NMR and Mössbauer Studies Reveal a Temperature-Dependent Switch from S = 1 to 2 in a Nonheme Oxoiron(IV) Complex with Faster C-H Bond Cleavage Rates.

S = 2 FeIV═O centers generated in the active sites of nonheme iron oxygenases cleave substrate C-H bonds at rates significantly faster than most known synthetic FeIV═O complexes. Unlike the majority of the latter, which are S = 1 complexes, [FeIV(O)(tris(2-quinolylmethyl)amine)(MeCN)]2+ (3) is a rare example of a synthetic S = 2 FeIV═O complex that cleaves C-H bonds 1000-fold faster than the related [FeIV(O)(tris(pyridyl-2-methyl)amine)(MeCN)]2+ complex (0). To rationalize this significant difference, a systematic comparison of properties has been carried out on 0 and 3 as well as related complexes 1 and 2 with mixed pyridine (Py)/quinoline (Q) ligation. Interestingly, 2 with a 2-Q-1-Py donor combination cleaves C-H bonds at 233 K with rates approaching those of 3, even though Mössbauer analysis reveals 2 to be S = 1 at 4 K. At 233 K however, 2 becomes S = 2, as shown by its 1H NMR spectrum. These results demonstrate a unique temperature-dependent spin-state transition from triplet to quintet in oxoiron(IV) chemistry that gives rise to the high C-H bond cleaving reactivity observed for 2.

Prognostic significance of perihematomal edema in basal ganglia hemorrhage after minimally invasive endoscopic evacuation.

OBJECTIVE: Spontaneous basal ganglia hemorrhage is a common type of intracerebral hemorrhage (ICH) with no definitive treatment. Minimally invasive endoscopic evacuation is a promising therapeutic approach for ICH. In this study the authors examined prognostic factors associated with long-term functional dependence (modified Rankin Scale [mRS] score ≥ 4) in patients who had undergone endoscopic evacuation of basal ganglia hemorrhage. METHODS: In total, 222 consecutive patients who underwent endoscopic evacuation between July 2019 and April 2022 at four neurosurgical centers were enrolled prospectively. Patients were dichotomized into functionally independent (mRS score ≤ 3) and functionally dependent (mRS score ≥ 4) groups. Hematoma and perihematomal edema (PHE) volumes were calculated using 3D Slicer software. Predictors of functional dependence were assessed using logistic regression models. RESULTS: Among the enrolled patients, the functional dependence rate was 45.50%. Factors independently associated with long-term functional dependence included female sex, older age (≥ 60 years), Glasgow Coma Scale score ≤ 8, larger preoperative hematoma volume (OR 1.02), and larger postoperative PHE volume (OR 1.03, 95% CI 1.01-1.05). A subsequent analysis evaluated the effect of stratified postoperative PHE volume on functional dependence. Specifically, patients with large (≥ 50 to < 75 ml) and extra-large (≥ 75 to 100 ml) postoperative PHE volumes had 4.61 (95% CI 0.99-21.53) and 6.75 (95% CI 1.20-37.85) times greater likelihood of long-term dependence, respectively, than patients with a small postoperative PHE volume (≥ 10 to < 25 ml). CONCLUSIONS: A large postoperative PHE volume is an independent risk factor for functional dependence among basal ganglia hemorrhage patients after endoscopic evacuation, especially with postoperative PHE volume ≥ 50 ml.

Long-term outcome of stereotactic aspiration, endoscopic evacuation, and open craniotomy for the treatment of spontaneous basal ganglia hemorrhage: a propensity score study of 703 cases.

BACKGROUND: To compare the long-term therapeutic effects of stereotactic aspiration (SA), endoscopic evacuation (EE), and open craniotomy (OC) in the surgical treatment of spontaneous basal ganglia hemorrhage and explore the appropriate clinical indications for each technique. METHODS: Multiple-treatment inverse probability of treatment weighting (IPTW)-adjusted logistic regression analysis was performed to evaluate the therapeutic effects of these techniques. The primary and secondary outcomes were 6-month modified Rankin Scale (mRS) and mortality rates, respectively. RESULTS: A total of 703 patients were ultimately enrolled. For the entire cohort, the 6-month mortality rate was significantly higher (OR 2.396, 95% CI: 1.865-3.080), and the 6-month functional outcome was significantly worse (OR 1.359, 95% CI: 1.091-1.692) for SA than that of EE. The 6-month mortality rate for OC was significantly higher (OR 1.395, 95% CI: 1.059-1.837) than that of EE. Further subgroup analysis was stratified by initial hematoma volume and Glasgow Coma Scale (GCS) score. The mortality rate for SA was significantly higher for patients with hematoma volume of 20-40 mL (OR 6.226, 95% CI: 3.848-10.075), 40-80 mL (OR 2.121, 95% CI: 1.492-3.016), and ≥80 mL (OR 5.544, 95% CI: 3.315-9.269) than in the same subgroups of EE. The functional outcomes for SA were significantly worse than that of EE for hematoma volume subgroups of 40-80 mL (OR 1.424, 95% CI: 1.039-1.951) and ≥80 mL (OR 4.224, 95% CI: 1.655-10.776). The mortality rate for SA was significantly higher than that of EE for the GCS score subgroups of 6-8 (OR 2.082, 95% CI: 1.410-3.076) and 3-5 (OR 2.985, 95% CI: 1.904-4.678). The mortality rate for OC was significantly higher for the GCS score of 3-5 subgroup (OR 1.718, 95% CI: 1.115-2.648), and a tendency for a higher mortality rate of 6-8 subgroup (OR 1.442, 95% CI: 0.965-2.156) than that of EE. CONCLUSIONS: EE can decrease the 6-month mortality rate and improve the 6-month functional outcomes of spontaneous basal ganglia hemorrhage in patients with a hematoma volume ≥40 mL. EE can decrease the 6-month mortality rate of spontaneous basal ganglia hemorrhage in patients with a GCS score of 3-8.

Stepwise nitrosylation of the nonheme iron site in an engineered azurin and a molecular basis for nitric oxide signaling mediated by nonheme iron proteins.

Mononitrosyl and dinitrosyl iron species, such as {FeNO}7, {FeNO}8 and {Fe(NO)2}9, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO}7 and {Fe(NO)2}9 species was achieved, and the elusive {FeNO}8 species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO}7 to {Fe(NO)2}9 species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO}7 → {FeNO}8 → {Fe(NO)2}9, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins.

Comparison of Long-Term Outcomes of Endoscopic and Minimally Invasive Catheter Evacuation for the Treatment of Spontaneous Cerebellar Hemorrhage.

Recently, minimally invasive techniques, including endoscopic evacuation and minimally invasive catheter (MIC) evacuation, have been used for the treatment of patients with spontaneous cerebellar hemorrhage (SCH). However, credible evidence is still needed to validate the effects of these techniques. To explore the long-term outcomes of both surgical techniques in the treatment of SCH. Fifty-two patients with SCH who received endoscopic evacuation or MIC evacuation were retrospectively reviewed. Six-month mortality and the modified Rankin Scale (mRS) score were the primary and secondary outcomes, respectively. A multivariate logistic regression model was used to assess the effects of the different surgical techniques on patient outcomes. In the present study, the mortality rate for the entire cohort was 34.6%. Univariate analysis showed that the surgical technique and preoperative Glasgow Coma Scale (GCS) score affected 6-month mortality. However, no variables were found to be correlated with 6-month mRS scores. Further multivariate analysis demonstrated that 6-month mortality in the endoscopic evacuation group was significantly lower than that in the MIC evacuation group (OR = 4.346, 95% CI 1.056 to 17.886). The 6-month mortality rate in the preoperative GCS 9-14 group was significantly lower than that in the GCS 3-8 group (OR = 7.328, 95% CI 1.723 to 31.170). Compared with MIC evacuation, endoscopic evacuation significantly decreased 6-month mortality in SCH patients. These preliminary results warrant further large, prospective, randomized studies.

Long-Term Effect of Endoscopic Evacuation for Large Basal Ganglia Hemorrhage With GCS Scores ≦ 8.

Aims: The surgical evacuation, including stereotactic aspiration, endoscopic evacuation, and craniotomy, is the most effective way to reduce the volume of intracerebral hemorrhage. However, credible evidence for the effects of these techniques is still insufficient. The present study explored the long-term outcomes of these techniques in the treatment of basal ganglia hematoma with low Glasgow Coma Scale (GCS) scores (≤8) and large-volume (≥40 ml), which were predictors of high mortality. Methods: Two hundred and fifty-eight consecutive patients were reviewed retrospectively. The primary and secondary outcomes were 6-months mortality and 6-months modified Rankin Scale score, which were assessed by a multivariate logistic regression model. Results: Compared with the endoscopic evacuation group, the mortality was significantly higher in the stereotactic aspiration group (OR 6.858, 95% CI 3.146-14.953) and open craniotomy group (OR 3.315, 95% CI 1.497-7.341). Age (OR = 2.237, 95% CI 1.290-3.877) and herniation (OR = 2.257, 95% CI 1.172-4.348) were independent predictors for mortality. No significant difference in the neurological functional outcome was found in the stereotactic aspiration group (OR 0.501, 95% CI 0.192-1.308) and the craniotomy group (OR 0.774, 95% CI 0.257-2.335) compared with the endoscopic evacuation group. Conclusion: Endoscopic evacuation significantly decreased the 6-months mortality in patients with hemorrhage ≥40 ml and GCS ≤ 8.

Effects of Noncovalent Interactions on High-Spin Fe(IV)-Oxido Complexes.

High-valent nonheme FeIV-oxido species are key intermediates in biological oxidation, and their properties are proposed to be influenced by the unique microenvironments present in protein active sites. Microenvironments are regulated by noncovalent interactions, such as hydrogen bonds (H-bonds) and electrostatic interactions; however, there is little quantitative information about how these interactions affect crucial properties of high valent metal-oxido complexes. To address this knowledge gap, we introduced a series of FeIV-oxido complexes that have the same S = 2 spin ground state as those found in nature and then systematically probed the effects of noncovalent interactions on their electronic, structural, and vibrational properties. The key design feature that provides access to these complexes is the new tripodal ligand [poat]3-, which contains phosphinic amido groups. An important structural aspect of [FeIVpoat(O)]- is the inclusion of an auxiliary site capable of binding a Lewis acid (LAII); we used this unique feature to further modulate the electrostatic environment around the Fe-oxido unit. Experimentally, studies confirmed that H-bonds and LAII s can interact directly with the oxido ligand in FeIV-oxido complexes, which weakens the Fe═O bond and has an impact on the electronic structure. We found that relatively large vibrational changes in the Fe-oxido unit correlate with small structural changes that could be difficult to measure, especially within a protein active site. Our work demonstrates the important role of noncovalent interactions on the properties of metal complexes, and that these interactions need to be considered when developing effective oxidants.

Sc3+-Promoted O-O Bond Cleavage of a (µ-1,2-Peroxo)diiron(III) Species Formed from an Iron(II) Precursor and O2 to Generate a Complex with an FeIV2(µ-O)2 Core.

Soluble methane monooxygenase (sMMO) carries out methane oxidation at 4 °C and under ambient pressure in a catalytic cycle involving the formation of a peroxodiiron(III) intermediate (P) from the oxygenation of the diiron(II) enzyme and its subsequent conversion to Q, the diiron(IV) oxidant that hydroxylates methane. Synthetic diiron(IV) complexes that can serve as models for Q are rare and have not been generated by a reaction sequence analogous to that of sMMO. In this work, we show that [FeII(Me3NTB)(CH3CN)](CF3SO3)2 (Me3NTB = tris((1-methyl-1H-benzo[d]imidazol-2-yl)methyl)amine) (1) reacts with O2 in the presence of base, generating a (µ-1,2-peroxo)diiron(III) adduct with a low O-O stretching frequency of 825 cm-1 and a short Fe···Fe distance of 3.07 Å. Even more interesting is the observation that the peroxodiiron(III) complex undergoes O-O bond cleavage upon treatment with the Lewis acid Sc3+ and transforms into a bis(µ-oxo)diiron(IV) complex, thus providing a synthetic precedent for the analogous conversion of P to Q in the catalytic cycle of sMMO.

Spectroscopic and Reactivity Comparisons between Nonheme Oxoiron(IV) and Oxoiron(V) Species Bearing the Same Ancillary Ligand.

This work directly compares the spectroscopic and reactivity properties of an oxoiron(IV) and an oxoiron(V) complex that are supported by the same neutral tetradentate N-based PyNMe3 ligand. A complete spectroscopic characterization of the oxoiron(IV) species (2) reveals that this compound exists as a mixture of two isomers. The reactivity of the thermodynamically more stable oxoiron(IV) isomer (2b) is directly compared to that exhibited by the previously reported 1e--oxidized analogue [FeV(O)(OAc)(PyNMe3)]2+ (3). Our data indicates that 2b is 4 to 5 orders of magnitude slower than 3 in hydrogen atom transfer (HAT) from C-H bonds. The origin of this huge difference lies in the strength of the O-H bond formed after HAT by the oxoiron unit, the O-H bond derived from 3 being about 20 kcal·mol-1 stronger than that from 2b. The estimated bond strength of the FeIVO-H bond of 100 kcal·mol-1 is very close to the reported values for highly active synthetic models of compound I of cytochrome P450. In addition, this comparative study provides direct experimental evidence that the lifetime of the carbon-centered radical that forms after the initial HAT by the high valent oxoiron complex depends on the oxidation state of the nascent Fe-OH complex. Complex 2b generates long-lived carbon-centered radicals that freely diffuse in solution, while 3 generates short-lived caged radicals that rapidly form product C-OH bonds, so only 3 engages in stereoretentive hydroxylation reactions. Thus, the oxidation state of the iron center modulates not only the rate of HAT but also the rate of ligand rebound.

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.

Structural implications of the paramagnetically shifted NMR signals from pyridine H atoms on synthetic nonheme FeIV=O complexes.

Oxoiron(IV) motifs are found in important intermediates in many enzymatic cycles that involve oxidations. Over half of the reported synthetic nonheme oxoiron(IV) analogs incorporate heterocyclic donors, with a majority of them comprising pyridines. Herein, we report 1H-NMR studies of oxoiron(IV) complexes containing pyridines that are arranged in different configurations relative to the Fe = O unit and give rise to paramagnetically shifted resonances that differ by as much as 50 ppm. The strong dependence of 1H-NMR shifts on the different configurations and orientation of pyridines relative to the oxoiron(IV) unit demonstrates how unpaired electronic spin density of the iron center affects the chemical shifts of these protons.

Oxidative Decarboxylase UndA Utilizes a Dinuclear Iron Cofactor.

UndA is a nonheme iron enzyme that activates oxygen to catalyze the decarboxylation of dodecanoic acid to undecene and carbon dioxide. We report the first optical and Mössbauer spectroscopic characterization of UndA, revealing that the enzyme harbors a coupled dinuclear iron cluster. Single turnover studies confirm that the reaction of the diferrous enzyme with dioxygen produces stoichiometric product per cluster. UndA is the first characterized example of a diiron decarboxylase, thus expanding the repertoire of reactions catalyzed by dinuclear iron enzymes.

NMR Reveals That a Highly Reactive Nonheme FeIV =O Complex Retains Its Six-Coordinate Geometry and S=1 State in Solution.

The [FeIV (O)(Me3 NTB)]2+ (Me3 NTB=tris[(1-methyl-benzimidazol-2-yl)methyl]amine) complex 1 has been shown by Mössbauer spectroscopy to have an S=1 ground state at 4 K, but is proposed to become an S=2 trigonal-bipyramidal species at higher temperatures based on a DFT model to rationalize its very high C-H bond-cleavage reactivity. In this work, 1 H NMR spectroscopy was used to determine that 1 does not have C3 -symmetry in solution and is not an S=2 species. Our results show that 1 is unique among nonheme FeIV =O complexes in retaining its S=1 spin state and high reactivity at 193 K, providing evidence that S=1 FeIV =O complexes can be as reactive as their S=2 counterparts. This result emphasizes the need to identify factors besides the ground spin state of the FeIV =O center to rationalize nonheme oxoiron(IV) reactivity.

Repurposing Nonheme Iron Hydroxylases To Enable Catalytic Nitrile Installation through an Azido Group Assistance.

Three mononuclear nonheme iron and 2-oxoglutarate dependent enzymes, l-Ile 4-hydroxylase, l-Leu 5-hydroxylase and polyoxin dihydroxylase, are previously reported to catalyze the hydroxylation of l-isoleucine, l-leucine, and l-α-amino-δ-carbamoylhydroxyvaleric acid (ACV). In this study, we showed that these enzymes can accommodate leucine isomers and catalyze regiospecific hydroxylation. On the basis of these results, as a proof-of-concept, we demonstrated that the outcome of the reaction can be redirected by installation of an assisting group within the substrate. Specifically, instead of canonical hydroxylation, these enzymes can catalyze non-native nitrile group installation when an azido group is introduced. The reaction is likely to proceed through C-H bond activation by an Fe(IV)-oxo species, followed by azido-directed C≡N bond formation. These results offer a unique opportunity to investigate and expand the reaction repertoire of Fe/2OG enzymes.

Crystallographic Evidence for a Sterically Induced Ferryl Tilt in a Non-Heme Oxoiron(IV) Complex that Makes it a Better Oxidant.

Oxoiron(IV) units are often implicated as intermediates in the catalytic cycles of non-heme iron oxygenases and oxidases. The most reactive synthetic analogues of these intermediates are supported by tetradentate tripodal ligands with N-methylbenzimidazole or quinoline donors, but their instability precludes structural characterization. Herein we report crystal structures of two [FeIV (O)(L)]2+ complexes supported by pentadentate ligands incorporating these heterocycles, which show longer average Fe-N distances than the complex with only pyridine donors. These longer distances correlate linearly with log k2 ' values for O- and H-atom transfer rates, suggesting that weakening the ligand field increases the electrophilicity of the Fe=O center. The sterically bulkier quinoline donors are also found to tilt the Fe=O unit away from a linear N-Fe=O arrangement by 10°.

Spectroscopic and DFT Characterization of a Highly Reactive Nonheme FeV-Oxo Intermediate.

The reaction of [(PyNMe3)FeII(CF3SO3)2], 1, with excess peracetic acid at -40 °C generates a highly reactive intermediate, 2b(PAA), that has the fastest rate to date for oxidizing cyclohexane by a nonheme iron species. It exhibits an intense 490 nm chromophore associated with an S = 1/2 EPR signal having g-values at 2.07, 2.01, and 1.94. This species was shown to be in a fast equilibrium with a second S = 1/2 species, 2a(PAA), assigned to a low-spin acylperoxoiron(III) center. Unfortunately, contaminants accompanying the 2(PAA) samples prevented determination of the iron oxidation state by Mössbauer spectroscopy. Use of MeO-PyNMe3 (an electron-enriched version of PyNMe3) and cyclohexyl peroxycarboxylic acid as oxidant affords intermediate 3b(CPCA) with a Mössbauer isomer shift δ = -0.08 mm/s that indicates an iron(V) oxidation state. Analysis of the Mössbauer and EPR spectra, combined with DFT studies, demonstrates that the electronic ground state of 3b(CPCA) is best described as a quantum mechanical mixture of [(MeO-PyNMe3)FeV(O)(OC(O)R)]2+ (∼75%) with some FeIV(O)(•OC(O)R) and FeIII(OOC(O)R) character. DFT studies of 3b(CPCA) reveal that the unbound oxygen of the carboxylate ligand, O2, is only 2.04 Å away from the oxo group, O1, corresponding to a Wiberg bond order for the O1-O2 bond of 0.35. This unusual geometry facilitates reversible O1-O2 bond formation and cleavage and accounts for the high reactivity of the intermediate when compared to the rates of hydrogen atom transfer and oxygen atom transfer reactions of FeIII(OC(O)R) ferric acyl peroxides and FeIV(O) complexes. The interaction of O2 with O1 leads to a significant downshift of the Fe-O1 Raman frequency (815 cm-1) relative to the 903 cm-1 value predicted for the hypothetical [(MeO-PyNMe3)FeV(O)(NCMe)]3+ complex.

Characterization of the Fleeting Hydroxoiron(III) Complex of the Pentadentate TMC-py Ligand.

Nonheme mononuclear hydroxoiron(III) species are important intermediates in biological oxidations, but well-characterized examples of synthetic complexes are scarce due to their instability or tendency to form µ-oxodiiron(III) complexes, which are the thermodynamic sink for such chemistry. Herein, we report the successful stabilization and characterization of a mononuclear hydroxoiron(III) complex, [FeIII(OH)(TMC-py)]2+ (3; TMC-py = 1-(pyridyl-2'-methyl)-4,8,11-trimethyl-1,4,8,11-tetrazacyclotetradecane), which is directly generated from the reaction of [FeIV(O)(TMC-py)]2+ (2) with 1,4-cyclohexadiene at -40 °C by H-atom abstraction. Complex 3 exhibits a UV spectrum with a λmax at 335 nm (ε ≈ 3500 M-1 cm-1) and a molecular ion in its electrospray ionization mass spectrum at m/z 555 with an isotope distribution pattern consistent with its formulation. Electron paramagnetic resonance and Mössbauer spectroscopy show 3 to be a high-spin Fe(III) center that is formed in 85% yield. Extended X-ray absorption fine structure analysis reveals an Fe-OH bond distance of 1.84 Å, which is also found in [(TMC-py)FeIII-O-CrIII(OTf)3]+ (4) obtained from the reaction of 2 with Cr(OTf)2. The S = 5/2 spin ground state and the 1.84 Å Fe-OH bond distance are supported computationally. Complex 3 reacts with 1-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOH) at -40 °C with a second-order rate constant of 7.1 M-1 s-1 and an OH/OD kinetic isotope effect value of 6. On the basis of density functional theory calculations, the reaction between 3 and TEMPOH is classified as a proton-coupled electron transfer as opposed to a hydrogen-atom transfer.

CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates.

CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate P is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of P in the absence of substrates (decay t1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlIred) can react with NH2-CAM and O2 in either order to form a P-NH2-CAM intermediate. P-NH2-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlIox) complex with the aryl-hydroxylamine [NH(OH)-CAM] pathway intermediate. CmlIox-NH(OH)-CAM undergoes a rapid internal redox reaction to form a CmlIred-nitroso-CAM (NO-CAM) complex. O2 binding results in formation of P-NO-CAM that converts to CmlIox-CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site.

The Two Faces of Tetramethylcyclam in Iron Chemistry: Distinct Fe-O-M Complexes Derived from [FeIV(Oanti/syn)(TMC)]2+ Isomers.

Tetramethylcyclam (TMC, 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) exhibits two faces in supporting an oxoiron(IV) moiety, as exemplified by the prototypical [(TMC)FeIV(Oanti)(NCCH3)](OTf)2, where anti indicates that the O atom is located on the face opposite all four methyl groups, and the recently reported syn isomer [(TMC)FeIV(Osyn)(OTf)](OTf). The ability to access two isomers of [(TMC)FeIV(Oanti/syn)] raises the fundamental question of how ligand topology can affect the properties of the metal center. Previously, we have reported the formation of [(CH3CN)(TMC)FeIII-Oanti-CrIII(OTf)4(NCCH3)] (1) by inner-sphere electron transfer between Cr(OTf)2 and [(TMC)FeIV(Oanti)(NCCH3)](OTf)2. Herein we demonstrate that a new species 2 is generated from the reaction between Cr(OTf)2 and [(TMC)FeIV(Osyn)(NCCH3)](OTf)2, which is formulated as [(TMC)FeIII-Osyn-CrIII(OTf)4(NCCH3)] based on its characterization by UV-vis, resonance Raman, Mössbauer, and X-ray absorption spectroscopic methods, as well as electrospray mass spectrometry. Its pre-edge area (30 units) and Fe-O distance (1.77 Å) determined by X-ray absorption spectroscopy are distinctly different from those of 1 (11-unit pre-edge area and 1.81 Å Fe-O distance) but more closely resemble the values reported for [(TMC)FeIII-Osyn-ScIII(OTf)4(NCCH3)] (3, 32-unit pre-edge area and 1.75 Å Fe-O distance). This comparison suggests that 2 has a square pyramidal iron center like 3, rather than the six-coordinate center deduced for 1. Density functional theory calculations further validate the structures for 1 and 2. The influence of the distinct TMC topologies on the coordination geometries is further confirmed by the crystal structures of [(Cl)(TMC)FeIII-Oanti-FeIIICl3] (4Cl) and [(TMC)FeIII-Osyn-FeIIICl3](OTf) (5). Complexes 1-5 thus constitute a set of complexes that shed light on ligand topology effects on the coordination chemistry of the oxoiron moiety.