Mössbauer characterization of an unusual high-spin side-on peroxo-Fe3+ species in the active site of superoxide reductase from Desulfoarculus Baarsii. Density functional calculations on related models.

Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H(2)O(2). Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron-peroxo species when quickly reacted with H(2)O(2) [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966-4967]. To further investigate this non-heme peroxo-iron species, we have carried out a Mössbauer study of the (57)Fe-enriched E47A SOR from D. baarsii reacted quickly with H(2)O(2). Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo-Fe(3+) complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: delta(/Fe) = 0.54 (1) mm/s, DeltaE(Q) = -0.80 (5) mm/s, and the asymmetry parameter eta = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499-2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH(3)-S](1)(-) group has been replaced by the neutral [NH(3)](0) group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829-12848]. Both models 2 and 3 contain a formally high-spin Fe(3+) ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction ( approximately 0.6 for 2, approximately 0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe(3+) mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe(3+) ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61-0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278-3283], the isomer shift value for a high-spin eta(2)-O(2) Fe(3+) complex with an axial thiolate group can be estimated to be in the 0.54-0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo-Fe(3+) species in another biological system [Karlsson et al. (2003) Science 299, 1039-1042].

Spectroscopic description of the two nitrosyl-iron complexes responsible for fur inhibition by nitric oxide.

Ferric uptake regulation protein (Fur) is a global regulator, ubiquitous in Gram negative bacteria, that acts as a transcriptional repressor when it binds ferrous ion. Fur is involved in responses to several types of stress related to iron metabolism, such as stress induced by nitric oxide (NO) generated by macrophages against bacterial invasion. NO was recently shown to react with Fe(2+) ions in FeFur (iron substituted Fur protein) leading to an Fur bound iron-nitrosyl complex, unable to bind DNA, and characterized by a g = 2.03 EPR signal, associated with an S = (1)/(2) ground state. This electronic configuration could arise from either a mononitrosyl-iron [Fe(NO)](7) or a dinitrosyl-iron [Fe(NO)(2)](9) complex. The use of several spectroscopic tools such as EPR, ENDOR, FTIR, Mössbauer, and UV-visible spectroscopies as well as mass spectrometry analysis was necessary to characterize the iron-nitrosyl species in Fur. Furthermore, changes of C132 and C137 into glycines by site directed mutagenesis reveal that neither of the two cysteines is required for the formation of the g = 2.03 signal. Altogether, we found that two species are responsible for Fur inhibition in NO stress conditions: the major species, S(1/2), is an [Fe(NO)(2)](9) (S = (1)/(2)) complex without bound thiolate and the minor species is probably a diamagnetic [Fe(NO)(2)](8) (S = 0) complex. This is the first characterization of these physiologically relevant species potentially linking iron metabolism and the response to NO toxicity in bacteria.

Spectroscopic and electrochemical characterization of an aqua ligand exchange and oxidatively induced deprotonation in diiron complexes.

Reaction of the unsymmetrical phenol ligand 2-((bis(2-pyridylmethyl)amino)methyl)-6-(((2-pyridylmethyl)benzylamino)methyl)-4-methylphenol (HL-Bn) or its 2,6-dichlorobenzyl analogue (HL-BnCl(2)) with Fe(H(2)O)(6)(ClO(4))(2) in the presence of disodium m-phenylenedipropionate (Na(2)(mpdp)) followed by exposure to atmosphere affords the diiron(II,III) complexes [Fe(2)(L-Bn)(mpdp)(H(2)O)](ClO(4))(2) and [Fe(2)(L-BnCl(2))(mpdp)(CH(3)OH)](ClO(4))(2), respectively. The latter complex has been characterized by X-ray crystallography. It crystallizes in the monoclinic system, space group P2(1)/n, with a = 13.3095(14) A, b = 20.1073(19) A, c = 19.4997(19) A, alpha = 90 degrees, beta = 94.471(2) degrees, gamma = 90 degrees, V = 5202.6(9) A(3), and Z = 4. The structure of the compound is very similar to that of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) determined earlier, except for the replacement of a water by a methanol on the ferrous site. Magnetic measurements of [Fe(2)(L-Bn)(mpdp)(H(2)O)](BPh(4))(2) reveal that the two high-spin Fe ions are moderately antiferromagnetically coupled (J = -3.2(2) cm(-)(1)). Upon dissolution in acetonitrile the terminal ligand on the ferrous site is replaced by a solvent molecule. The acetonitrile-water exchange has been investigated by various spectroscopic techniques (UV-visible, NMR, Mössbauer) and electrochemistry. The substitution of acetonitrile by water is clearly evidenced by Mössbauer spectroscopy by a reduction of the quadrupole splitting value from 3.14 to 2.41 mm/s. In addition, it causes a 210 mV downshift of the oxidation potential of the ferrous site and a similar reduction of the stability domain of the mixed-valence state. Exhaustive electrolysis of a solution of [Fe(2)(L-Bn)(mpdp)(H(2)O)](2+) shows that the aqua diferric species is not stable and undergoes a chemical reaction which can be partly reversed by reduction to the mixed-valent state. This and other electrochemical observations suggest that upon oxidation of the diiron center to the diferric state the aqua ligand is deprotonated to a hydroxo. This hypothesis is supported by Mössbauer spectroscopy. Indeed, this species possesses a large quadrupole splitting value (DeltaE(Q) >or= 1.0 mm.s(-)(1)) similar to that of analogous complexes with a terminal phenolate ligand. This study illustrates the drastic effects of aqua ligand exchange and deprotonation on the electronic structure and redox potentials of diiron centers.

Iron carbonyl, nitrosyl, and nitro complexes of a tetrapodal pentadentate amine ligand: synthesis, electronic structure, and nitrite reductase-like reactivity.

The tetrapodal pentaamine 2,6-C5H3N[CMe(CH2NH2)2]2 (pyN4, 1) forms a series of octahedral iron(II) complexes of general formula [Fe(L)(1)]Xn with a variety of small-molecule ligands L at the sixth coordination site (L = X = Br, n = 1 (2); L = CO, X = Br, n = 2 (3); L = NO, X = Br, n = 2 (4); L = NO+, X = Br, n = 3 (5); L = NO2-, X = Br, n = 1 (6)). The bromo complex, which is remarkably stable towards hydrolysis and oxidation, serves as the precursor for all other complexes, which may be obtained by ligand exchange, employing CO, NO, NOBF4, and NaNO2, respectively. All complexes have been fully characterised, including solid-state structures in most cases. Attempts to obtain single crystals of 6 produced the dinuclear complex [Fe2[mu 2-(eta 1-N: eta 1-O)-NO2](1)2]Br2PF6 (7), whose bridging NO2- unit, which is unsupported by bracketing ligands, is without precedent in the coordination chemistry of iron. Compound 2 has a high-spin electronic configuration with four unpaired electrons (S = 2), while the carbonyl complex 3 is low-spin (S = 0), as are complexes 5, 6 and 7 (S = 0 in all cases); the 19 valence electron nitrosyl complex 4 has S = 1/2. Complex 4 and its oxidation product, 5 ([Fe(NO)]7 and [Fe(NO)]6 in the Feltham-Enemark notation) may be interconverted by a one-electron redox process. Both complexes are also accessible from the mononuclear nitro complex 6: Treatment with acid produces the 18 valence electron NO+ complex 5, whereas hydrolysis in the absence of added protons (in methanolic solution) gives the 19 valence electron NO. complex 4, with formal reduction of the NO2- ligand. This reactivity mimicks the function of certain heme-dependent nitrite reductases. Density functional calculations for complexes 3, 4 and 5 provide a description of the electronic structures and are compatible with the formulation of iron(II) in all cases; this is derived from the careful analysis of the combined IR, ESR and Mössbauer spectroscopic data, as well as structural parameters.