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 Nomega-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, Ndelta-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.

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.

Oxoferryl porphyrin π-cation radical active sites of compound I intermediates which are found in enzymes such as peroxidases and catalases have been extensively modeled by oxidized synthetic metalloporphyrins. The electronic symmetry states of these compounds were initially assigned on the basis of electronic absorption data. In recent years new experimental and theoretical results have become available which have led to a re-evaluation and modification of the original assignments. A historical perspective of these developments is provided in the context of recent NMR, resonance Raman, and other spectroscopic data and theoretical calculations for the synthetic models and enzymatic systems.

Freeze‐quenched intermediates of substrate‐free cytochrome 57 Fe‐P450 cam in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mössbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of ∼90% of ferric low‐spin iron with g ‐factors and hyperfine parameters of the starting material; the remaining ∼10% are identified as a free radical ( S ′=1/2) by its EPR and as an iron(IV) ( S =1) species by its Mössbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mössbauer 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 P450 cam to iron(IV).

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 FeIV=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 Mössbauer 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: FeIVO(tmp)Cl (3): Fe–O=1.66(1), Fe–Cl=2.39(2) and Fe–Np=1.99(1) Å; FeIV=O(tmp)Br (4): Fe–O=1.65(1), Fe–Br=2.93(2), Fe–Np=2.02(1) Å. 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.

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 H2O2 from H218O2. 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, H2O2, and oxygen exchange by axially ligated porphyrins can be explained by a mechanism involving the reversibility of compound I formation.

The hydroxylation of aniline with H2O2 as the oxidant has been utilized to probe the catalytic activity of the water-insoluble model porphyrin in a water-soluble MCM-41 system and to investigate the catalytic activity and operational stability of microperoxidase-8 upon its incorporation into MCM-41. Our results demonstrate that metalloporphyrins supported on MCM-41 can catalyze cytochrome P450-like reactions in aqueous solutions but point out limitations in the use of MCM-41 as a solid support for heterogeneous catalysts.

Despite similar ring deformations in solution and in the solid state, the chloroiron(III) derivative of 2,3,7,8,12,13,17,18-octaethyl-5,10,15,20-tetraphenylporphyrin ([FeCl(oetpp)], shown schematically) prepared in this study exhibits only a very weak quantum-mechanical admixture of spin S=3/2 (only 4-10 %) with spin S=5/2. In contrast, for the variety of [FeCl(oetpp)] studied earlier by other researchers a 40 % contribution of the S=3/2 state was found.

Nuclear resonant forward scattering (NFS) of synchrotron radiation was employed as Mössbauer spectroscopy in the time domain only recently, while conventional Mössbauer spectroscopy in the energy domain is widely applied since its discovery in 1958. Experimental setup and theoretical background required for NFS are explained, and examples are given for detecting quadrupole splitting, isomer shift, thickness effect and magnetic hyperfine interaction. Nuclear inelastic scattering provides the possibility to detect molecular vibrations.

The mechanism of spin‐lattice relaxation has been investigated in the “picket‐fence” porphyrin [Fe(CH 3 COO)(TP piv P)] - , a high‐spin iron(II) complex with unusual large quadrupole splitting of 4.25 mm s -1 , by conventional Mössbauer spectroscopy as well as by nuclear resonant forward scattering (NFS). Superparamagnetism with a blocking temperature of about 8 K is observable by both methods in the spectra of bacterioferritin from S. olivaceus. From these two examples general conclusions about the merits of both methods can be drawn.

Nuclear resonant forward scattering (NFS) of synchrotron radiation represents Mossbauer spectroscopy in the time domain. This new technique complements the conventional nuclear resonance absorption, e.g, Mossbauer spectroscopy 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 bents, i.e. as modulation of the time-dependent intensity of the transmitted radiation, which is delayed with respect to the incoming synchrotron pulse. We have investigated Fe-57-enriched iron porphyrins to test NFS for first biophysical applications. NFS spectra of the diamagnetic porphyrin FeO2(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.

The tetraaza macrocycles 2-oxa-3-oxotetramesitylporphine (|H(2) 1|) and 2-oxa-3-oxotetrakis(2,6-dichlorophenyl)porphine (|H(2) 2|) and the corresponding iron complexes (|Fe(III)(X) 1| and |Fe(III)(X) 2|; X= Cl(-), OH(-), or SO(3)CF(3)(-)) have been synthesized. These macrocycles are derived from porphyrins by transformation of one pyrrole ring to an oxazolone ring. The resulting lactone functionality serves to restrict but not completely block pi-conjugation around the periphery. These complexes thus share properties with both porphyrins and chlorins. The ferric and high-valent iron complexes have been characterized by a variety of spectroscopic techniques. The molecular structure of |Fe(III)(Cl) 2| has been obtained by X-ray crystallography and shows that the structural changes at the macrocycle periphery do not perturb the coordination sphere of iron relative to the corresponding porphyrin complexes. This is illustrated by the observation that Fe-O frequencies in the resonance Raman spectra of the porpholactone analogues of compounds I and II are not substantially different from those of porphyrins and by the axial appearance of the EPR signals of the high-spin ferric complexes. This is consistent with reports that the Fe=O unit of oxidized porphyrins and chlorins is relatively insensitive to alteration of macrocycle symmetry. Nevertheless, probes of properties of the porpholactone macrocycle ((1)H NMR, resonance Raman skeletal modes) show effects of the asymmetry induced by the oxazolone ring. On the basis of (1)H NMR, EPR, Mössbauer, and resonance Raman data, the singly occupied molecular orbital of oxoferryl porpholactone pi-cation radicals correlates with the a(1u) molecular orbital of porphyrins under D(4)(h)() symmetry. Moreover, the paramagnetic properties and the intramolecular exchange interaction of ferryl iron and the porpholactone pi-radical have been characterized by EPR and magnetic Mössbauer measurements and spin-Hamiltonian analyses. The values J(0) = 17 cm(-)(1) and J(0) = 11 cm(-)(1) obtained for the exchange coupling constants of the oxoferryl porpholactone pi-cation radical complexes |Fe(IV)=O 1|(+) and |Fe(IV)=O 2|(+), respectively, are among the lowest found for synthetic compound I analogues.

The molecular structure of the chloroiron(III) porphyrinate [(FeCl)-Cl-III-(tmtmp)] (1) is described. The doming of the bifacially encumbered tmtmp dianionic ligand present in 1 is characterized by a separation of 0.09 (1) Angstrom between the mean plane of the four pyrrole nitrogens (4N(p)) and that of the porphyrin core. The coordination polyhedron of the five-coordinate iron atom is a square pyramid of C-4v symmetry. The metal is displaced by 0.44 (1) Angstrom from the 4N(p) mean plane towards the axial chloro ligand, The Fe-N-p and Fe-Cl bond lengths are 2.057 (6) and 2.223 (5) Angstrom , respectively, Upon treatment of [Fe(III)Tf(tmtmp)] (2) with m-chloroperoxybenzoic acid, the green oxoferryl pi radical cation complex [Fe-IV = O(tmtmp(.))](+) (3) is obtained. It has been studied by EPR, Mossbauer, and resonance Raman spectroscopy. It exhibits an EPR spectrum strikingly similar to those of compounds I of Micrococcus lysodeikticus catalase (MLC-I) and ascorbate peroxidase (APX-I). The exchange interactions between the spins of the radical cation and the ferryl iron are of the ferromagnetic type and are the weakest ever found for a synthetic compound I model. The shift of + 22 cm(-1) in the radical marker band v(2) in the resonance Raman spectra upon oxidation of 2 to 3 confirms that the electronic state of the porphyrin pi radical cation is predominantly (2)A(1u) this tmtmp compound I model.

A series of (oxoferryl)porphyrin pi-cation radicals generated from porphyrins substituted at the meso positions with highly electron-withdrawing aryl groups has been characterized: tetrakis-5,10,15,20-(2,6-dichlorophenyl)-, 5-(2-chloro-6-nitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)-, and 5-(2,6-dinitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)porphyrins (porphyrins 1-3, respectively). The physical-chemical properties of the oxidized complexes of 1-3 are compared to those of two (oxoferryl)porphyrin pi-cation radical complexes substituted with electron-releasing aryl groups: tetramesitylporphyrin (TMP) and 2-iodotetramesitylporphyrin (2-iodoTMP). While all of the complexes examined show close correspondance in a number of spectroscopic parameters, some significant differences were observed. In contrast to observations for the oxidized complexes of TMP and 2-iodoTMP, the resonance Raman marker bands nu(2) and nu(11), which are indicators of symmetry state of porphyrin pi-cation radicals of 1-3, do not show the expected downfrequency shifts for oxidation to compound I analogs in a(2u) symmetry states. The upfield hyperfine NMR shifts of the pyrrole beta-proton signals of the compound I analogs of 1-3 are much larger than those for TMP and 2-iodoTMP. These data may be explained by admixture of some a(1u) character into the ground state of radical cations of 1-3, consistent with the hypothesis that electron-withdrawing meso substituents lower the energy of the a(2u) molecular orbital, favoring an a(1u) admixture.