Spectroscopic and Kinetic Characterization of Peroxidase-Like π-Cation Radical Pinch-Porphyrin-Iron(III) Reaction Intermediate Models of Peroxidase Enzymes

Abstract

The spectroscopic and kinetic characterization of two intermediates from the H₂O₂ oxidation of three dimethyl ester [(proto), (meso), (deuteroporphyrinato) (picdien)]Fe(III) complexes ([FePPPic], [FeMPPic] and [FeDPPic], respectively) pinch-porphyrin peroxidase enzyme models, with s = 5/2 and 3/2 Fe(III) quantum mixed spin (qms) ground states is described herein. The kinetic study by UV/Vis at λmax = 465 nm showed two different types of kinetics during the oxidation process in the guaiacol test for peroxidases (1-3 + guaiacol + H₂O₂ → oxidation guaiacol products). The first intermediate was observed during the first 24 s of the reaction. When the reaction conditions were changed to higher concentration of pinch-porphyrins and hydrogen peroxide only one type of kinetics was observed. Next, the reaction was performed only between pinch-porphyrins-Fe(III) and H₂O₂, resulting in only two types of kinetics that were developed during the first 0-4 s. After this time a self-oxidation process was observed. Our hypotheses state that the formation of the π-cation radicals, reaction intermediates of the pinch-porphyrin-Fe(III) family with the ligand picdien [N,N'-bis-pyridin-2-ylmethyl-propane-1,3-diamine], occurred with unique kinetics that are different from the overall process and was involved in the oxidation pathway. UV-Vis, ¹H-NMR and ESR spectra confirmed the formation of such intermediates. The results in this paper highlight the link between different spectroscopic techniques that positively depict the kinetic traits of artificial compounds with enzyme-like activity.

Keywords: peroxidases models; pinch-porphyrins; spectroscopic studies; π-cation radical.

Figure 1

Pinch-porphyrins. When R = –CH=CH₂ FePPPic; R = –CH₂–CH₃ FeMPPic and -H FeDPPic. Pr = methylpropionate.

Figure 2

(a) Single kinetics showing only the guaiacol oxidation products; (b) Two types of kinetics, oxidation of A pinch-porphyrin and B guaiacol, or three in some cases, were observed when 2.5 mM [H₂O₂] was added, one magnitude order lower than for activity catalytic studies.

Figure 3

Kinetic pathway of the reaction [FeDPPic]_(MeOH) + H₂O_2(aq) at the first four seconds of reaction and at 500 nm ($\mathbf{―}$). The blue line ($\mathbf{―}$) corresponds to the condition with the H₂O_2(aq) concentration one magnitude order higher.

Scheme 1

The normal peroxidase cycle. HRP Horseradish peroxidase, HRP---O₂H₂ Compound 0, HRP-I compound-I, HRP-II compound-II.

Figure 4

Snapshots of [FePPPic]^+. formation observed for 25 min. Soret bands decrease indicating that the [FePPPic] is being auto-oxidized because of the absence of guaiacol in the reaction media, similar to HRP-I (compound I of horseradish peroxidase) behavior in an excess of hydrogen peroxide, which reduces compound I. d-d transitions energetically change showing a new spectrum.

Figure 5

Precursors of the Fe(III)-dimethyl ester porphyrin and 1,9-bis-(2-piridyl)-2,5,8-triazanonane (Picdien). Protoporphyrin R = –CH=CH₂, Mesoporphyrin R = –CH₂–CH₃, Deuteroporphyrin-H.

Figure 6

(a) ¹H-NMR of [FeDPPic] with chemical shifts of meso-protons at 110.78 ppm and methyl-protons at 63.89 and 58.96 ppm; (b) ¹H-NMR of [DPPicFe^IV=O]^+· with the highest chemical shifts at 22.67 ppm and with no possibilities of assignation.

Figure 7

Top: ESR spectrum of [FeMPPic] at 77 K in methanol solution with two species of Fe(III) with qms; s = 3/2, S = 5/2, species with g = 5.22, 1.99 contains more s = 5/2 in qms; species with g = 4.06, 1.95, contains more s = 3/2 in qms. Bottom: [FeMPPic]-I with two species of iron ion, one with g = 5.74 and higher percentage of this species in qms but in less proportion than the second species (g = 3.97) with s = 3/2. At g = 1.93 the radical signal is present.

Figure 8

ESR axial spectra of [PPicdienFe^IV=O]^+· with species of iron ion at higher energy with qms (g > 5.7), proportion decrease from [FePPPic]-I > [FeMPPic]-I > [FeDPPic]-I. Species with s = 3/2 (g ~ 4) and with the highest proportion in each sample. The s = 3/2 corresponds to three unpaired spins, which stem of the Fe(IV) ion low spin (s = 1) ferromagnetically coupled with radical with s = 1/2, giving a total spin of s = 3/2.

Scheme 2

The intermediate compound 0 formed faster if the Fe (III) has less stability to receive less electronic contribution of its porphyrin macroligand.

Figure 9

Comparison of the activation energies from three compounds with and without the pinch-axial-ligand.