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Nitric oxide (NO) is a potent intra- and intercellular messenger involved in the control of vascular tone, neuronal signalling and host response to infection. In mammals, NO is synthesized by oxidation of l-arginine catalysed by hemeproteins called NO-synthases with intermediate formation of Nω-hydroxy-l-arginine (NOHA). NOHA and some hydroxyguanidines have been shown to be able to deliver nitrogen oxides including NO in the presence of various oxidative systems. In this study, NOHA and a model compound, N-(4-chlorophenyl)-N′-hydroxyguanidine, were tested for their ability to generate NO in the presence of a haemprotein model, microperoxidase 8 (MP8), and hydrogen peroxide. Nitrite and nitrate production along with selective formation of 4-chlorophenylcyanamide was observed from incubations of N-(4-chlorophenyl)-N′-hydroxyguanidine in the presence of MP8 and hydrogen peroxide. In the case of NOHA, the corresponding cyanamide, Nδ-cyano-L-ornithine, was too unstable under the conditions used and l-citrulline was the only product identified. A NO-specific conversion of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide to 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl and formation of MP8–Fe–NO complexes were observed by EPR spectroscopy and low-temperature UV/visible spectroscopy, respectively. These results clearly demonstrate the formation of nitrogen oxides including NO from the oxidation of exogenous hydroxyguanidines by hydrogen peroxide in the presence of a minienzyme such as MP8. The importance of the bioactivation of endogenous (NOHA) or exogenous N-hydroxyguanidines by peroxidases of physiological interest remains to be established in vivo.

This study describes the catalytic properties of manganese microperoxidase 8 [Mn(III)MP8] compared to iron microperoxidase 8 [Fe(III)MP8]. The mini-enzymes were tested for pH-dependent activity and operational stability in peroxidase-type conversions, using 2-methoxyphenol and 3,3'-dimethoxybenzidine, and in a cytochrome P450-like oxygen transfer reaction converting aniline to para-aminophenol. For the peroxidase type of conversions the Fe to Mn replacement resulted in a less than 10-fold decrease in the activity at optimal pH, whereas the aniline para-hydroxylation is reduced at least 30-fold. In addition it was observed that the peroxidase type of conversions are all fully blocked by ascorbate and that aniline para-hydroxylation by Fe(III)MP8 is increased by ascorbate whereas aniline para-hydroxylation by Mn(III)MP8 is inhibited by ascorbate. Altogether these results indicate that different types of reactive metal oxygen intermediates are involved in the various conversions. Compound I/II, scavenged by ascorbate, may be the reactive species responsible for the peroxidase reactions, the polymerization of aniline and (part of) the oxygen transfer to aniline in the absence of ascorbate. The para-hydroxylation of aniline by Fe(III)MP8, in the presence of ascorbate, must be mediated by another reactive iron-oxo species which could be the electrophilic metal(III) hydroperoxide anion of microperoxidase 8 [M(III)OOH MP8]. The lower oxidative potential of Mn, compared to Fe, may affect the reactivity of both compound I/II and the metal(III) hydroperoxide anion intermediate, explaining the differential effect of the Fe to Mn substitution on the pH-dependent behavior, the rate of catalysis and the operational stability of MP8.

A comparison of the exchange interactions arising in the peroxidase and catalase Compound I intermediates and their iron(IV)-oxo porphyrin π-cation radical models, both of which are two oxidizing equivalents above the ferric state, suggests that in the models the oxidizing equivalent is localized on the porphyrin ring, while in the proteins it is partly delocalized onto the proximal ligand. Thus, the proximal axial ligand of iron participates indirectly in the oxidation reactions catalyzed by the enzymes. Possible roles of the axial ligand in the catalytic mechanism of these heme-based enzymes are discussed.

Mild conversion of the nitrile substituent into an amide functional group is observed upon coordination of CNTPA [CNTPA = (6-cyano 2-pyridylmethyl)bis(2-pyridylmethyle)amine] to FeCl2 followed by reaction with water. The crystal structure of CNTPAFeCl2 is reported and reveals that the nitrile group does not coordinate but is activated in the vicinity of the ferrous center. Upon treatment with water under anaerobic conditions, CNTPAFeCl2 converts into H2NCOTPAFeCl2, [H2NCOTPA = (6-carboxamido 2-pyridylmethyl)bis(2-pyridylmethyle)amine], the X-ray crystal structure of which is reported. The new ligand H2NCOTPA can easily be obtained upon decomplexation.

Kinetic studies were performed with microperoxidase-8 (Fe(III)MP-8), the proteolytic breakdown product of horse heart cytochrome c containing an octapeptide linked to an iron protoporphyrin IX. Mn(III) was substituted for Fe(III) in Mn(III)MP-8. The mechanism of formation of the reactive metal-oxo and metal-hydroperoxo intermediates of M(III)MP-8 upon reaction of H(2)O(2) with Fe(III)MP-8 and Mn(III)MP-8 was investigated by rapid-scan stopped-flow spectroscopy and transient EPR. Two steps (k(obs1) and k(obs2)) were observed and analyzed for the reaction of hydrogen peroxide with both catalysts. The plots of k(obs1) as function of [H(2)O(2)] at pH 8.0 and pH 9.1 for Fe(III)MP-8, and at pH 10.2 and pH 10.9 for Mn(III)MP-8, exhibit saturation kinetics, which reveal the accumulation of an intermediate. Double reciprocal plots of 1/k(obs1) as function of 1/[H(2)O(2)] at different pH values reveal a competitive effect of protons in the oxidation of M(III)MP-8. This effect of protons is confirmed by the linear dependence of 1/k(obs1) on [H(+)] showing that k(obs1) increases with the pH. The UV-visible spectra of the intermediates formed at the end of the first step (k(obs1)) exhibit a spectrum characteristic of a high-valent metal-oxo intermediate for both catalysts. Transient EPR of Mn(III)MP-8 incubated with an excess of H(2)O(2), at pH 11.5, shows the detection of a free radical signal at g approximately equal to 2 and of a resonance at g approximately equal to 4 characteristic of a Mn(IV) (S = 3/2) species. On the basis of these results, the following mechanism is proposed: (i) M(III)MP-8-OH(2) is deprotonated to M(III)MP-8-OH in a rapid preequilibrium step, with a pK(a) = 9.2 +/- 0.9 for Fe(III)MP-8 and a pK(a) = 11.2 +/- 0.3 for Mn(III)MP-8; (ii) M(III)MP-8-OH reacts with H(2)O(2) to form Compound 0, M(III)MP8-OOH, with a second-order rate constant k(1) = (1.3 +/- 0.6) x 10(6) M(-1) x s(-1) for Fe(III)MP-8 and k(1) = (1.6 +/- 0.9) x 10(5) M(-1) x s(-1) for Mn(III)MP-8; (iii) this metal-hydroperoxo intermediate is subsequently converted to a high-valent metal-oxo species, M(IV)MP-8=O, with a free radical on the peptide (R(*+)). The first-order rate constants for the cleavage of the hydroperoxo group are k(2) = 165 +/- 8 s(-1) for Fe(III)MP-8 and k(2) = 145 +/- 7 s(-1) for Mn(III)MP-8; and (iv) the proposed M(IV)MP-8=O(R(*+)) intermediate slowly decays (k(obs2)) with a rate constant of k(obs2) = 13.1 +/- 1.1 s(-)(1) for Fe(III)MP-8 and k(obs2) = 5.2 +/- 1.2 s(-1) for Mn(III)MP-8. The results show that Compound 0 is formed prior to what is analyzed as a high-valent metal-oxo peptide radical intermediate.

The generation of six-coordinate oxoiron(IV) tetramesitylporphyrin π-cation radical complexes by m -CPBA ( meta -chloroperbenzoic acid) oxidation of ferric tetramesitylporphyrin derivatives in butyronitrile at −78 °C was investigated. UV–Vis and EPR spectroscopies indicate that the axial ligand present in the ferric starting derivatives is retained in the high-valent iron complexes. Indirect evidence for the formation of six-coordinate oxoiron(IV) tetramesitylporphyrin complexes Fe IV =O(tmp )X (X=Cl − , Br − ) by m -CPBA oxidation of FeX(tmp) (X=Cl − , Br − ) in butyronitrile at −78 °C was also obtained by Mossbauer spectroscopy. Direct confirmation of the presence of a halide ion as second axial ligand of iron in these high-valent iron species was obtained by X-ray absorption spectroscopy. The EXAFS spectra of the samples obtained by m -CPBA oxidation of FeX(tmp) (X=Cl − , Br − ) were refined using two different coordination models including both four porphyrinato-nitrogens and the axial oxo group. The two models include (model I) or exclude (model II) the axial halogen. The statistical tests indicate the presence of a halide ion as second axial ligand of iron in both derivatives. The refinements led to the following bond distances: Fe IV O(tmp )Cl ( 3 ): Fe–O=1.66(1), Fe–Cl=2.39(2) and Fe–N p =1.99(1) A; Fe IV =O(tmp )Br ( 4 ): Fe–O=1.65(1), Fe–Br=2.93(2), Fe–N p =2.02(1) A. The lengthening of the Fe–X (X=Cl − , Br − ) distances relative to those occurring in the ferric precursor porphyrins is, most probably, related to the strong trans influence of the oxoiron(IV) fragment present in 3 and 4 .

Freeze-quenched intermediates of substrate-free cyto- chrome 57 Fe-P450cam in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mo «ssbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of V V90% of ferric low-spin iron with g-factors and hyperfine parameters of the starting material ; the remaining V10% are identified as a free radical (SP = 1/2) by its EPR and as an iron(IV) (S = 1) species by its Mo «ssbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mo «ssbauer and EPR-spectra exhibit 100% of the starting material. We note that the spin-Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr-96) into a tyrosine radical (Tyr- 96), and the iron(III) center of substrate-free P450cam to iron(IV). fl 2000 Federation of European Biochemical Soci- eties. Published by Elsevier Science B.V. All rights reserved.

Oxygen exchange between high-valent metal–oxo complexes and bulk water has been monitored for nonligated model porphyrins (hemin, FeTDCPPS, MnTMPyP) and the axially ligated microperoxidase-8 (MP-8). Exchange extents up to 90% were measured for MP-8 in spite of the presence of an axial histidine ligand and accompanied by the formation of nonlabelled H 2 O 2 from H 2 18 O 2 . These results point to the existence of a mechanism for oxygen exchange between the high-valent iron–oxo complex and the solvent different from the so-called “oxo-hydroxo tautomerism.” Regeneration of the primary oxidant, H 2 O 2 , and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.

Oxoferrylporphyrin cation radical complexes were generated by m-chloroperoxybenzoic acid oxidation of the chloro and trifluoromethanesulfonato complexes of tetramesitylporphyrinatoiron(III) [(TMP)Fe] and the trifluoromethanesulfonato complex of tetra(2,6-dichlorophenyl)porphyrinatoiron(III) [TPP(2,6-Cl)Fe]. Coupling between ferryl iron (S = 1) and porphyrin radical (S' = 1/2) spin systems was investigated by Mössbauer and EPR spectroscopy. The oxoferrylporphyrin cation radical systems generated from the TMP complexes show strong ferromagnetic coupling. Analysis of the magnetic Mössbauer spectra, using a spin Hamiltonian explicitly including a coupling tensor J, suggests an exchange-coupling constant J greater than 80 cm-1. The EPR spectra show non-zero rhombicity, the origin of which is discussed in terms of contributions from the usual zero-field effects of iron and from iron-radical spin-dipolar interaction. A consistent estimate of zero-field splitting parameter D approximately + 6 cm-1 was obtained by EPR and Mössbauer measurements. EPR and Mössbauer parameters are shown to be slightly dependent on solvent, but not on the axial ligand in the starting (TMP)Fe complex. In contrast to the TMP complex, the oxoferrylporphyrin cation radical system generated from [TPP(2,6-Cl)FeOSO2CF3] exhibits Mössbauer and EPR spectra consistent with weak iron-porphyrin radical coupling of magnitude of J approximately 1 cm-1.

Nuclear resonant forward scattering (NFS) of synchrotron radiation re- presents Mossbauer spectroscopy in the time domain. This new technique com- plements the conventional nuclear resonance absorption, e.g. Mossbauer spectro- scopy in the energy domain, by supplying highly brilliant, polarized, collimated and timed radiation. In NFS the hyperfine interaction of coherently excited nuclei manifests itself as quantum beats, i.e. as modulation of the time-dependent inten- sity of the transmitted radiation, which is delayed with respect to the incoming synchrotron pulse. We have investigated 57Fe-enriched iron porphyrins to test NFS for first biophysical applications. NFS spectra of the diamagnetic porphyrin Fe02(SC6HF4)(TPpivP) and of the paramagnetic porphyrin (Fe(CH3COO)(TPpiVP))- were recorded at various temperatures, with and without reference scatterer, with and without applied field , Dynamic molecular properties, e.g. dynamic structural disorder or spin-lattice relaxation document as variation of the time-delayed count rate. Measured NFS spectra were analysed theoretically by programs which are basically the analogue in the time domain compared to the usual calculations in the energy domain.

Complexation of C 6 Me 6 by Fe 2+ in (C 6 Me 6 ) 2 Fe 2+ followed by (i) addition of hydride, (ii) subsequent addition of a carbanion, (iii) removal of hydride by Ph 3 C + by an electron-transfer pathway, (iv) deprotonation, (v) acylation, (vi) deprotonation, and (vii) decomplexation by Al 2 O 3 /O 2 gives a regio- and stereo-specifically controlled cyclohexadiene.

The synthesis of the complexes [Fe(η 6 -arene) 2 ] 2+ (PF 6 − ) 2 (arene = benzene ( 1a ), toluene ( 1b ), p -xylene ( 1c ), mesitylene ( 1d ) and hexamethylbenzene ( 1e ) has been reexamined. Isomerization occurs, especially in the case of toluene. Reactions of 1a–e with 1 eq. NaBH 4 give [Fe(η 5 -cyclohexadienyl)(η 6 -arene)] + cations 2a–e . Reactions of 1a and 1e with C and O nucleophiles give mainly electron transfer, which inhibits the formation of CC or CO bonds. The labile complexes [Fe(η 4 -C 6H 8 )(η 6 -C 6 Me 6 )] ( 3 ) and [Fe(η 4 - exo -(C 6 H 5 ) 2  C 6 H 4 Me 2 )(η 6 - p -C 6 H 4 Me 2 )] ( 5 ) have, however, been synthesized, but in low yields.

[Mo(C 22 H 22 N 4 )] 2 PF 6 •CH 2 Cl 2 cristallise dans le systeme monoclinique avec le groupe d'espace C2/c. Affinement de la structure jusqu'a 0,054