Nitrogen Fixation via a Terminal Fe(IV) Nitride

Terminal iron nitrides (FeN) have been proposed as intermediates of (bio)­catalytic nitrogen fixation, yet experimental evidence to support this hypothesis has been lacking. In particular, no prior synthetic examples of terminal FeN species have been derived from N₂. Here we show that a nitrogen-fixing Fe–N₂ catalyst can be protonated to form a neutral Fe­(NNH₂) hydrazido(2−) intermediate, which, upon further protonation, heterolytically cleaves the N–N bond to release [Fe^IVN]⁺ and NH₃. These observations provide direct evidence for the viability of a Chatt-type (distal) mechanism for Fe-mediated N₂-to-NH₃ conversion. The physical oxidation state range of the Fe complexes in this transformation is buffered by covalency with the ligand, a feature of possible relevance to catalyst design in synthetic and natural systems that facilitate multiproton/multielectron redox processes

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P‑Cluster

Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, βSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av βSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that βSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of βSer188 both in vivo and in vitro by characterizing the ability of Av βSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of βSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that βSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters

Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process

Direct Observation of Oxygen Rebound with an Iron-Hydroxide Complex

The rebound mechanism for alkane hydroxylation was invoked over 40 years ago to help explain reactivity patterns in cytochrome P450, and subsequently has been used to provide insight into a range of biological and synthetic systems. Efforts to model the rebound reaction in a synthetic system have been unsuccessful, in part because of the challenge in preparing a suitable metal-hydroxide complex at the correct oxidation level. Herein we report the synthesis of such a complex. The reaction of this species with a series of substituted radicals allows for the direct interrogation of the rebound process, providing insight into this uniformly invoked, but previously unobserved process

Setting an Upper Limit on the Myoglobin Iron(IV)Hydroxide p<i>K</i>_a: Insight into Axial Ligand Tuning in Heme Protein Catalysis

To provide insight into the iron­(IV)­hydroxide p<i>K</i>_a of histidine ligated heme proteins, we have probed the active site of myoglobin compound II over the pH range of 3.9–9.5, using EXAFS, Mössbauer, and resonance Raman spectroscopies. We find no indication of ferryl protonation over this pH range, allowing us to set an upper limit of 2.7 on the iron­(IV)­hydroxide p<i>K</i>_a in myoglobin. Together with the recent determination of an iron­(IV)­hydroxide p<i>K</i>_a ∼ 12 in the thiolate-ligated heme enzyme cytochrome P450, this result provides insight into Nature’s ability to tune catalytic function through its choice of axial ligand

Spectroscopic Investigations of Catalase Compound II: Characterization of an Iron(IV) Hydroxide Intermediate in a Non-thiolate-Ligated Heme Enzyme

We report on the protonation state of <i>Helicobacter pylori</i> catalase compound II. UV/visible, Mössbauer, and X-ray absorption spectroscopies have been used to examine the intermediate from pH 5 to 14. We have determined that HPC-II exists in an iron­(IV) hydroxide state up to pH 11. Above this pH, the iron­(IV) hydroxide complex transitions to a new species (p<i>K</i>_a = 13.1) with Mössbauer parameters that are indicative of an iron­(IV)-oxo intermediate. Recently, we discussed a role for an elevated compound II p<i>K</i>_a in diminishing the compound I reduction potential. This has the effect of shifting the thermodynamic landscape toward the two-electron chemistry that is critical for catalase function. In catalase, a diminished potential would increase the selectivity for peroxide disproportionation over off-pathway one-electron chemistry, reducing the buildup of the inactive compound II state and reducing the need for energetically expensive electron donor molecules

A 2.8 Å Fe–Fe Separation in the Fe₂^III/IV Intermediate, X, from <i>Escherichia coli</i> Ribonucleotide Reductase

A class Ia ribonucleotide reductase (RNR) employs a μ-oxo-Fe₂^III/III/tyrosyl radical cofactor in its β subunit to oxidize a cysteine residue ∼35 Å away in its α subunit; the resultant cysteine radical initiates substrate reduction. During self-assembly of the <i>Escherichia coli</i> RNR-β cofactor, reaction of the protein’s Fe₂^II/II complex with O₂ results in accumulation of an Fe₂^III/IV cluster, termed <b>X</b>, which oxidizes the adjacent tyrosine (Y₁₂₂) to the radical (Y₁₂₂^•) as the cluster is converted to the μ-oxo-Fe₂^III/III product. As the first high-valent non-heme-iron enzyme complex to be identified and the key activating intermediate of class Ia RNRs, <b>X</b> has been the focus of intensive efforts to determine its structure. Initial characterization by extended X-ray absorption fine structure (EXAFS) spectroscopy yielded a Fe–Fe separation (<i>d</i>_Fe–Fe) of 2.5 Å, which was interpreted to imply the presence of three single-atom bridges (O^2–, HO^–, and/or μ-1,1-carboxylates). This short distance has been irreconcilable with computational and synthetic models, which all have <i>d</i>_Fe–Fe ≥ 2.7 Å. To resolve this conundrum, we revisited the EXAFS characterization of <b>X</b>. Assuming that samples containing increased concentrations of the intermediate would yield EXAFS data of improved quality, we applied our recently developed method of generating O₂ <i>in situ</i> from chlorite using the enzyme chlorite dismutase to prepare <b>X</b> at ∼2.0 mM, more than 2.5 times the concentration realized in the previous EXAFS study. The measured <i>d</i>_Fe–Fe = 2.78 Å is fully consistent with computational models containing a (μ-oxo)₂-Fe₂^III/IV core. Correction of the <i>d</i>_Fe–Fe brings the experimental data and computational models into full conformity and informs analysis of the mechanism by which <b>X</b> generates Y₁₂₂^•

Characterization of a Cross-Linked Protein–Nucleic Acid Substrate Radical in the Reaction Catalyzed by RlmN

RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical <i>S</i>-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein–nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, ¹³C hyperfine interactions between the radical and the methylene carbon of the formerly [<i>methyl</i>-¹³C]­Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process

Oxygen-Atom Transfer Reactivity of Axially Ligated Mn(V)–Oxo Complexes: Evidence for Enhanced Electrophilic and Nucleophilic Pathways

Addition of anionic donors to the manganese­(V)–oxo corrolazine complex Mn^V(O)­(TBP₈Cz) has a dramatic influence on oxygen-atom transfer (OAT) reactivity with thioether substrates. The six-coordinate anionic [Mn^V(O)­(TBP₈Cz)­(X)]⁻ complexes (X = F^–, N₃^–, OCN^–) exhibit a ∼5 cm^–1 downshift of the Mn–O vibrational mode relative to the parent Mn^V(O)­(TBP₈Cz) complex as seen by resonance Raman spectroscopy. Product analysis shows that the oxidation of thioether substrates gives sulfoxide product, consistent with single OAT. A wide range of OAT reactivity is seen for the different axial ligands, with the following trend determined from a comparison of their second-order rate constants for sulfoxidation: five-coordinate ≈ thiocyanate ≈ nitrate < cyanate < azide < fluoride ≪ cyanide. This trend correlates with DFT calculations on the binding of the axial donors to the parent Mn^V(O)­(TBP₈Cz) complex. A Hammett study was performed with <i>p</i>-X-C₆H₄SCH₃ derivatives and [Mn^V(O)­(TBP₈Cz)­(X)]⁻ (X = CN^– or F^–) as the oxidant, and unusual “V-shaped” Hammett plots were obtained. These results are rationalized based upon a change in mechanism that hinges on the ability of the [Mn^V(O)­(TBP₈Cz)­(X)]⁻ complexes to function as either an electrophilic or weak nucleophilic oxidant depending upon the nature of the <i>para</i>-X substituents. For comparison, the one-electron-oxidized cationic Mn^V(O)­(TBP₈Cz^•+) complex yielded a linear Hammett relationship for all substrates (ρ = −1.40), consistent with a straightforward electrophilic mechanism. This study provides new, fundamental insights regarding the influence of axial donors on high-valent Mn^V(O) porphyrinoid complexes

O₂-Evolving Chlorite Dismutase as a Tool for Studying O₂-Utilizing Enzymes

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas’s modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, <i>in situ</i> generation of O₂ at concentrations far exceeding 2 mM. This method was used to define the O₂ concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i>, in which the enzyme’s Mn^IV/Fe^III cofactor forms from a Mn^II/Fe^II complex and O₂ via a Mn^IV/Fe^IV intermediate, at effective O₂ concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O₂ adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C–H-bond-cleaving Fe^IV–oxo complex, <b>J</b>, in taurine:α-ketoglutarate dioxygenase and superoxo–Fe₂^III/III complex, <b>G</b>, in <i>myo</i>-inositol oxygenase, and the tyrosyl-radical-generating Fe₂^III/IV intermediate, <b>X</b>, in <i>Escherichia coli</i> RNR, were all accumulated to yields more than twice those previously attained. This means of <i>in situ</i> O₂ evolution permits a >5 mM “pulse” of O₂ to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O₂ addition steps