Correlating Valence and 2p3d RIXS Spectroscopies: A Ligand-Field Study of Spin-Crossover Iron(II).

The molecular spin-crossover phenomenon between high-spin (HS) and low-spin (LS) states is a promising route to next-generation information storage, sensing applications, and molecular spintronics. Spin-crossover complexes also provide a unique opportunity to study the ligand field (LF) properties of a system in both HS and LS states while maintaining the same ligand environment. Presently, we employ complementing valence and core-level spectroscopic methods to probe the electronic excited-state manifolds of the spin-crossover complex [FeII(H2B(pz)2)2phen]0. Light-induced excited spin-state trapping (LIESST) at liquid He temperatures is exploited to characterize magnetic and spectroscopic properties of the photoinduced HS state using SQUID magnetometry and magnetic circular dichroism spectroscopy. In parallel, Fe 2p3d RIXS spectroscopy is employed to examine the ΔS = 0, 1 excited LF states. These experimental studies are combined with state-of-the-art CASSCF/NEVPT2 and CASCI/NEVPT2 calculations characterizing the ground and LF excited states. Analysis of the acquired LF information further supports the notion that the spin-crossover of [FeII(H2B(pz)2)2phen]0 is asymmetric, evidenced by a decrease in eπ in the LS state. The results demonstrate the power of cross-correlating spectroscopic techniques with high and low LF information content to make accurate excited-state assignments, as well as the current capabilities of ab initio theory in interpreting these electronic properties.

HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens.

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote Nε nitrogens and (ii) the amide Np using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the CuII site and coordinating ligands. Furthermore, our calculations support the assignment of Np to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

A designed Copper Histidine-brace enzyme for oxidative depolymerization of polysaccharides as a model of lytic polysaccharide monooxygenase.

The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His)2 with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.

Primary and Secondary Coordination Sphere Effects on the Structure and Function of S-Nitrosylating Azurin.

Much progress has been made in understanding the roles of the secondary coordination sphere (SCS) in tuning redox potentials of metalloproteins. In contrast, the impact of SCS on reactivity is much less understood. A primary example is how copper proteins can promote S-nitrosylation (SNO), which is one of the most important dynamic post-translational modifications, and is crucial in regulating nitric oxide storage and transportation. Specifically, the factors that instill CuII with S-nitrosylating capabilities and modulate activity are not well understood. To address this issue, we investigated the influence of the primary and secondary coordination sphere on CuII-catalyzed S-nitrosylation by developing a series of azurin variants with varying catalytic capabilities. We have employed a multidimensional approach involving electronic absorption, S and Cu K-edge XAS, EPR, and resonance Raman spectroscopies together with QM/MM computational analysis to examine the relationships between structure and molecular mechanism in this reaction. Our findings have revealed that kinetic competency is correlated with three balancing factors, namely Cu-S bond strength, Cu spin localization, and relative S(ps) vs S(pp) contributions to the ground state. Together, these results support a reaction pathway that proceeds through the attack of the Cu-S bond rather than electrophilic addition to CuII or radical attack of SCys. The insights gained from this work provide not only a deeper understanding of SNO in biology but also a basis for designing artificial and tunable SNO enzymes to regulate NO and prevent diseases due to SNO dysregulation.

Structural correlations of nitrogenase active sites using nuclear resonance vibrational spectroscopy and QM/MM calculations.

The biological conversion of N2 to NH3 is accomplished by the nitrogenase family, which is collectively comprised of three closely related but unique metalloenzymes. In the present study, we have employed a combination of the synchrotron-based technique of 57Fe nuclear resonance vibrational spectroscopy together with DFT-based quantum mechanics/molecular mechanics (QM/MM) calculations to probe the electronic structure and dynamics of the catalytic components of each of the three unique M N2ase enzymes (M = Mo, V, Fe) in both the presence (holo-) and absence (apo-) of the catalytic FeMco clusters (FeMoco, FeVco and FeFeco). The results described herein provide vibrational mode assignments for important fingerprint regions of the FeMco clusters, and demonstrate the sensitivity of the calculated partial vibrational density of states (PVDOS) to the geometric and electronic structures of these clusters. Furthermore, we discuss the challenges that are faced when employing NRVS to investigate large, multi-component metalloenzymatic systems, and outline the scope and limitations of current state-of-the-art theory in reproducing complex spectra.

Stepwise assembly of the active site of [NiFe]-hydrogenase.

[NiFe]-hydrogenases are biotechnologically relevant enzymes catalyzing the reversible splitting of H2 into 2e- and 2H+ under ambient conditions. Catalysis takes place at the heterobimetallic NiFe(CN)2(CO) center, whose multistep biosynthesis involves careful handling of two transition metals as well as potentially harmful CO and CN- molecules. Here, we investigated the sequential assembly of the [NiFe] cofactor, previously based on primarily indirect evidence, using four different purified maturation intermediates of the catalytic subunit, HoxG, of the O2-tolerant membrane-bound hydrogenase from Cupriavidus necator. These included the cofactor-free apo-HoxG, a nickel-free version carrying only the Fe(CN)2(CO) fragment, a precursor that contained all cofactor components but remained redox inactive and the fully mature HoxG. Through biochemical analyses combined with comprehensive spectroscopic investigation using infrared, electronic paramagnetic resonance, Mössbauer, X-ray absorption and nuclear resonance vibrational spectroscopies, we obtained detailed insight into the sophisticated maturation process of [NiFe]-hydrogenase.

Sulfur-Ligated [2Fe-2C] Clusters as Synthetic Model Systems for Nitrogenase.

Metal clusters featuring carbon and sulfur donors have coordination environments comparable to the active site of nitrogenase enzymes. Here, we report a series of di-iron clusters supported by the dianionic yldiide ligands, in which the Fe sites are bridged by two µ2-C atoms and four pendant S donors.The [L2Fe2] (L = {[Ph2P(S)]2C}2-) cluster is isolable in two oxidation levels, all-ferrous Fe2II and mixed-valence FeIIFeIII. The mixed-valence cluster displays two peaks in the Mössbauer spectra, indicating slow electron transfer between the two sites. The addition of the Lewis base 4-dimethylaminopyridine to the Fe2II cluster results in coordination with only one of the two Fe sites, even in the presence of an excess base. Conversely, the cluster reacts with 8 equiv of isocyanide tBuNC to give a monometallic complex featuring a new C-C bond between the ligand backbone and the isocyanide. The electronic structure descriptions of these complexes are further supported by X-ray absorption and resonant X-ray emission spectroscopies.

Tryptophan Can Promote Oxygen Reduction to Water in a Biosynthetic Model of Heme Copper Oxidases.

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An X-ray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H2O2. We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

Designing Artificial Metalloenzymes by Tuning of the Environment beyond the Primary Coordination Sphere.

Metalloenzymes catalyze a variety of reactions using a limited number of natural amino acids and metallocofactors. Therefore, the environment beyond the primary coordination sphere must play an important role in both conferring and tuning their phenomenal catalytic properties, enabling active sites with otherwise similar primary coordination environments to perform a diverse array of biological functions. However, since the interactions beyond the primary coordination sphere are numerous and weak, it has been difficult to pinpoint structural features responsible for the tuning of activities of native enzymes. Designing artificial metalloenzymes (ArMs) offers an excellent basis to elucidate the roles of these interactions and to further develop practical biological catalysts. In this review, we highlight how the secondary coordination spheres of ArMs influence metal binding and catalysis, with particular focus on the use of native protein scaffolds as templates for the design of ArMs by either rational design aided by computational modeling, directed evolution, or a combination of both approaches. In describing successes in designing heme, nonheme Fe, and Cu metalloenzymes, heteronuclear metalloenzymes containing heme, and those ArMs containing other metal centers (including those with non-native metal ions and metallocofactors), we have summarized insights gained on how careful controls of the interactions in the secondary coordination sphere, including hydrophobic and hydrogen bonding interactions, allow the generation and tuning of these respective systems to approach, rival, and, in a few cases, exceed those of native enzymes. We have also provided an outlook on the remaining challenges in the field and future directions that will allow for a deeper understanding of the secondary coordination sphere a deeper understanding of the secondary coordintion sphere to be gained, and in turn to guide the design of a broader and more efficient variety of ArMs.

A conformational role for NifW in the maturation of molybdenum nitrogenase P-cluster.

Reduction of dinitrogen by molybdenum nitrogenase relies on complex metalloclusters: the [8Fe:7S] P-cluster and the [7Fe:9S:Mo:C:homocitrate] FeMo-cofactor. Although both clusters bear topological similarities and require the reductive fusion of [4Fe:4S] sub-clusters to achieve their respective assemblies, P-clusters are assembled directly on the NifD2K2 polypeptide prior to the insertion of FeMo-co, which is fully assembled separately from NifD2K2. P-cluster maturation involves the iron protein NifH2 as well as several accessory proteins, whose role has not been elucidated. In the present work, two NifD2K2 species bearing immature P-clusters were isolated from an Azotobacter vinelandii strain in which the genes encoding NifH and the accessory protein NifZ were deleted, and characterized by X-ray absorption spectroscopy and EPR. These analyses showed that both NifD2K2 complexes harbor clusters that are electronically and structurally similar, with each NifDK unit containing two [4Fe:4S]2+/+ clusters. Binding of the accessory protein NifW parallels a decrease in the distance between these clusters, as well as a subtle change in their coordination. These results support a conformational role for NifW in P-cluster biosynthesis, bringing the two [4Fe:4S] precursors closer prior to their fusion, which may be crucial in challenging cellular contexts.

Organometallic Synthesis of Bimetallic Cobalt-Rhodium Nanoparticles in Supported Ionic Liquid Phases (Cox Rh100- x @SILP) as Catalysts for the Selective Hydrogenation of Multifunctional Aromatic Substrates.

The synthesis, characterization, and catalytic properties of bimetallic cobalt-rhodium nanoparticles of defined Co:Rh ratios immobilized in an imidazolium-based supported ionic liquid phase (Cox Rh100- x @SILP) are described. Following an organometallic approach, precise control of the Co:Rh ratios is accomplished. Electron microscopy and X-ray absorption spectroscopy confirm the formation of small, well-dispersed, and homogeneously alloyed zero-valent bimetallic nanoparticles in all investigated materials. Benzylideneacetone and various bicyclic heteroaromatics are used as chemical probes to investigate the hydrogenation performances of the Cox Rh100- x @SILP materials. The Co:Rh ratio of the nanoparticles is found to have a critical influence on observed activity and selectivity, with clear synergistic effects arising from the combination of the noble metal and its 3d congener. In particular, the ability of Cox Rh100- x @SILP catalysts to hydrogenate 6-membered aromatic rings is found to experience a remarkable sharp switch in a narrow composition range between Co25 Rh75 (full ring hydrogenation) and Co30 Rh70 (no ring hydrogenation).

Preparation and spectroscopic characterization of lyophilized Mo nitrogenase.

Mo nitrogenase is the primary source of biologically fixed nitrogen, making this system highly interesting for developing new, energy efficient ways of ammonia production. Although heavily investigated, studies of the active site of this enzyme have generally been limited to spectroscopic methods that are compatible with the presence of water and relatively low protein concentrations. One method of overcoming this limitation is through lyophilization, which allows for measurements to be performed on solvent free, high concentration samples. This method also has the potential for allowing efficient protein storage and solvent exchange. To investigate the viability of this preparatory method with Mo nitrogenase, we employ a combination of electron paramagnetic resonance, Mo and Fe K-edge X-ray absorption spectroscopy, and acetylene reduction assays. Our results show that while some small distortions in the metallocofactors occur, oxidation and spin states are maintained through the lyophilization process and that reconstitution of either lyophilized protein component into buffer restores acetylene reducing activity.

The Spectroscopy of Nitrogenases.

Nitrogenases are responsible for biological nitrogen fixation, a crucial step in the biogeochemical nitrogen cycle. These enzymes utilize a two-component protein system and a series of iron-sulfur clusters to perform this reaction, culminating at the FeMco active site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different spectroscopic approaches have shed light on various aspects of these enzymes, including their structure, mechanism, alternative reactivity, and maturation. Synthetic model chemistry and theory have also played significant roles in developing our present understanding of these systems and are discussed in the context of their contributions to interpreting the nature of nitrogenases. Despite years of significant progress, there is still much to be learned from these enzymes through spectroscopic means, and we highlight where further spectroscopic investigations are needed.

Mechanism of L2,3-edge x-ray magnetic circular dichroism intensity from quantum chemical calculations and experiment-A case study on V(IV)/V(III) complexes.

In this work, we present a combined experimental and theoretical study on the V L2,3-edge x-ray absorption (XAS) and x-ray magnetic circular dichroism (XMCD) spectra of VIVO(acac)2 and VIII(acac)3 prototype complexes. The recorded V L2,3-edge XAS and XMCD spectra are richly featured in both V L3 and L2 spectral regions. In an effort to predict and interpret the nature of the experimentally observed spectral features, a first-principles approach for the simultaneous prediction of XAS and XMCD spectra in the framework of wavefunction based ab initio methods is presented. The theory used here has previously been formulated for predicting optical absorption and MCD spectra. In the present context, it is applied to the prediction of the V L2,3-edge XAS and XMCD spectra of the VIVO(acac)2 and VIII(acac)3 complexes. In this approach, the spin-free Hamiltonian is computed on the basis of the complete active space configuration interaction (CASCI) in conjunction with second order N-electron valence state perturbation theory (NEVPT2) as well as the density functional theory (DFT)/restricted open configuration interaction with singles configuration state functions based on a ground state Kohn-Sham determinant (ROCIS/DFT). Quasi-degenerate perturbation theory is then used to treat the spin-orbit coupling (SOC) operator variationally at the many particle level. The XAS and XMCD transitions are computed between the relativistic many particle states, considering their respective Boltzmann populations. These states are obtained from the diagonalization of the SOC operator along with the spin and orbital Zeeman operators. Upon averaging over all possible magnetic field orientations, the XAS and XMCD spectra of randomly oriented samples are obtained. This approach does not rely on the validity of low-order perturbation theory and provides simultaneous access to the calculation of XMCD A, B, and C terms. The ability of the method to predict the XMCD C-term signs and provide access to the XMCD intensity mechanism is demonstrated on the basis of a generalized state coupling mechanism based on the type of the excitations dominating the relativistically corrected states. In the second step, the performance of CASCI, CASCI/NEVPT2, and ROCIS/DFT is evaluated. The very good agreement between theory and experiment has allowed us to unravel the complicated XMCD C-term mechanism on the basis of the SOC interaction between the various multiplets with spin S' = S, S ± 1. In the last step, it is shown that the commonly used spin and orbital sum rules are inadequate in interpreting the intensity mechanism of the XAS and XMCD spectra of the VIVO(acac)2 and VIII(acac)3 complexes as they breakdown when they are employed to predict their magneto-optical properties. This conclusion is expected to hold more generally.

Planar three-coordinate iron sulfide in a synthetic [4Fe-3S] cluster with biomimetic reactivity.

Iron-sulfur clusters are emerging as reactive sites for the reduction of small-molecule substrates. However, the four-coordinate iron sites of typical iron-sulfur clusters rarely react with substrates, implicating three-coordinate iron. This idea is untested because fully sulfide-coordinated three-coordinate iron is unprecedented. Here we report a new type of [4Fe-3S] cluster that features an iron centre with three bonds to sulfides, and characterize examples of the cluster in three oxidation levels using crystallography, spectroscopy, and ab initio calculations. Although a high-spin electronic configuration is characteristic of other iron-sulfur clusters, the three-coordinate iron centre in these clusters has a surprising low-spin electronic configuration due to the planar geometry and short Fe-S bonds. In a demonstration of biomimetic reactivity, the [4Fe-3S] cluster reduces hydrazine, a natural substrate of nitrogenase. The product is the first example of NH2 bound to an iron-sulfur cluster. Our results demonstrate that three-coordinate iron supported by sulfide donors is a plausible precursor to reactivity in iron-sulfur clusters like the FeMoco of nitrogenase.

Nitrogenase-Relevant Reactivity of a Synthetic Iron-Sulfur-Carbon Site.

Simple synthetic compounds with only S and C donors offer a ligation environment similar to the active site of nitrogenase (FeMoco) and thus demonstrate reasonable mechanisms and geometries for N2 binding and reduction in nature. We recently reported the first example of N2 binding at a mononuclear iron site supported by only S and C donors. In this work, we report experiments that examine the mechanism of N2 binding in this system. The reduction of an iron(II) tris(thiolate) complex with 1 equiv of KC8 leads to a thermally unstable intermediate, and a combination of Mössbauer, EPR, and X-ray absorption spectroscopies identifies it as a high-spin (S = 3/2) iron(I) species that maintains coordination of all three sulfur atoms. DFT calculations suggest that this iron(I) intermediate has a pseudotetrahedral geometry that resembles the S3C iron coordination environment of the belt iron sites in the resting state of the FeMoco. Further reduction to the iron(0) oxidation level under argon causes the dissociation of one of the thiolate donors and gives an η6-arene species which reacts with N2. Thus, in this system the loss of thiolate and binding of N2 require reduction beyond the iron(I) level to the iron(0) level. Further reduction of the iron(0)-N2 complex gives a reactive, formally iron(-I) species. Treatment of the putative iron(-I) complex with weak acids gives low yields of ammonia and hydrazine, demonstrating that these nitrogenase products can be generated from N2 at a synthetic Fe-S-C site. Catalytic N2 reduction is not observed, which is attributed to protonation of the supporting ligand and degradation of the complex via ligand dissociation. Identification of the challenges in this system gives insight into the design features needed for functional biomimetic complexes.

Spectroscopic Description of the E1 State of Mo Nitrogenase Based on Mo and Fe X-ray Absorption and Mössbauer Studies.

Mo nitrogenase (N2ase) utilizes a two-component protein system, the catalytic MoFe and its electron-transfer partner FeP, to reduce atmospheric dinitrogen (N2) to ammonia (NH3). The FeMo cofactor contained in the MoFe protein serves as the catalytic center for this reaction and has long inspired model chemistry oriented toward activating N2. This field of chemistry has relied heavily on the detailed characterization of how Mo N2ase accomplishes this feat. Understanding the reaction mechanism of Mo N2ase itself has presented one of the most challenging problems in bioinorganic chemistry because of the ephemeral nature of its catalytic intermediates, which are difficult, if not impossible, to singly isolate. This is further exacerbated by the near necessity of FeP to reduce native MoFe, rendering most traditional means of selective reduction inept. We have now investigated the first fundamental intermediate of the MoFe catalytic cycle, E1, as prepared both by low-flux turnover and radiolytic cryoreduction, using a combination of Mo Kα high-energy-resolution fluorescence detection and Fe K-edge partial-fluorescence-yield X-ray absorption spectroscopy techniques. The results demonstrate that the formation of this state is the result of an Fe-centered reduction and that Mo remains redox-innocent. Furthermore, using Fe X-ray absorption and 57Fe Mössbauer spectroscopies, we correlate a previously reported unique species formed under cryoreducing conditions to the natively formed E1 state through annealing, demonstrating the viability of cryoreduction in studying the catalytic intermediates of MoFe.

X-ray Magnetic Circular Dichroism Spectroscopy Applied to Nitrogenase and Related Models: Experimental Evidence for a Spin-Coupled Molybdenum(III) Center.

Nitrogenase enzymes catalyze the reduction of atmospheric dinitrogen to ammonia utilizing a Mo-7Fe-9S-C active site, the so-called FeMoco cluster. FeMoco and an analogous small-molecule (Et4 N)[(Tp)MoFe3 S4 Cl3 ] cubane have both been proposed to contain unusual spin-coupled MoIII sites with an S(Mo)=1/2 non-Hund configuration at the Mo atom. Herein, we present Fe and Mo L3 -edge X-ray magnetic circular dichroism (XMCD) spectroscopy of the (Et4 N)[(Tp)MoFe3 S4 Cl3 ] cubane and Fe L2,3 -edge XMCD spectroscopy of the MoFe protein (containing both FeMoco and the 8Fe-7S P-cluster active sites). As the P-clusters of MoFe protein have an S=0 total spin, these are effectively XMCD-silent at low temperature and high magnetic field, allowing for FeMoco to be selectively probed by Fe L2,3 -edge XMCD within the intact MoFe protein. Further, Mo L3 -edge XMCD spectroscopy of the cubane model has provided experimental support for a local S(Mo)=1/2 configuration, demonstrating the power and selectivity of XMCD.

Methylated Histidines Alter Tautomeric Preferences that Influence the Rates of Cu Nitrite Reductase Catalysis in Designed Peptides.

Copper proteins have the capacity to serve as both redox active catalysts and purely electron transfer centers. A longstanding question in this field is how the function of histidine ligated Cu centers are modulated by δ vs ε-nitrogen ligation of the imidazole. Evaluating the impact of these coordination modes on structure and function by comparative analysis of deposited crystal structures is confounded by factors such as differing protein folds and disparate secondary coordination spheres that make direct comparison of these isomers difficult. Here, we present a series of de novo designed proteins using the noncanonical amino acids 1-methyl-histidine and 3-methyl-histidine to create Cu nitrite reductases where δ- or ε-nitrogen ligation is enforced by the opposite nitrogen's methylation as a means of directly comparing these two ligation states in the same protein fold. We find that ε-nitrogen ligation allows for a better nitrite reduction catalyst, displaying 2 orders of magnitude higher activity than the δ-nitrogen ligated construct. Methylation of the δ nitrogen, combined with a secondary sphere mutation we have previously published, has produced a new record for efficiency within a homogeneous aqueous system, improving by 1 order of magnitude the previously published most efficient construct. Furthermore, we have measured Michaelis-Menten kinetics on these highly active constructs, revealing that the remaining barriers to matching the catalytic efficiency ( kcat/ KM) of native Cu nitrite reductase involve both substrate binding ( KM) and catalysis ( kcat).

Resolving the structure of the E1 state of Mo nitrogenase through Mo and Fe K-edge EXAFS and QM/MM calculations.

Biological nitrogen fixation is predominately accomplished through Mo nitrogenase, which utilizes a complex MoFe7S9C catalytic cluster to reduce N2 to NH3. This cluster requires the accumulation of three to four reducing equivalents prior to binding N2; however, despite decades of research, the intermediate states formed prior to N2 binding are still poorly understood. Herein, we use Mo and Fe K-edge X-ray absorption spectroscopy and QM/MM calculations to investigate the nature of the E1 state, which is formed following the addition of the first reducing equivalent to Mo nitrogenase. By analyzing the extended X-ray absorption fine structure (EXAFS) region, we provide structural insight into the changes that occur in the metal clusters of the protein when forming the E1 state, and use these metrics to assess a variety of possible models of the E1 state. The combination of our experimental and theoretical results supports that formation of E1 involves an Fe-centered reduction combined with the protonation of a belt-sulfide of the cluster. Hence, these results provide critical experiment and computational insight into the mechanism of this important enzyme.