Chem. Res. Toxicol. 1990, 3, 289-291
289
Communications Redox Reactivity of Iron( III)Porphyrins and Heme Proteins with Nitric Oxide. Nitrosyl Transfer to Carbon, Oxygen, Nitrogen, and Sulfur Sir: Nitrosamines are well-known carcinogens (1). They are reported to be denitrosated by mammalian cytochrome P-450 (2-11), and the nitrosyl adduct of the P-450 heme has been detected (2). As a part of our study of these and related reactions, we expected high-spin hemes to readily cleave the N-N bond of nitrosamines to generate the enormously stable heme-NO adducts (12-17):
+ ONNRz + H+
PFe(N0) + PFenl + HNRz (1) This reaction, however, did not readily 0ccur.l As a consequence, we wondered whether a reverse nitrosation reaction with iron(II1) complexes and nitric oxide was feasible. Precedence for this hypothesis lies in the spectroscopic work of Wayland and Olson (18).The reversibly formed NO adduct of CIFemTPP in toluene solution was formulated as an entity possessing substantial iron(II) NO+ character (PFemNO PFenNO+). However, gassing the chloroiron(II1) porphyrin with nitric oxide in methanol/ toluene resulted in the iron(I1)-NO complex (and presumably methyl nitrite). In related work (19, 20) with hemoglobin and myoglobin, Chien had demonstrated that treatment of either the iron(II1) or iron(I1) proteins with nitric oxide produced the iron(I1)-NO adducts. With methemoglobin (191, the reaction could be conducted in the crystalline state. We report herein that nitric oxide is indeed redox activated by iron(II1) porphyrins and heme proteins to a nitrosating species having the character of nitrosonium ion. This reactivity has special significance because it has recently been recognized that the endogenous generation of nitric oxide occurs within a variety of mammalian cell types (21,221. Most notably, the "endothelium derived relaxing factor" has been identified as nitric oxide. Moreover, NO has been implicated in the cytotoxicity of phagocytic cells and in cell to cell communication in the nervous system (21). The molecular means by which this substance is activated in vivo may well entail reaction pathways similar to those described herein. Results and Discussion. Representative rections with metmyoglobin [(4-5) X lo4 MI under argon in 0.01 M argon purged phosphate buffer and 0.1 M KC1, pH 7.4, M) are illustrated saturated with nitric oxide (-2 X in Scheme I. The heme-NO adduct is produced quantitatively in all cases (eq 2). Under these anaerobic con2PFe"
-
Mb+ + 2 N 0 + Nuc-H
-
Nuc-NO
+ MbNO + H+ (2)
ditions, blank runs in the absence of heme proteins produced no nitrosated products. Water competes with the organics as a nucleophile in these reactions, and the nitrite ion produced accounts for a material balance. Thus, with proline at 0.2 M, a 15% yield of N-nitrosoproline and an 85% yield of nitrite ion are obtained. Except for the thiol, Homogeneous solutions of hemes
(lo4 M) and nitrosamines
(10-9-10-oM)in N-methylpyrrolidoneaceticacid exhibited no spectral
change in days. The nitrosamine was unreacted (GC).
Scheme I (Mb'
NO)
t
Hp 0 I OH-
> HONO+
H t + NO; (100%)
(?y 16\ p x
OH
8 NO
(100%)
0
NO (15%)
ON\S&
NH
J , 0
OH
(85%)
Table I. Yields of Nitrosophenol from Heme Proteins, in 0.01 M Phosphate Buffer and 0.1 M KCl, pH 7.4 phenol yield8 of concn, nitrosoFeIU protein concn, mol/L mol/L phenol, % myoglobin" 4.9 x 10-4 8.0 x 10-9 100 catalaseb 3.7 x 10-5 2.0 x 10-3 88h peroxidase' 4.7 x 10-5 2.0 x 10-3 50 hemoglobind 1.3 X lo4 4.0 X 5 cyt ~-450-e 7.7 x 10-5 4.0 x 10-3 5 cytochrome d 6.3 X 2.0 X 0 none 2.0 x 10-3 0 a Horse. Bovine liver. Horseradish. dHuman. e Bacterial. /Horse heart. 8 100 X nitrosophenol/iron(III) protein. Calculated on the basis of one reactive iron(II1) porphyrin per tetramer.
N-acetylcysteine,nitrite ion itself is not a nitrosating agent for these substrates under the conditions employed. The 5'-nitroso derivative decomposes in solution, and the yield reported for it is a minimum. With iron(II1) porphyrins, reactions can be conducted in the absence of water and high yields of nitrosated organics can be obtained. As an example, quantitative yields of N-nitrosodiethylamine and nitrosylheme are obtained by gassing, under argon, methylene chloride solutions of the amine with nitric oxide in the presence of chloroiron(II1) octaethylporphyrin.2 The high yields of nitrosophenol with myoglobin prompted a comparison of the reactivity of a variety of heme proteins to nitric oxide and phenol under anaerobic conditions. The results are presented in Table I. The yield of protein iron(I1)-NO is quantitative for all iron porphyrin active sites except catalase? However, the yield of nitrosophenol varies widely and is least for the most rapidly reacting protein, iron(II1) cytochrome c. With this protein, nitrite ion and iron(I1) nitrosylcytochrome c are obtained but phenol is not nitrosated. We shall report upon the iron porphyrin reactions separately because they can be more complex and entail a further reduction of some of the nitroeated organics. Only one heme of the catalase tetramer is active in the demmpmition of H20%Accordingly, the yield of this case is calculated baed upon the reactivity of only one iron(II1) p o r p p The visible spectrum of the product is a composite of native Fe and Fe"N0 species.
0893-228x/90/2103-0289$02.50/00 1990 American Chemical Society
290 Chem. Res. Toricol., Vol. 3, No. 4 , 1990
Communications
The data suggest that substrate binding near, or accessibility to, the active site must be a dominant factor in determining the degree of nitrosation. Thus, the C conformation of cytochrome c should preclude (23) ready access of phenol to an iron-bound NO, but the G conformation of myoglobin should allow it. Moreover, in addition to the gross steric constraints imposed by these proteins, a favorable binding of phenol near the active site is indicated for myoglobin, catalase, and peroxidase. Clearly, the result are not compatible with the generation of a free nitrosonium ion. If free NO+ were the reactive species, equal ratios of nitrosophenol/N02- should be produced at fixed substrate concentration. This is not the case. These results accord with a competitive attack between water and organic nucleophile upon a transient "iron(II1)-NO" adduct (eq 3) and are consistent with the formulation proPFem
+ NO
-
[PFe(NO)]+5 NOz-
References
+ PFeU k
N
u
c
t
4
(3)
0
PFe'(N0)
posed for the adduct by Wayland and Chien. The results are also consistent with the inability of nitrosylhemoglobin itself to nitrosate proline but the capacity of nitosyliron(I1) octaethylporphyrin to bring about some nitrosation of diphenylamine in air at 60 "C as reported4 by Bonnett and colleagues (24). Finally, the nitrosation of diethylamine brought about by ntiric oxide and cupric chloride (25,26) and the generation of methyl nitrite by reaction of nitric oxide and methanol with cobalt(I1)-TMEDA complexes (27)may entail some mechanistic analogy to the processes reported here. Most importantly, our findings coupled with the endogenous generation of nitric oxide in endothelial cells (21, 22) and other cell types suggest an alternate means for the generation of carcinogenic nitroso compounds and other physiologically active substances in vivo. It is quite plausible that myoblobin and other heme proteins play a significant role in these reactions. Experimental Section. In a typical reaction, 2.5 mL of a buffered solution of myoglobin and phenol was purged with argon for 1 h a t room temperature in a 1-cm spectrophotometric cuvette. The cuvette was equipped with a serum-capped stopcock. Gases were passed in and out via hypodermic tubing. The outlet was connected to a mercury trap. Reaction was commenced by the injection of 1 mL of nitric oxide through the solution and monitored by UV-vis. The stopcock was closed under slight NO pressure. The iron proteins were pure and were obtained as previously described (28,29). T h e iron(I1)-NO product spectra obtained matched the literature in all cases and were identical with the spectra obtained by gassing standard iron(I1) proteins with NO. The A- and molar c used for these quantitations and determined in our milieu were as follows (PFenNO): myoglobin, 548 nm, 12.5 x 109; hemoglobin, 541 nm, 12.0 x lo3; cyt c, 530 nm, 11.2 x 103; catalase, 539 nm, 17.3 X 109; peroxidase, 533 nm, 13.3 X 109; and P-450,', 542 nm, 18.0 X 109. Before workup reaction mixtures were again purged with argon to remove any excess nitric oxide. Nitrite ion was analyzed by diazotizing sulfanilamide and coupling with 1-naphthylethylenediamineto the colored azo compound. The latter was read spectrophotometridy at its A,-, 543 nm (30). Nitrosophenol was isolated by extraction of acidified (pH 2) reaction mixtures with ether. The ether solution was back-extracted with p H 8.5 buffer to separate the nitroso derivative from the parent phenol. Nitrosophenol was determined spectrophotometrically by its characteristic spectrum in acid (A, 300 nm) and base (Amm 400 nm). The substance was also confirmed by
'The nitrosatingspecies in the air oxidation process may have been
N20,; cf. ref 1, Chapter 1.
HPLC analysis (silica ODS column, 1:l CH2C12/MeOH, 2 mL/ min, 12.1 min). N-Nitrosoproline was qualitatively detected by GC (2' Porapak P, 175O) by direct analysis of the reaction mixture and as ita derivative methyl ester. The substance was quantitated spectrophotometrically a t 238 nm from ether extracts of acidified reaction mixtures (31). S-Nitroso-N-acetylcysteine is unstable. The substance was separated from myoglobin-NO on a Sephadex G-25 column and determined spectrophotometrically at 335 nm (cf. ref 1, p 174). In situ generation of authentic material gave cm as lo00 in agreement with Williams. Registry No. Cyt P-450,9035-51-2; HzO, 7732-18-5; HONO, 7782-77-6; proline, 147-85-3; N-nitrosoproline, 7519-36-0; Nacetylcysteine, 616-91-1; S-nitroso-N-acetylcysteine,56577-02-7; 4-nitrosophenol, 104-91-6; phenol, 108-95-2; catalase, 9001-05-2; peroxidase, 9003-99-0.
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Communications porphinato) iron(II1)perchlorate. J. Am. Chem. SOC.106, 3191-3198. (18) Wayland, B. B., and Olson, L. W. (1974) Spectroscopic studies and binding model for nitric oxide complexes of iron porphyrins. J. Am. Chem. SOC. 96,603743041. (19) Chien, J. C. W. (1969) Reactions of nitric oxide with methemoglobin. J. Am. Chem. SOC. 91,2166-2168. (20) Dickson, L. C. W., and Chien, J. C. W. (1971) An electron paramagnetic resonance study of nitrosylmyoglobin. J. Am. Chem. SOC. 93,5036-5040. (21) Moncada, S., Palmer, R. M. J., and Higgs, A. E. (1989) Biosynthesis of nitric oxide from L-arginine. A pathway for the regulation of cell function and communication. Biochem. Pharmacol. 38, 1709-1715. (22) Marletta, M. A. (1989) Nitric oxide: biosynthesis and biological significance. Trends Biochem. Sci. 14,486492. (23) Castro, C. E. (1978) Routes of electron transfer. In The Porphyrins (Dolphin, D., Ed.) Vol. V, pp 1-27, Academic Press, New York. (24) Bonnett, R., Charalambides, A,, and Martin, R. A. (1978) Nitrosation and nitrosylation of haemoproteins and related compounds. Part 2. Porphyrins and metallo porphyrins. J. Chem. SOC., Perkin Trans., 974-980. (25) Brackman, W., and Smit, P. J. (1985) Homogeneous catalysis. Kinetics and mechanism of the copper-catalyzed reaction of nitric
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Ruth S. Wade, C. E. Castro* Department of Nematology University of California Riverside, California 92521 Received February 15, 1990
