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Cysteinate oxygenation is intimately tied to the function of both cysteine dioxygenases (CDOs) and nitrile hydratases (NHases), and yet the mechanisms by which sulfurs are oxidized by these enzymes are unknown, in part because intermediates have yet to be observed. Herein, we report a five-coordinate bis-thiolate ligated Fe(III) complex, [FeIII(S2Me2N3(Pr,Pr))]+ (2), that reacts with oxo atom donors (PhIO, IBX-ester, and H2O2) to afford a rare example of a singly oxygenated sulfenate, [FeIII(η2-SMe2O)(SMe2)N3(Pr,Pr)]+ (5), resembling both a proposed intermediate in the CDO catalytic cycle and the essential NHase Fe-S(O)Cys114 proposed to be intimately involved in nitrile hydrolysis. Comparison of the reactivity of 2 with that of a more electron-rich, crystallographically characterized derivative, [FeIIIS2Me2NMeN2amide(Pr,Pr)]- (8), shows that oxo atom donor reactivity correlates with the metal ion's ability to bind exogenous ligands. Density functional theory calculations suggest that the mechanism of S-oxygenation does not proceed via direct attack at the thiolate sulfurs; the average spin-density on the thiolate sulfurs is approximately the same for 2 and 8, and Mulliken charges on the sulfurs of 8 are roughly twice those of 2, implying that 8 should be more susceptible to sulfur oxidation. Carboxamide-ligated 8 is shown to be unreactive towards oxo atom donors, in contrast to imine-ligated 2. Azide (N3-) is shown to inhibit sulfur oxidation with 2, and a green intermediate is observed, which then slowly converts to sulfenate-ligated 5. This suggests that the mechanism of sulfur oxidation involves initial coordination of the oxo atom donor to the metal ion. Whether the green intermediate is an oxo atom donor adduct, Fe-O═I-Ph, or an Fe(V)═O remains to be determined.

Nitric oxide (NO) is frequently used to probe the substrate-binding site of "spectroscopically silent" non-heme Fe2+; sites of metalloenzymes, such as superoxide reductase (SOR). Herein we use NO to probe the superoxide binding site of our thiolate- ligated biomimetic SOR model [FeII(SMe2N4(tren))]+ (1). Like NO-bound trans-cysteinate-ligated SOR (SOR-NO), the rhombic S = 3/2 EPR signal of NO-bound cis-thiolate-ligated [Fe(SMe2N4-(tren)(NO)]+ (2; g = 4.44, 3.54, 1.97), the isotopically sensitive vNO(v15NO) stretching frequency (1685(1640) cm-1), and the 0.05 A decrease in Fe-S bond length are shown to be consistent with the oxidative addition of NO to Fe(II) to afford an Fe(III)-NO-{FeNO}7 species containing high-spin (S = 5/2) Fe(III) antiferro-magnetically coupled to NO- (S = 1). The cis versus trans positioning of the thiolate does not appear to influence these properties. Although it has yet to be crystallographically characterized, SOR-NO is presumed to possess a bent Fe-NO similar to that of 2 (Fe- N-O = 151.7(4)°). The N-O bond is shown to be more activated in 2 relative to N- and O-ligated {FeNO}7 complexes, and this is attributed to the electron-donating properties of the thiolate ligand. Hydrogen-bonding to the cysteinate sulfur attenuates N-O bond activation in SOR, as shown by its higher VNO frequency (1721 cm-1). In contrast, the vO-O frequency of the SOR peroxo intermediate and its analogues is not affected by H-bonds to the cysteinate sulfur or other factors influencing the Fe-SR bond strength; these only influence the vNO frequency. Reactions between 1 and NO2 are shown to result in the proton-dependent heterolytic cleavage of an N-O bond. The mechanism of this reaction is proposed to involve both FeII-NO2- and {FeNO}6 intermediates similar to those implicated in the mechanism of NiR-promoted NO2- reduction. [ABSTRACT FROM AUTHOR]

Abstract: Mechanistic pathways of metalloenzymes are controlled by the metal ion’s electronic and magnetic properties, which are tuned by the coordinated ligands. The functional advantage gained by incorporating cysteinates into the active site of non-heme iron enzymes such as superoxide reductase (SOR) is not entirely understood. Herein, we compare the structural and redox properties of a series of structurally-related thiolate, alkoxide, and amine-ligated Fe(II) complexes in order to determine how the thiolate influences properties critical to function. Thiolates are shown to reduce metal ion Lewis acidity relative to alkoxides and amines, and have a strong trans influence thereby helping to maintain an open coordination site. Comparison of the redox potentials of the structurally analogous compounds described herein shows that alkoxide ligands favor the higher-valent Fe3+ oxidation state, amine ligands favor the reduced Fe2+ oxidation state, and thiolates fall somewhere in between. These properties provide a functional advantage for substrate reducing enzymes in that they provide a site at the metal ion for substrate to bind, and a moderate potential that facilitates both substrate reduction and regeneration of the catalytically active reduced state. Redox potentials for structurally-related Co(II) complexes are shown to be cathodically-shifted relative to their Fe(II) analogues, making them ineffective reducing agents for substrates such as superoxide. [Copyright &y& Elsevier]

S K-edge X-ray absorption spectroscopy (XAS) is a direct experimental probe of metal ion electronic structure as the pre-edge energy reflects its oxidation state, and the energy splitting pattern of the pre-edge transitions reflects its spin state. The combination of sulfur K-edge XAS and density functional theory (DFT) calculations indicates that the electronic structures of {FeNO}7 (S = 3/2) (SMe2N4(tren)Fe(NO), complex I) and {FeNO}7 (S = 1/2) ((bme-daco)Fe(NO), complex II) are FeIII(S = 5/2)-NO-(S = 1) and FeIII(S = 3/2)-NO-(S = 1), respectively. When an axial ligand is computationally added to complex II, the electronic structure becomes FeII(S = 0)-NO•(S = 1/2). These studies demonstrate how the ligand field of the Fe center defines its spin state and thus changes the electron exchange, an important factor in determining the electron distribution over {FeNO}7 and {FeO2}8 sites. [ABSTRACT FROM AUTHOR]

Herein, we describe an alkyl thiolate-ligated iron complex that reacts with dioxygen to form an unprecedented example of an iron superoxo (O 2 •- ) intermediate, [Fe III (S 2 Me2 N 3 (Pr,Pr))(O 2 )] (4), which is capable of cleaving strong C-H bonds. A cysteinate-ligated iron superoxo intermediate is proposed to play a key role in the biosynthesis of β-lactam antibiotics by isopenicillin N-synthase (IPNS). Superoxo 4 converts to a metastable putative Fe(III)-OOH intermediate, at rates that are dependent on the C-H bond strength of the H atom donor, with a kinetic isotope effect ( k H / k D = 4.8) comparable to that of IPNS ( k H / k D = 5.6). The bond dissociation energy of the C-H bonds cleaved by 4 (92 kcal/mol) is comparable to C-H bonds cleaved by IPNS (93 kcal/mol). Both the calculated and experimental electronic absorption spectra of 4 are comparable to those of the putative IPNS superoxo intermediate, and are shown to involve RS - → Fe-O 2 •- and O 2 •- → Fe charge transfer transitions. The π-back-donation by the electron-rich alkyl thiolate presumably facilitates this reactivity by increasing the basicity of the distal oxygen. The frontier orbitals of 4 are shown to consist of two strongly coupled unpaired electrons of opposite spin, one in a superoxo π*(O-O) orbital, and the other in an Fe(d xy ) orbital.

Nitric oxide (NO) is frequently used to probe the substrate-binding site of "spectroscopically silent" non-heme Fe(2+) sites of metalloenzymes, such as superoxide reductase (SOR). Herein we use NO to probe the superoxide binding site of our thiolate-ligated biomimetic SOR model [Fe(II)(S(Me(2))N(4)(tren))](+) (1). Like NO-bound trans-cysteinate-ligated SOR (SOR-NO), the rhombic S = 3/2 EPR signal of NO-bound cis-thiolate-ligated [Fe(S(Me(2))N(4)(tren)(NO)](+) (2; g = 4.44, 3.54, 1.97), the isotopically sensitive ν(NO)(ν((15)NO)) stretching frequency (1685(1640) cm(-1)), and the 0.05 Å decrease in Fe-S bond length are shown to be consistent with the oxidative addition of NO to Fe(II) to afford an Fe(III)-NO(-) {FeNO}(7) species containing high-spin (S = 5/2) Fe(III) antiferromagnetically coupled to NO(-) (S = 1). The cis versus trans positioning of the thiolate does not appear to influence these properties. Although it has yet to be crystallographically characterized, SOR-NO is presumed to possess a bent Fe-NO similar to that of 2 (Fe-N-O = 151.7(4)°). The N-O bond is shown to be more activated in 2 relative to N- and O-ligated {FeNO}(7) complexes, and this is attributed to the electron-donating properties of the thiolate ligand. Hydrogen-bonding to the cysteinate sulfur attenuates N-O bond activation in SOR, as shown by its higher ν(NO) frequency (1721 cm(-1)). In contrast, the ν(O-O) frequency of the SOR peroxo intermediate and its analogues is not affected by H-bonds to the cysteinate sulfur or other factors influencing the Fe-SR bond strength; these only influence the ν(Fe-O) frequency. Reactions between 1 and NO(2)(-) are shown to result in the proton-dependent heterolytic cleavage of an N-O bond. The mechanism of this reaction is proposed to involve both Fe(II)-NO(2)(-) and {FeNO}(6) intermediates similar to those implicated in the mechanism of NiR-promoted NO(2)(-) reduction.

S K-edge X-ray absorption spectroscopy (XAS) is a direct experimental probe of metal ion electronic structure as the pre-edge energy reflects its oxidation state, and the energy splitting pattern of the pre-edge transitions reflects its spin state. The combination of sulfur K-edge XAS and density functional theory (DFT) calculations indicates that the electronic structures of {FeNO}(7) (S = 3/2) (S(Me2)N4(tren)Fe(NO), complex I) and {FeNO}(7) (S = 1/2) ((bme-daco)Fe(NO), complex II) are Fe(III)(S = 5/2)-NO(-)(S = 1) and Fe(III)(S = 3/2)-NO(-)(S = 1), respectively. When an axial ligand is computationally added to complex II, the electronic structure becomes Fe(II)(S = 0)-NO•(S = 1/2). These studies demonstrate how the ligand field of the Fe center defines its spin state and thus changes the electron exchange, an important factor in determining the electron distribution over {FeNO}(7) and {FeO2}(8) sites.