A Nonheme Iron(III) Superoxide Complex Leads to Sulfur Oxygenation.

A new alkylthiolate-ligated nonheme iron complex, FeII(BNPAMe2S)Br (1), is reported. Reaction of 1 with O2 at -40 °C, or reaction of the ferric form with O2•- at -80 °C, gives a rare iron(III)-superoxide intermediate, [FeIII(O2)(BNPAMe2S)]+ (2), characterized by UV-vis, 57Fe Mössbauer, ATR-FTIR, EPR, and CSIMS. Metastable 2 then converts to an S-oxygenated FeII(sulfinate) product via a sequential O atom transfer mechanism involving an iron-sulfenate intermediate. These results provide evidence for the feasibility of proposed intermediates in thiol dioxygenases.

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins.

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with the full-length diheme c4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains, and isolated c4-A is folded. In contrast, the isolated c4-B is partially unfolded, and the interface with c4-A guides folding of this domain. The c4-A and c4-B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation.

Direct Reduction of NO to N2O by a Mononuclear Nonheme Thiolate Ligated Iron(II) Complex via Formation of a Metastable {FeNO}7 Complex.

Addition of NO to a nonheme dithiolate-ligated iron(II) complex, FeII(Me3TACN)(S2SiMe2) (1), results in the generation of N2O. Low-temperature spectroscopic studies reveal a metastable six-coordinate {FeNO}7 intermediate (S = 3/2) that was trapped at -135 °C and was characterized by low-temperature UV-vis, resonance Raman, EPR, Mössbauer, XAS, and DFT studies. Thermal decay of the {FeNO}7 species leads to the evolution of N2O, providing a rare example of a mononuclear thiolate-ligated {FeNO}7 that mediates NO reduction to N2O without the requirement of any exogenous electron or proton sources.

An Iron(III) Superoxide Corrole from Iron(II) and Dioxygen.

A new structurally characterized ferrous corrole [FeII (ttppc)]- (1) binds one equivalent of dioxygen to form [FeIII (O2-. )(ttppc)]- (2). This complex exhibits a 16/18 O2 -isotope sensitive ν(O-O) stretch at 1128 cm-1 concomitantly with a single ν(Fe-O2 ) at 555 cm-1 , indicating it is an η1 -superoxo ("end-on") iron(III) complex. Complex 2 is the first well characterized Fe-O2 corrole, and mediates the following biologically relevant oxidation reactions: dioxygenation of an indole derivative, and H-atom abstraction from an activated O-H bond.

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.

Artificial Metalloproteins with Dinuclear Iron-Hydroxido Centers.

Dinuclear iron centers with a bridging hydroxido or oxido ligand form active sites within a variety of metalloproteins. A key feature of these sites is the ability of the protein to control the structures around the Fe centers, which leads to entatic states that are essential for function. To simulate this controlled environment, artificial proteins have been engineered using biotin-streptavidin (Sav) technology in which Fe complexes from adjacent subunits can assemble to form [FeIII-(µ-OH)-FeIII] cores. The assembly process is promoted by the site-specific localization of the Fe complexes within a subunit through the designed mutation of a tyrosinate side chain to coordinate the Fe centers. An important outcome is that the Sav host can regulate the Fe···Fe separation, which is known to be important for function in natural metalloproteins. Spectroscopic and structural studies from X-ray diffraction methods revealed uncommonly long Fe···Fe separations that change by less than 0.3 Å upon the binding of additional bridging ligands. The structural constraints imposed by the protein host on the di-Fe cores are unique and create examples of active sites having entatic states within engineered artificial metalloproteins.

Activation of Dioxygen by a Mononuclear Nonheme Iron Complex: Sequential Peroxo, Oxo, and Hydroxo Intermediates.

The activation of dioxygen by FeII(Me3TACN)(S2SiMe2) (1) is reported. Reaction of 1 with O2 at -135 °C in 2-MeTHF generates a thiolate-ligated (peroxo)diiron complex FeIII2(O2)(Me3TACN)2(S2SiMe2)2 (2) that was characterized by UV-vis (λmax = 300, 390, 530, 723 nm), Mössbauer (δ = 0.53, |ΔEQ| = 0.76 mm s-1), resonance Raman (RR) (ν(O-O) = 849 cm-1), and X-ray absorption (XAS) spectroscopies. Complex 2 is distinct from the outer-sphere oxidation product 1ox (UV-vis (λmax = 435, 520, 600 nm), Mössbauer (δ = 0.45, |ΔEQ| = 3.6 mm s-1), and EPR (S = 5/2, g = [6.38, 5.53, 1.99])), obtained by one-electron oxidation of 1. Cleavage of the peroxo O-O bond can be initiated either photochemically or thermally to produce a new species assigned as an FeIV(O) complex, FeIV(O)(Me3TACN)(S2SiMe2) (3), which was identified by UV-vis (λmax = 385, 460, 890 nm), Mössbauer (δ = 0.21, |ΔEQ| = 1.57 mm s-1), RR (ν(FeIV═O) = 735 cm-1), and X-ray absorption spectroscopies, as well as reactivity patterns. Reaction of 3 at low temperature with H atom donors gives a new species, FeIII(OH)(Me3TACN)(S2SiMe2) (4). Complex 4 was independently synthesized from 1 by the stoichiometric addition of a one-electron oxidant and a hydroxide source. This work provides a rare example of dioxygen activation at a mononuclear nonheme iron(II) complex that produces both FeIII-O-O-FeIII and FeIV(O) species in the same reaction with O2. It also demonstrates the feasibility of forming Fe/O2 intermediates with strongly donating sulfur ligands while avoiding immediate sulfur oxidation.

Tuning the Geometric and Electronic Structure of Synthetic High-Valent Heme Iron(IV)-Oxo Models in the Presence of a Lewis Acid and Various Axial Ligands.

High-valent ferryl species (e.g., (Por)FeIV═O, Cmpd-II) are observed or proposed key oxidizing intermediates in the catalytic cycles of heme-containing enzymes (P-450s, peroxidases, catalases, and cytochrome c oxidase) involved in biological respiration and oxidative metabolism. Herein, various axially ligated iron(IV)-oxo complexes were prepared to examine the influence of the identity of the base. These were generated by addition of various axial ligands (1,5-dicyclohexylimidazole (DCHIm), a tethered-imidazole system, and sodium derivatives of 3,5-dimethoxyphenolate and imidazolate). Characterization was carried out via UV-vis, electron paramagnetic resonance (EPR), 57Fe Mössbauer, Fe X-ray absorption (XAS), and 54/57Fe resonance Raman (rR) spectroscopies to confirm their formation and compare the axial ligand perturbation on the electronic and geometric structures of these heme iron(IV)-oxo species. Mössbauer studies confirmed that the axially ligated derivatives were iron(IV) and six-coordinate complexes. XAS and 54/57Fe rR data correlated with slight elongation of the iron-oxo bond with increasing donation from the axial ligands. The first reported synthetic H-bonded iron(IV)-oxo heme systems were made in the presence of the protic Lewis acid, 2,6-lutidinium triflate (LutH+), with (or without) DCHIm. Mössbauer, rR, and XAS spectroscopic data indicated the formation of molecular Lewis acid ferryl adducts (rather than full protonation). The reduction potentials of these novel Lewis acid adducts were bracketed through addition of outer-sphere reductants. The oxidizing capabilities of the ferryl species with or without Lewis acid vary drastically; addition of LutH+ to F8Cmpd-II (F8 = tetrakis(2,6-difluorophenyl)porphyrinate) increased its reduction potential by more than 890 mV, experimentally confirming that H-bonding interactions can increase the reactivity of ferryl species.

Structural and Spectroscopic Characterization of a Product Schiff Base Intermediate in the Reaction of the Quinoprotein Glycine Oxidase, GoxA.

The LodA-like proteins make up a recently identified family of enzymes that rely on a cysteine tryptophylquinone cofactor for catalysis. They differ from other tryptophylquinone enzymes in that they are oxidases rather than dehydrogenases. GoxA is a member of this family that catalyzes the oxidative deamination of glycine. Our previous work with GoxA from Pseudoalteromonas luteoviolacea demonstrated that this protein forms a stable intermediate upon anaerobic incubation with glycine. The spectroscopic properties of this species were unique among those identified for tryptophylquinone enzymes characterized to date. Here we use X-ray crystallography and resonance Raman spectroscopy to identify the GoxA catalytic intermediate as a product Schiff base. Structural work additionally highlights features of the active site pocket that confer substrate specificity, intermediate stabilization, and catalytic activity. The unusual properties of GoxA are discussed within the context of the other tryptophylquinone enzymes.

A Six-Coordinate Peroxynitrite Low-Spin Iron(III) Porphyrinate Complex-The Product of the Reaction of Nitrogen Monoxide (·NO(g)) with a Ferric-Superoxide Species.

Peroxynitrite (-OON═O, PN) is a reactive nitrogen species (RNS) which can effect deleterious nitrative or oxidative (bio)chemistry. It may derive from reaction of superoxide anion (O2•-) with nitric oxide (·NO) and has been suggested to form an as-yet unobserved bound heme-iron-PN intermediate in the catalytic cycle of nitric oxide dioxygenase (NOD) enzymes, which facilitate a ·NO homeostatic process, i.e., its oxidation to the nitrate anion. Here, a discrete six-coordinate low-spin porphyrinate-FeIII complex [(PIm)FeIII(-OON═O)] (3) (PIm; a porphyrin moiety with a covalently tethered imidazole axial "base" donor ligand) has been identified and characterized by various spectroscopies (UV-vis, NMR, EPR, XAS, resonance Raman) and DFT calculations, following its formation at -80 °C by addition of ·NO(g) to the heme-superoxo species, [(PIm)FeIII(O2•-)] (2). DFT calculations confirm that 3 is a six-coordinate low-spin species with the PN ligand coordinated to iron via its terminal peroxidic anionic O atom with the overall geometry being in a cis-configuration. Complex 3 thermally transforms to its isomeric low-spin nitrato form [(PIm)FeIII(NO3-)] (4a). While previous (bio)chemical studies show that phenolic substrates undergo nitration in the presence of PN or PN-metal complexes, in the present system, addition of 2,4-di-tert-butylphenol (2,4DTBP) to complex 3 does not lead to nitrated phenol; the nitrate complex 4a still forms. DFT calculations reveal that the phenolic H atom approaches the terminal PN O atom (farthest from the metal center and ring core), effecting O-O cleavage, giving nitrogen dioxide (·NO2) plus a ferryl compound [(PIm)FeIV═O] (7); this rebounds to give [(PIm)FeIII(NO3-)] (4a).The generation and characterization of the long sought after ferriheme peroxynitrite complex has been accomplished.

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.

Why copper is preferred over iron for oxygen activation and reduction in haem-copper oxidases.

Haem-copper oxidase (HCO) catalyses the natural reduction of oxygen to water using a haem-copper centre. Despite decades of research on HCOs, the role of non-haem metal and the reason for nature's choice of copper over other metals such as iron remains unclear. Here, we use a biosynthetic model of HCO in myoglobin that selectively binds different non-haem metals to demonstrate 30-fold and 11-fold enhancements in the oxidase activity of Cu- and Fe-bound HCO mimics, respectively, as compared with Zn-bound mimics. Detailed electrochemical, kinetic and vibrational spectroscopic studies, in tandem with theoretical density functional theory calculations, demonstrate that the non-haem metal not only donates electrons to oxygen but also activates it for efficient O-O bond cleavage. Furthermore, the higher redox potential of copper and the enhanced weakening of the O-O bond from the higher electron density in the d orbital of copper are central to its higher oxidase activity over iron. This work resolves a long-standing question in bioenergetics, and renders a chemical-biological basis for the design of future oxygen-reduction catalysts.

Effect of Outer-Sphere Side Chain Substitutions on the Fate of the trans Iron-Nitrosyl Dimer in Heme/Nonheme Engineered Myoglobins (Fe(B)Mbs): Insights into the Mechanism of Denitrifying NO Reductases.

Denitrifying NO reductases are transmembrane protein complexes that utilize a heme/nonheme diiron center at their active sites to reduce two NO molecules to the innocuous gas N2O. Fe(B)Mb proteins, with their nonheme iron sites engineered into the heme distal pocket of sperm whale myoglobin, are attractive models for studying the molecular details of the NO reduction reaction. Spectroscopic and structural studies of Fe(B)Mb constructs have confirmed that they reproduce the metal coordination spheres observed at the active site of the cytochrome c-dependent NO reductase from Pseudomonas aeruginosa. Exposure of Fe(B)Mb to excess NO, as examined by analytical and spectroscopic techniques, results primarily in the formation of a five-coordinate heme-nitrosyl complex without N2O production. However, substitution of the outer-sphere residue Ile107 with a glutamic acid (i.e., I107E) decreases the formation rate of the five-coordinate heme-nitrosyl complex and allows for the substoichiometric production of N2O. Here, we aim to better characterize the formation of the five-coordinate heme-nitrosyl complex and to explain why the level of N2O production increases with the I107E substitution. We follow the formation of the five-coordinate heme-nitrosyl inhibitory complex through the sequential exposure of Fe(B)Mb to different NO isotopomers using rapid-freeze-quench resonance Raman spectroscopy. The data show that the complex is formed by the displacement of the proximal histidine by a new NO molecule after the weakening of the Fe(II)-His bond in the intermediate six-coordinate low-spin (6cLS) heme-nitrosyl complex. These results lead us to explore diatomic migration within the scaffold of myoglobin and whether substitutions at residue 107 can be sufficient to control access to the proximal heme cavities. Results on a new Fe(B)Mb construct with an I107F substitution (Fe(B)Mb3) show an increased rate for the formation of the five-coordinate low-spin heme-nitrosyl complex without N2O production. Taken together, our results suggest that production of N2O from the [6cLS heme {FeNO}(7)/{Fe(B)NO}(7)] trans iron-nitrosyl dimer intermediate requires a proton transfer event facilitated by an outer-sphere residue such as E107 in Fe(B)Mb2 and E280 in P. aeruginosa cNOR.

Light-induced N2O production from a non-heme iron-nitrosyl dimer.

Two non-heme iron-nitrosyl species, [Fe2(N-Et-HPTB)(O2CPh)(NO)2](BF4)2 (1a) and [Fe2(N-Et-HPTB)(DMF)2(NO)(OH)](BF4)3 (2a), are characterized by FTIR and resonance Raman spectroscopy. Binding of NO is reversible in both complexes, which are prone to NO photolysis under visible light illumination. Photoproduction of N2O occurs in high yield for 1a but not 2a. Low-temperature FTIR photolysis experiments with 1a in acetonitrile do not reveal any intermediate species, but in THF at room temperature, a new {FeNO}(7) species quickly forms under illumination and exhibits a ν(NO) vibration indicative of nitroxyl-like character. This metastable species reacts further under illumination to produce N2O. A reaction mechanism is proposed, and implications for NO reduction in flavodiiron proteins are discussed.

Thioether-ligated iron(II) and iron(III)-hydroperoxo/alkylperoxo complexes with an H-bond donor in the second coordination sphere.

The non-heme iron complexes, [Fe(II)(N3PySR)(CH3CN)](BF4)2 () and [Fe(II)(N3Py(amide)SR)](BF4)2 (), afford rare examples of metastable Fe(iii)-OOH and Fe(iii)-OOtBu complexes containing equatorial thioether ligands and a single H-bond donor in the second coordination sphere. These peroxo complexes were characterized by a range of spectroscopic methods and density functional theory studies. The influence of a thioether ligand and of one H-bond donor on the stability and spectroscopic properties of these complexes was investigated.

The production of nitrous oxide by the heme/nonheme diiron center of engineered myoglobins (Fe(B)Mbs) proceeds through a trans-iron-nitrosyl dimer.

Denitrifying NO reductases are transmembrane protein complexes that are evolutionarily related to heme/copper terminal oxidases. They utilize a heme/nonheme diiron center to reduce two NO molecules to N2O. Engineering a nonheme Fe(B) site within the heme distal pocket of sperm whale myoglobin has offered well-defined diiron clusters for the investigation of the mechanism of NO reduction in these unique active sites. In this study, we use FTIR spectroscopy to monitor the production of N2O in solution and to show that the presence of a distal Fe(B)(II) is not sufficient to produce the expected product. However, the addition of a glutamate side chain peripheral to the diiron site allows for 50% of a productive single-turnover reaction. Unproductive reactions are characterized by resonance Raman spectroscopy as dinitrosyl complexes, where one NO molecule is bound to the heme iron to form a five-coordinate low-spin {FeNO}(7) species with ν(FeNO)(heme) and ν(NO)(heme) at 522 and 1660 cm(-1), and a second NO molecule is bound to the nonheme Fe(B) site with a ν(NO)(FeB) at 1755 cm(-1). Stopped-flow UV-vis absorption coupled with rapid-freeze-quench resonance Raman spectroscopy provide a detailed map of the reaction coordinates leading to the unproductive iron-nitrosyl dimer. Unexpectedly, NO binding to Fe(B) is kinetically favored and occurs prior to the binding of a second NO to the heme iron, leading to a (six-coordinate low-spin heme-nitrosyl/FeB-nitrosyl) transient dinitrosyl complex with characteristic ν(FeNO)(heme) at 570 ± 2 cm(-1) and ν(NO)(FeB) at 1755 cm(-1). Without the addition of a peripheral glutamate, the dinitrosyl complex is converted to a dead-end product after the dissociation of the proximal histidine of the heme iron, but the added peripheral glutamate side chain in Fe(B)Mb2 lowers the rate of dissociation of the promixal histidine which in turn allows the (six-coordinate low-spin heme-nitrosyl/Fe(B)-nitrosyl) transient dinitrosyl complex to decay with production of N2O at a rate of 0.7 s(-1) at 4 °C. Taken together, our results support the proposed trans mechanism of NO reduction in NORs.

Secondary coordination sphere influence on the reactivity of nonheme iron(II) complexes: an experimental and DFT approach.

The new biomimetic ligands N4Py(2Ph) (1) and N4Py(2Ph,amide) (2) were synthesized and yield the iron(II) complexes [Fe(II)(N4Py(2Ph))(NCCH3)](BF4)2 (3) and [Fe(II)(N4Py(2Ph,amide))](BF4)2 (5). Controlled orientation of the Ph substituents in 3 leads to facile triplet spin reactivity for a putative Fe(IV)(O) intermediate, resulting in rapid arene hydroxylation. Addition of a peripheral amide substituent within hydrogen-bond distance of the iron first coordination sphere leads to stabilization of a high-spin Fe(III)OOR species which decays without arene hydroxylation. These results provide new insights regarding the impact of secondary coordination sphere effects at nonheme iron centers.

Proximal ligand electron donation and reactivity of the cytochrome P450 ferric-peroxo anion.

CYP125 from Mycobacterium tuberculosis catalyzes sequential oxidation of the cholesterol side-chain terminal methyl group to the alcohol, aldehyde, and finally acid. Here, we demonstrate that CYP125 simultaneously catalyzes the formation of five other products, all of which result from deformylation of the sterol side chain. The aldehyde intermediate is shown to be the precursor of both the conventional acid metabolite and the five deformylation products. The acid arises by protonation of the ferric-peroxo anion species and formation of the ferryl-oxene species, also known as Compound I, followed by hydrogen abstraction and oxygen transfer. The deformylation products arise by addition of the same ferric-peroxo anion to the aldehyde intermediate to give a peroxyhemiacetal that leads to C-C bond cleavage. This bifurcation of the catalytic sequence has allowed us to examine the effect of electron donation by the proximal ligand on the properties of the ferric-peroxo anion. Replacement of the cysteine thiolate iron ligand by a selenocysteine results in UV-vis, EPR, and resonance Raman spectral changes indicative of an increased electron donation from the proximal selenolate ligand to the iron. Analysis of the product distribution in the reaction of the selenocysteine substituted enzyme reveals a gain in the formation of the acid (Compound I pathway) at the expense of deformylation products. These observations are consistent with an increase in the pK(a) of the ferric-peroxo anion, which favors its protonation and, therefore, Compound I formation.

Phenol nitration induced by an {Fe(NO)2}(10) dinitrosyl iron complex.

Cellular dinitrosyl iron complexes (DNICs) have long been considered NO carriers. Although other physiological roles of DNICs have been postulated, their chemical functionality outside of NO transfer has not been demonstrated thus far. Here we report the unprecedented dioxygen reactivity of a N-bound {Fe(NO)(2)}(10) DNIC, [Fe(TMEDA)(NO)(2)] (1). In the presence of O(2), 1 becomes a nitrating agent that converts 2,4,-di-tert-butylphenol to 2,4-di-tert-butyl-6-nitrophenol via formation of a putative iron-peroxynitrite [Fe(TMEDA)(NO)(ONOO)] (2) that is stable below -80 °C. Iron K-edge X-ray absorption spectroscopy on 2 supports a five-coordinated metal center with a bound peroxynitrite in a cyclic bidentate fashion. The peroxynitrite ligand of 2 readily decays at increased temperature or under illumination. These results suggest that DNICs could have multiple physiological or deleterious roles, including that of cellular nitrating agents.

Influence of the nitrogen donors on nonheme iron models of superoxide reductase: high-spin Fe(III)-OOR complexes.

A new five-coordinate, (N(4)S(thiolate))Fe(II) complex, containing tertiary amine donors, [Fe(II)(Me(4)[15]aneN(4))(SPh)]BPh(4) (2), was synthesized and structurally characterized as a model of the reduced active site of superoxide reductase (SOR). Reaction of 2 with tert-butyl hydroperoxide (tBuOOH) at -78 degrees C led to the generation of the alkylperoxo-iron(III) complex [Fe(III)(Me(4)[15]aneN(4))(SPh)(OOtBu)](+) (2a). The nonthiolate-ligated complex, [Fe(II)(Me(4)[15]aneN(4))(OTf)(2)] (3), was also reacted with tBuOOH and yielded the corresponding alkylperoxo complex [Fe(III)(Me(4)[15]aneN(4))(OTf)(OOtBu)](+) (3a) at an elevated temperature of -23 degrees C. These species were characterized by low-temperature UV-vis, EPR, and resonance Raman spectroscopies. Complexes 2a and 3a exhibit distinctly different spectroscopic signatures than the analogous alkylperoxo complexes [Fe(III)([15]aneN(4))(SAr)(OOR)](+), which contain secondary amine donors. Importantly, alkylation at nitrogen leads to a change from low-spin (S = 1/2) to high-spin (S = 5/2) of the iron(III) center. The resonance Raman data reveal that this change in spin state has a large effect on the nu(Fe-O) and nu(O-O) vibrations, and a comparison between 2a and the nonthiolate-ligated complex 3a shows that axial ligation has an additional significant impact on these vibrations. To our knowledge this study is the first in which the influence of a ligand trans to a peroxo moiety has been evaluated for a structurally equivalent pair of high-spin/low-spin peroxo-iron(III) complexes. The implications of spin state and thiolate ligation are discussed with regard to the functioning of SOR.