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The catalytic Zn(II) ion of horse liver alcohol dehydrogenase (EE isozyme) was replaced by Fe(II), providing a novel iron protein with the unusual coordination of Fe(II) to two cysteines residues, one histine residue and water. The electronic structure of iron in this system was characterized by Mrssbauer spectroscopy at various temperatures as well as applied magnetic fields and analysed in terms of the spin Hamiltonian formalism. The novelty we found is an unusually weak spin coupling (¦J¦ < 0.1 cm-1) of a paramagnet (S = 1) with iron (S = 2). From EPR and biochemical studies we conclude that the corresponding chemical species is triplet oxygen (O2). The quantitative determination of the coupling energy was possible utilizing the competition between Zeemann interaction and spin coupling at weak magnetic fields and low temperature. Oxidation experiments followed by Mössbauer spectroscopy showed that the spin-coupled system is an outer-sphere Fe(II)·(O2)aq complex occurring as an intermediate during a Fe(II)-catalyzed dioxygen activation. We observed two additional Fe(II) species after treatment with O2 and dithionite. The spin Hamiltonian parameters of iron in the coupled system are presented. The results are compared with those of iron in other non-heme iron proteins. [ABSTRACT FROM AUTHOR]

Two dioxygen adducts of thiolato‐iron(II) porphyrins, [K(⊂ 222)][Fe(TPpivP)(SC6HF4)(O2)] 1a and [Na(⊂ 18c.6)][Fe(TPpivP)(SC6HF4)(O2)] 2 were synthesized by reaction of O2 with five‐coordinate, high‐spin, cryptated alkali metal thiolato‐iron(II) ‘picket fence’ porphyrinate. They were characterized by visible and infrared spectroscopy: λmax (log ɛ) = 360 nm (4), 427 nm (4.69), 560 nm (3.69), 610 nm (3.40) for both compounds; v(16O‐16O) = 1139 cm−1 in chlorobenzene and fluorobenzene for 1a and 2. Single crystals of composition [K(⊂ 222)][Fe(TPpivP)(SC6HF4)(O2)] · [K(⊂ 222)](SC6HF4)(C6H5Cl)(H2O) 1b were obtained by diffusion of pentane/xylene mixtures into chlorobenzene solutions of 1a at – 5°C. Single crystals of composition [Na(⊂ 18c.6)][Fe(TPpivP)(SC6HF4)(O2)] were obtained by slow diffusion of pentane into benzene solutions of 2. Structures of 1b and 2 were studied at 20°C (1b) and – 100°C (1b and 2). 1b: space group P21/c (monoclinic), a= 16.806(5) Å (1.6806 nm), b= 14.331(4) Å (1.4331 nm), c= 52.000(15) Å (5.2000 nm), β= 92.95(2)°, V= 12507 Å3 (12.507 nm3), Z= 4, Dcal= 1.28 g · cm−3 (t= 20°C). The final R1 factor was 0.085 for 5238 reflections having I > 3 σ(I). 2: space group P21/c (monoclinic), a= 13.107(3) Å (1.3107 nm), b= 27.055(4) Å (2.7055 nm), c= 25.029(4) Å (2.5029 nm), β= 96.84(2)°, V= 8812 Å3 (8.812 nm3), Z= 4, Dcal= 1.18 g · cm−3 (t=−100°C). The final R1 factor was 0.088 for 6587 reflections having I > 3 σ(I). The iron atom is, in both compounds, bonded to the four porphyrinato nitrogens (Np), the sulfur atom of the axial thiolate and one oxygen atom of the axially end‐on bonded dioxygen molecule. The average Fe‐Np distance found in 1b [1.994(4) Å, 0.1994 nm] is not significantly different from that found in 2 [1.993(3) Å, 0.1993 nm]. The Fe‐S bond length is 2.367(3) Å (0.2367 nm) in 1b and 2.365(2) Å (0.2365 nm) in 2. The Fe‐O1 distances with the oxygen atom of O2 bonded to iron are respectively 1.837(9) Å (0.1837 nm) and 1.850(4) Å (0.1850 nm). The end‐on bonded O2 molecule is disordered in both complexes 1b and 2. This disorder is, however, slightly different from one structure to the other. Whereas the terminal oxygen atom of O2 occupies two positions in 1b [τ(O2A) = 78%, τ(O2B) = 22% at 20°C], it occupies three sites in 2 [τ(O2A) = 51%, τ(O2B) =τ(O2C) = 24.5% at – 100°C] (τ= occupancy). The estimate of the magnitude of the non‐bonded interactions between O2 and its environment located in a sphere of 7‐Å (0.7‐nm) radius in 1b and 2 is consistent with a static disorder of O2 in 1b and a dynamic distribution in 2. Mössbauer measurements were performed with polycrystals of 1b and 2 in the temperature ranges 5 – 197 K and 4.2 – 295 K, respectively, and under externally applied magnetic field (6.76 T) at 4.2 K (1b and 2) and at 173 K (2). The temperature variation of the Lamb‐Mössbauer factor indicates that the iron atom in these oxycytochrome P450 models is not very firmly bonded, more like that in pentacoordinated than in other hexacoordinated hemes. From the specific temperature behavior of quadrupole splitting, asymmetry parameter and line width obtained for 2, the structural disorder of the terminal oxygen atom of O2 is identified as dynamic distribution over three different sites. Corresponding values obtained for 1b are consistent with a static disorder of O2. Magnetic spectra reveal that the oxycytochrome P450 models are purely diamagnetic. [ABSTRACT FROM AUTHOR]

Mononuclear non-heme cofactor iron of putidamonooxin has been investigated in the binary oxidized ‘enzyme · substrate’ complex and in the ternary ‘enzyme · substrate · NO’ complex via Mössbauer spectroscopy. The experimental spectra were analyzed on the basis of the spin-Hamiltonian formalism. The resulting fine and hyperfine structure parameters are compared with literature values of similar compounds. From this comparison we conclude that in the binary complex (reduced and oxidized) the mononuclear non-heme cofactor iron has a coordination number higher than four. Additionally, the cofactor iron shows remarkable spectral similarities with iron in protocatechuate 3.4-dioxygenase, though the catalytic properties of the iron sites in the two proteins are different. The data obtained form the ternary ‘enzyme · substrate · NO’ complex indicate that the cofactor iron (a) is in the ferric intermediate spin state (S = 3/2) and (b) is pentacoordinated, which means that upon NO binding to the reduced cofactor iron at least one ligand has to be released. Comparing our data with literature values suggests that the cofactor iron in the binary as well as in the ternary NO complex is not directly bound to a sulfur atom, though biochemical arguments seem to indicate the opposite. [ABSTRACT FROM AUTHOR]