Nitroxyl-adducts

Other enzymes that are not obviously related to the dioxygenases have at least superficially similar metal sites. The fatty acid desaturase of the endoplasmic reticuluum is a nonheme iron protein and requires both oxygen and reducing equivalents for activity (Strittmatter and Enoch, 1978). It is not known whether this enzyme forms a nitroxyl complex, but rat liver microsomes containing the enzyme form an S = nitroxyl adduct when treated with nitrite and dithionite. 

AU the nitroxyl adducts I-V were generated [1 ] pulseradiolytically in aqueous solution from nitrobenzenes and the corresponding a-hydroxyethyl, o.Jl-dihydrox-yethyl, 6-methyluracil-6-yl, 6-methyl-/-cytosinc-6-yl and 6-methyldihydrouracil-6-yl radicals. Production of a-hydroxyethyl, a,3-dihydroxyethyl, 6-rncthyluracil-6-yl, 6-methyl-/-cytosine-6-yl and 6-methyldihydrouracil-6-yl radicals were described elsewhere [1—4]. The heterolysis rate constants, and all activation free energy changes were taken from those references. The LFER plot for the heterolysis reaction of the nitroxyl adducts 1 and V is linear (Fig. 1) (r = 0.99). The slope of this plot, which should be equal to the ratio of the Hammett p values for the heterolysis of the two, adducts 1 and V, results as 1.0, in agreement with the individually determined p values (both 1.5). Also the solvent isotope effect (SIE) on of adduct V, that is... 

Fig- 1 Correlation of free energies of activation of Nitroxyl adducts I and V... 

In contrast, the LFER plot for the heterolysis reaction of the nitroxyl adducts III... 

DNA base radicals induced by OH radicals are also a possible target for interactions with nitro-aromatics [117]. For instance, the OH adducts of the nucleobases with reducing properties interact with nitro-aromatics to form nitroxyl adducts [118]. The rate constants for this interaction show only a weak dependence on the one electron reduction potential of the nitro-aromatic. However, using poly C as a model for a biopolymer, the nitroxyl radical adducts formed through addition of nitrofurantoin to the C-6 position of the C(5)-OH... 

The mechanism of ssb enhancement and radiosensitisation may be related in some way to those oxidants which form adducts with the DNA radicals. With the nitroaromatics a plausible enhancement of ssb is the possible H-atom abstraction by the base nitroxyl radical. The reactivity of the nitroxyl adducts with thiols or other reductants has not been studied in detail as there are at least two possible interactions where competition between the nitro-aromatic and a thiol may occur as shown in reactions (11-15). 

As our intent in this review is to address questions of NO c reductions at heme active sites from a mechanistic inorganic perspective, the use of simple heme model compounds is quite useful and will be thoroughly described. Here similar issues of solubility and reactivity come into play, as biomimetic NO c catalysis requires either water-soluble porphyrins or surface-adsorbed catalysts. As might be expected, questions of intersite reactivity come into play both in the reactivity of transient intermediate species such as nitroxyl-adducts, and in multielectron reactivity required for assimilatory reductase activity. We will begin with brief descriptions of the known families of heme-based NOjc reductases and the all-too-few electrochemical investigations of these native enzymes. 

The products of the aqueous catalytic reduction, as determined by tandem bulk electrolysis and mass spectrometry, were mainly NH2OH, NH3, and N2O with very minor amounts of N2. Meyer attributed the N2O formation as evidence of a bimolecular dimerization between reduced nitroxyl adducts(Fe -NO ) as shown in Eqs. (4.9)-(4.10) and Figure 4.7. Su and coworkers utilized Co tetra(Af-methyl-4-pyridyl)porphyrin (TMPyP) as an electrocatalyst to examine the pH dependence for nitrite reduction in aqueous solution. At low pH, both hydroxy-lamine and ammonia are formed at high pH only hydroxylamine is detected with no observable N-N coupled products. 

Important early electrochemical characterizations of nitrosyl adducts of porphyrin complexes were performed by the Kadish group. Voltammetric studies of the nitrosyl adduct of Fe (TPP) demonstrated an electrochemically reversible reduction which was substantially positive to that of the parent Fe (TPP). The product of the reduction, a nitroxyl adduct formulated as Fe (TPP)(NO) , could be generated in situ and its absorbance spectra observed reduction of the dinitro-syl, Fe (TPP)(NO)2, who s formation is characterized by a positive 1/2 shift. Table 4.2, also forms the same product. Nevertheless, attempts to prepare the reduced nitrosyl product via bulk electrolysis were unsuccessful. Up to ten reducing equivalents were passed through a sample solution electrolytically, but the major species in solution remained the ferrous nitrosyl, i.e., the nitroxyl apparently decomposed via some unspecified reaction to regenerate the more stable nitrosyl. 

Significantly, voltammetry at fast scan rates (>1 s) under NO gas yielded anodic currents attributed to the re-oxidation of the Fe nitroxyl complex, which implied that a nitroxyl adduct had a measurable lifetime during the catalytic reaction. A similar voltammetric fingerprint was apparent after succes-... 

Is Fe -NO a kinetic trap A common mechanistic quandary in both NiR and NoR chemistries is the possible inhibition of catalysis due to formation of a stable ferrous nitrosyl. Electrochemical studies of heme model systems suggest that the potential needed to reduce these species are well out of the normal range of biological reductants. Is the Fe -NO state truly a kinetic trap, and if so, how is it avoided during biological catalysis There is most obviously a correlation between macrocyclic structure and the potential of the ferrous NO adduct, and therefore structural even for the isobacteriochlorin model complex would require a powerful reductant to generate a nitroxyl adduct. 

What is the reactivity of the nitroxyladduct As suggested above, the nitroxyl adduct plays a central role in the several NO reduction pathways. Recent results have shown that the N-N coupling reaction in P450nor enzymes proceeds through a nitroxyl intermediate, and that this species is formed by a hydride transfer to the coordinated ferric nitrosyl. Is this species protonated, as is the stable adduct of Mb And, following the work of Ryan, how does the p a of this species control its subsequent reactions, N-N coupling or further reduction ... 

What is the sequence of multielectron reductions in the NiRl There is as yet no consensus on the sequence of multielectron transformations between NO and NH3 in the aNiR, and likewise no identifiable Fe-bound species past the nitroxyl adduct. The two electron priming of the siroheme/FeS site as proposed by Cowan remains difficult to rationalize with the observed one and three electron reduction steps seen by Ryan for the simple model Fe(TPP)(NO). 

The polymer-nitroxyl adduct P-X reversibly dissociates thermally, in process 1 into the polymer radical P and the nitroxyl radical X. The rate constants of dissociation and combination are and kc, respectively. The, so-called, degenerative transfer takes place in process 11. The second-order rate constant for active species in either direction is k y. Here all the rate constants are assumed to be independent of chain length. Since the frequency of cleavage of the P-X bond is proportional to [P-X] in process 1 and to [P l [P -X]] in process 11, the overall frequency,/ per unit time and per unit volume, of the bond-cleaving or activation reactions, may be expressed by [277] ... 

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