfine tensors of several N and H nuclei in the radical were obtained. Density functional theory calculations at the PBE0/EPR-II level for H4B radical models predict different spin density distributions and g and hyperfine tensors for different protonation states. Comparison of the predicted and experimental values leads to the conclusion that the radical is cationic H4B•+, suggesting that NOS stabilizes this protonated form to utilize the cofactor in a unique dual one-electron redox role, where it can deliver an electron to the active site for reductive oxygen activation and also remove an electron from the active site to generate NO and not NO−. The protein environment also prevents further oxidation and subsequent loss of function of the cofactor, thus enabling the enzyme to perform the unusual catalytic one-electron chemistry.

Pulse electron paramagnetic resonance (EPR) spectroscopy is employed at two very different excitation frequencies, 9.77 and 30.67 GHz, in the study of the nitrogen coordination environment of the Mn(III)Mn(IV) state of the dimanganese-containing catalases from Lactobacillus plantarum and Thermus thermophilus. Consistent with previous studies, the lower-frequency results reveal one unique histidine nitrogen−Mn cluster interaction. For the first time, a second, more strongly hyperfine-coupled 14N atom is unambiguously observed through the use of higher frequency/higher field EPR spectroscopy. The low excitation frequency spectral features are rationalized as arising from the interaction of a histidine nitrogen that is bound to the Mn(IV) ion, and the higher excitation frequency features are attributed to the histidine nitrogen bound to the Mn(III) ion. These results allow for the computation of intrinsic hyperfine coupling constants, which range from 2.2 to 2.9 MHz, for sp2-hybridized nitrogens coordinating equatorially to high-valence Mn ions. The relevance of these findings is discussed in the context of recent results from analogous higher frequency EPR studies of the Mn cluster in photosystem II and other exchange-coupled, transition metal-containing systems.

Antiferromagnetically coupled MnIIIMnIV dimers have been commonly used to study biological systems that exhibit complex exchange interactions. Such is the case for the oxygen evolving complex (OEC) in photosystem II (PSII), where we have studied whether the C-terminal carboxylate of D1-Ala344 is directly bound to the Mn cluster. To probe these protein-derived carboxylate hyperfine interactions, which give direct bonding information, Q-band (34 GHz) Mims ENDOR was performed on a MnIIIMnIV dimer ([MnIIIMnIV(μ-O)2μ-OAc(TACN)2](BPh4)2) (1) that was labeled with 13C (I = 1/2) at the carboxylate position of the acetate bridge. Ãdip is computed based on atomic coordinates from available X-ray crystal structures to be [−2.4, −0.8, 3.2] MHz. The value for Aiso was determined based on simulation of the experimental ENDOR data, for complex 1 Aiso = −1 MHz. Similar studies were then performed on PSII from Synechocystis sp. PCC 6803, in which all alanine-derived C═O groups are labeled with 13C including the C-terminal α-COO− group of D1 (Ala344), as well as PSII proteins uniformly labeled with 13C. Using recent X-ray crystallography data from T. elongatus the values for Ãdip were calculated and simulations of the experimental data led to Aiso values of 1.2, 1, and 2 MHz, respectively. We infer from complex 1 that an Aiso significantly larger than 1.2 MHz for a Mn-coordinating carboxylate moiety is unlikely. Therefore, we support the closer arrangement of Ala344 suggested by the Loll and Guskov structures and conclude that the C-terminal carboxylate of D1 polypeptide is directly bound to the Mn cluster.

Multifrequency electron spin–echo envelope modulation (ESEEM) spectroscopy is employed to measure the strength of the hyperfine coupling of magnetic nuclei to the paramagnetic (S = 1/2) S2 form of photosystem II (PSII). Previous X-band-frequency ESEEM studies indicated that one or more histidine nitrogens are electronically coupled to the tetranuclear manganese cluster in the S2 state of PSII. However, the spectral resolution was relatively poor at the ∼9 GHz excitation frequency, precluding any in-depth analysis of the corresponding bonding interaction between the detected histidine and the manganese cluster. Here we report ESEEM experiments using higher X-, P-, and Ka-band microwave frequencies to target PSII membranes isolated from spinach. The X- to P-band ESEEM spectra suffer from the same poor resolution as that observed in previous experiments, while the Ka-band spectra show remarkably well-resolved features that allow for the direct determination of the nuclear quadrupolar couplings for a single I = 1 14N nucleus. The Ka-band results demonstrate that at an applied field of 1.1 T we are much closer to the exact cancelation limit (αiso = 2ν14N) that optimizes ESEEM spectra. These results reveal hyperfine (αiso = 7.3 ± 0.20 MHz and αdip = 0.50 ± 0.10 MHz) and nuclear quadrupolar (e2qQ = 1.98 ± 0.05 MHz and η = 0.84 ± 0.06) couplings for a single 14N nucleus magnetically coupled to the manganese cluster in the S2 state of PSII. These values are compared to the histidine imidazole nitrogen hyperfine and nuclear quadrupolar couplings found in superoxidized manganese catalase as well as 14N couplings in relevant manganese model complexes.

High-resolution X-ray structures of photosystem II reveal several potential substrate binding sites at the water-oxidizing/oxygen-evolving 4MnCa cluster. Aspartate-61 of the D1 protein hydrogen bonds with one such water (W1), which is bound to the dangler Mn4A of the oxygen-evolving complex. Comparison of pulse EPR spectra of 14NH3 and 15NH3 bound to wild-type Synechocystis PSII and a D1-D61A mutant lacking this hydrogen-bonding interaction demonstrates that ammonia binds as a terminal NH3 at this dangler Mn4A site and not as a partially deprotonated bridge between two metal centers. The implications of this finding on identifying the binding sites of the substrate and the subsequent mechanism of dioxygen formation are discussed.

Ribonucleotide reductases (RNRs) utilize radical chemistry to reduce nucleotides to deoxynucleotides in all organisms. In the class Ia and Ib RNRs, this reaction requires a stable tyrosyl radical (Y•) generated by oxidation of a reduced dinuclear metal cluster. The FeIII2-Y• cofactor in the NrdB subunit of the class Ia RNRs can be generated by self-assembly from FeII2-NrdB, O2, and a reducing equivalent. By contrast, the structurally homologous class Ib enzymes require a MnIII2-Y• cofactor in their NrdF subunit. MnII2-NrdF does not react with O2, but it binds the reduced form of a conserved flavodoxin-like protein, NrdIhq, which, in the presence of O2, reacts to form the MnIII2-Y• cofactor. Here we investigate the mechanism of assembly of the MnIII2-Y• cofactor in Bacillus subtilis NrdF. Cluster assembly from MnII2-NrdF, NrdIhq, and O2 has been studied by stopped flow absorption and rapid freeze quench EPR spectroscopies. The results support a mechanism in which NrdIhq reduces O2 to O2•– (40–48 s–1, 0.6 mM O2), the O2•– channels to and reacts with MnII2-NrdF to form a MnIIIMnIV intermediate (2.2 ± 0.4 s–1), and the MnIIIMnIV species oxidizes tyrosine to Y• (0.08–0.15 s–1). Controlled production of O2•– by NrdIhq during class Ib RNR cofactor assembly both circumvents the unreactivity of the MnII2 cluster with O2 and satisfies the requirement for an “extra” reducing equivalent in Y• generation.

Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolution of all of the principal components of the g tensors of the two radicals and revealed significant differences in the g tensor anisotropies. The spectra together with 2H ENDOR spectra and supporting DFT calculations show that the g tensor anisotropy is directly diagnostic of the presence or absence as well as the strength of a hydrogen bond to the indole nitrogen. The approach is a powerful one for identifying and characterizing hydrogen bonds that are critical in the regulation of tryptophan-assisted electron transfer and tryptophan-mediated redox chemistry in proteins.