Very general formation of tetrahydropterin cation radicals during reaction of iron porphyrins with tetrahydropterins: model for the corresponding NO-synthase reaction

Abstract

Electron transfer from tetrahydropterins to iron porphyrins, with formation of intermediate tetrahydropterin cation radicals, is a very general reaction that was shown to occur not only with tetrahydrobiopterin, as originally found in NO-synthases, but also with another important biological cofactor, tetrahydrofolate, and various iron porphyrins, either in their ferric state, or in the Fe^IIO₂ state, as in the first model of the corresponding NO-synthase reaction described in this paper.

---

Tetrahydropterins, such as tetrahydrobiopterin (H₄B) or tetrahydrofolate (H₄F) (Scheme 1), are important cofactors involved in many biological processes.¹ In nitric oxide synthase (NO-synthase), H₄B is an essential cofactor for the monooxygenation of L-arginine to L-citrulline and NO.² It has been recently shown that, during the NO-synthase-dependent monooxygenation, H₄B transfers one electron to the heme, a second cofactor which is present in the active site in close proximity to H₄B. The H₄B⁺˙ cation radical derived from this reaction has been detected by freeze–quench EPR methods.³ This is so far the only evidence for the intermediate formation of H₄B⁺˙ in a biological process. Moreover, in a more general manner, no data are presently available on electron transfer between tetrahydropterins and iron porphyrins. We have recently studied the reactions between various natural and synthetic tetrahydropterins and many iron porphyrins (Scheme 1) in order to mimic the electron transfer observed in NO-synthase and to ascertain whether this reaction is general or restricted to NO-synthase because of a special proximity and positioning of H₄B and the heme within the NO-synthase active site.

Scheme 1

Addition of one equivalent of H₄B to a solution of Fe^III[meso-tetra(pentafluorophenyl)-β-octabromo-porphyrin](Cl),⁴ Fe^III(TF₅PBr₈P)Cl (Scheme 1), in CH₃CN : H₂O (9 : 1), under anaerobic conditions, led to the immediate formation of Fe^II(TF₅PBr₈P), as shown by visible and EPR spectroscopy. Fast mixing (about 1 s) of these two reactants in an EPR Bruker AquaX cell at 20 °C led to signals centered at g = 2.003 (Fig. 1) very similar to those previously reported for H₄B⁺˙ obtained by chemical oxidation of H₄B.⁵ Simulation of this spectrum on the basis of literature data^3c,3d,5 gave a_N5, a_H5, a_N8 and a_H6 of 8.3, 9.7, 2.0 and 10.5 G respectively (for numbering of H₄B atoms, see Scheme 1). These values are similar to those previously reported for chemically generated H₄B⁺˙ ⁵ and comparable to those found for H₄B⁺˙ detected in NO-synthase,^3c,3d allowing for the different environments of H₄B⁺˙ in the chemical and enzymatic experiments.

Fig. 1 EPR spectrum observed during reaction of H₄B with Fe(TF₅PBr₈P)Cl. (—) experimental spectrum obtained 10 s after fast mixing of 2 mM Fe(TF₅PBr₈P)Cl and 2 mM H₄B in CH₃CN : H₂O (9 : 1) at 20 °C;([dash dash, graph caption]) simulated spectrum with g = 2.003 and a_N = 8.3 and 2.0 and a_H = 9.7 and 10.5 G.

Reaction of Fe^III(TF₅PBr₈P)Cl either with H₄F in DMF : H₂O 9 : 1, or with diMeH₄P under conditions identical to those used with H₄B, also resulted in fast reduction of the Fe^III porphyrin with intermediate formation of a species exhibiting EPR characteristics very similar to those of H₄B⁺˙.

Similar reactions were performed between diMeH₄P and seven other Fe^III porphyrins, Fe(TDCPN_xP)Cl, bearing from 0 to 7 β-nitro substituents and exhibiting redox potentials (vs. saturated calomel electrode) ranging from −225 to +560 mV for the Fe^III/Fe^II couple (in CH₂Cl₂).⁶ All reactions were done at 20 °C in CH₃CN : H₂O (9 : 1) under strictly anaerobic conditions, and were followed by UV–vis, ¹H NMR and EPR spectroscopy. Complete reduction of the starting Fe^III porphyrin occurred in less than 10 s after addition of one equiv. of diMeH₄P to Fe^III(TDCPN_xP)Cl with x > 2. In fact, addition of increasing amounts of diMeH₄P to Fe(TDCPN₅P)Cl showed that complete formation of Fe^II(TDCPN₅P) already occurred after addition of 0.5 equiv. of diMeH₄P, as expected if one considers that diMeH₄P is a two-electron reducing agent. Complete reduction of Fe(TDCPP)Cl, Fe(TDCPN₁P)Cl and Fe(TDCPN₂P)Cl required the addition of 5–10 equiv. of diMeH₄P. EPR studies of all these reactions after fast mixing of the reactants in an EPR AquaX cell at room temperature always showed the intermediate formation of signals at g = 2.003 which are characteristic of diMeH₄P⁺˙. Moreover, EPR studies of those reactions performed in the presence of various classical radical traps, such as α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone (POBN), always showed the appearance of the signals expected⁷ for the addition of a carbon-centered free radical derived from diMeH₄P⁺˙ to the spin trap. For instance, reaction of Fe(TF₅PBr₈P)Cl with diMeH₄P in the presence of POBN (1 : 1 : 100 molar ratio) produced a six-line spectrum centered at g = 2, with a_N = 14.5 G and a^β_H = 1.9 G, corresponding to the trapping by POBN of a tertiary carbon-centered radical derived from diMeH₄P⁺˙. A similar EPR spectrum has been recently observed during oxidation of H₄B by peroxynitrite in the presence of POBN.⁸

The above data showed that electron transfer from tetrahydropterins to iron (III) porphyrins, with intermediate formation of the corresponding tetrahydropterin cation radical, is a very general reaction [eqn. (1)].

diMeH₄P + (P)Fe^III → (P)Fe^II + diMeH₄P⁺˙ (1)

However, this electron transfer was not detected in the case of Fe^III porphyrins of very low redox potentials, such as Fe^III microperoxidase 11⁹ (∼ −375 mV vs. SCE) (data not shown), and does not occur between H₄B and the Fe^III heme (∼ −500 mV vs. SCE) in NO-synthase.² Indeed, the intermediate formation of H₄B⁺˙ in the NO-synthase reaction has been shown to result from an electron transfer from H₄B to the NO-synthase heme Fe^IIO₂ complex.³ In order to mimic that reaction, we have studied the reaction between diMeH₄P and one of the few stable (porphyrin)Fe^IIO₂ complexes reported in the literature, the Fe^II(T_pivPP)(O₂)(N–MeIm) complex (Scheme 1) obtained by exposure of the Fe^II(“picket-fence” porphyrin) to O₂ in the presence of N-methylimidazole.¹⁰

Fast mixing of solutions of Fe^II(T_pivPP)(O₂)(NMeIm) and diMeH₄P (2 equiv.) in acetone : H₂O 9 : 1 at 20 °C in an EPR AquaX cell led to the formation of a species having EPR characteristics identical to those of diMeH₄P⁺˙. The intensity of the diMeH₄P⁺˙ signal reached its maximum value in 10 s and then decreased with a half life of about 120 s. UV–vis studies of the same reaction at 20 °C showed the disappearance of the bands of the starting complex and the appearance of the bands of the Fe^III(T_pivPP)(OH) complex. EPR studies at 100 K of a similar reaction performed with addition of excess diMeH₄P, for 3 min at 180 K, led to signals at g = 2.33, 2.19 and 1.92. These g-values are highly similar to those previously reported for the Fe^III(TMP)(imidazole)(OOH) complex and for the hemoglobin–, P450cam– and heme oxygenase–Fe^IIIOOH complexes (Table 1); they are considered to be characteristic of hexacoordinated porphyrin Fe^IIIOOH complexes¹⁵ and are clearly different from the g-values observed for the corresponding Fe^IIIOO⁻ complexes.¹⁵ Further warming of the solution from 180 K to 293 K led to the disappearance of these signals at temperature higher than 210 K. These data indicate that diMeH₄P rapidly transfers an electron to the “picket-fence” porphyrin Fe^IIO₂ complex with intermediate formation of diMeH₄P⁺˙ and of a transient Fe^IIIOO⁻ species which should be rapidly protonated at 180 K in the acetone–H₂O medium with formation of the corresponding Fe^IIIOOH intermediate observed by EPR spectroscopy [eqn. (2)]. Such a protonation of the transient (“picket-fence” porphyrin)Fe^IIIOO⁻ species at 180 K is in complete agreement with results reported on intermediate HbFe^IIIOO⁻ generated by cryoreduction of HbFe^IIO₂ at 77 K, which leads to HbFe^IIIOOH upon warming at 180 K¹². Reaction of eqn. (2) mimics well the electron transfer from H₄B to heme Fe^IIO₂ in NO-synthase (NOS), that is supposed to occur with transient formation of NOSFe^IIIOOH¹⁵ [eqn. (3)].

Table 1 Comparison of the EPR data of the intermediate complex formed upon reaction of Fe(T_pivPP)(O₂)(NMeIm) and diMeH₄P with those previously reported for hexacoordinated porphyrin Fe^IIIOOH complexes

Complex^a	g-values			Ref.
a TMP, β-Hb, P450cam and HOx are used for meso-tetramesitylporphyrin, the β chain of hemoglobin, cytochrome P450 101 and heme oxygenase, respectively.
Fe(TpivPP)(O2)(NMeIm) + diMePH4 at 180K	2.33	2.19	1.92	This work
Fe^III(TMP)(Im)(OOH)	2.32	2.19	1.94	11
β-Hb Fe^IIIOOH	2.31	2.18	1.94	12
P450cam Fe^IIIOOH	2.29	2.17	1.96	13
HOx Fe^IIIOOH	2.37	2.19	1.92	14

---

Our data show that the transfer of an electron from tetrahydropterins to iron porphyrins, with intermediate formation of tetrahydropterin cation radicals, is a very general reaction. It was shown to occur not only with H₄B, as described for the first time in the case of NO-synthase,³ but also with diMeH₄P or another important biological cofactor, H₄F, and various iron porphyrins, either in their ferric state, or in the Fe^IIO₂ state, as in the first model of the NO-synthase reaction of that type described in this paper. These data suggest that such reactions should be found in enzymes using both a tetrahydropterin and a metallic centre in their active site; in that regard, it has been recently proposed that some bacterial NO-synthase could be associated with H₄F.¹⁶

Notes and references

1. R. L. Blakley, S. J. Benkovic, in Folates and Pterins, vol. 1 & 2, eds. R. L. Blakley and S. J. Benkovic, John Wiley & sons, New York, 1985.
2. S. Pfeiffer, B. Mayer and B. Hemmens, Angew. Chem., Int. Ed., 1999, 38, 1714.
3. (a) A. R. Hurshman, C. Krebs, D. E. Edmondson, B. H. Huynh and M. A. Marletta, Biochemistry, 1999, 38, 15689; (b) C-C. Wei, Z. Wang, Q. Wang, A. L. Meade, C. Hemann, R. Hille and D. J. Stuehr, J. Biol. Chem., 2001, 276, 1244; (c) P. P. Schmidt, R. Lange, A. C. F. Gorren, E. R. Werner, B. Mayer and K. K. Andersson, J. Biol. Inorg. Chem., 2001, 6, 151; (d) M. Du, H.-C. Yeh, V. Berka, L.-H. Wang and A.-L. Tsai, J. Biol. Chem., 2003, 278, 6002.
4. M. W. Grinstaff, M. G. Hill, E. R. Birnbaum, W. P. Schaefer, J. A. Labinger and H. B. Gray, Inorg. Chem., 1995, 34, 4896.
5. J. Vasquez-Vivar, J. Whitsett, P. Martasek, N. Hogg and B. Kalyanaraman, Free Radical Biol. Med., 2001, 31, 975.
6. J. F. Bartoli, K. Le Barch, M. Palacio, P. Battioni and D. Mansuy, Chem. Commun., 2001, 1718.
7. G. R. Buettner, Free Radical Biol. Med, 1987, 3, 259–303.
8. S. L. Kohnen, A. A. Mouithys-Mickalad, G. P. Deby-Dupont, C. M. T. Deby, M. L. Lamy and A. F. Noels, Free Radical Res., 2001, 35, 709.
9. D. A. Stotter, R. D. Thomas and M. T. Wilson, Bioinorg. Chem., 1977, 7, 87.
10. J. P. Collman, R. R. Gagne, C. A. Read, T. R. Halbert, G. Lang and W. T. Robinson, J. Am. Chem. Soc., 1975, 19, 1427.
11. K. Tajima, S. Oka, T. Edo, S. Miyake, H. Mano, K. Mukai, H. Sakurai and K. Ishizu, Chem. Commun., 1995, 1507.
12. R. Davydov, T. Yoshida, M. Ikeda-Saito and B. M. Hoffman, J. Am. Chem. Soc., 1999, 121, 10656.
13. R. Davydov, T. M. Makris, V. Kofman, D. E. Werst, S. G. Sligar and B. M. Hoffman, J. Am. Chem. Soc., 2001, 123, 1403.
14. R. Davydov, V. Kofman, H. Fujii, T. Yoshida, M. Ikeda-Saito and B. M. Hoffman, J. Am. Chem. Soc., 2002, 124, 1798.
15. R. Davydov, A. Ledbetter-Rogers, P. Martasek, M. Larukhin, M. Sono, J. D. Dawson, B. S. S. Masters and B. M. Hoffman, Biochemistry, 2002, 41, 10375.
16. S. Adak, A. M. Bilwes, K. Panda, D. Hosfield, K. S. Aulak, J. F. McDonald, J. A. Tainer, E. D. Getzhoff, B. R. Crane and D. J. Stuehr, Proc. Natl. Acad. Sci. USA, 2002, 41, 10375.

---