Researchin MAY / JUNE 1989 VOLUME 2. NUMBER 3 @Copyright 1989 by the American Chemical Society
Perspective Covalent Bonding of the Prosthetic Heme to Protein: A Potential Mechanism for the Suicide Inactivation or Activation of Hemoproteins Yoichi Osawa* and Lance R. Pohl Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 10, Room 8N110, Bethesda, Maryland 20892 Received May 3, 1989
Introduction
Scheme I. Mechanisms for Suicide Inactivation’
r
Since the first description of the phenomenon almost two decades ago, a vast array of enzymes have been doc+I umented to undergo suicide or mechanism-based inactivation (1-3). Among these are the P-450 cytochromes (P-450) ( 4 ) ,a family of hemoprotein monooxygenases that play a vital role in the metabolism of a variety of xenobiotics, including drugs and environmental pollutants, as well as endogenous compounds, such as steroids, prostaglandins, and fatty acids. The metabolites of P-450 are usually stable hydroxylated products; in certain cases, fhowever, depending on the structure of the substrates, highly reactive intermediates are formed, which are deleterious to the enzyme. During the past decade two pathways for the mechaCovdent MDdiliMion Covalent Mcdifiration Cwalsm Bonding nism-based destruction of liver microsomal P-450 cytoHeme oi Rotain of Heme fa Rmin chromes have been extensively characterized (Scheme I). ’I represents a reactive intermediate. One pathway involves the modification of the heme moiety to products that can dissociate from the protein; this is best The best example is the suicide inactivation caused by exemplified by the formation of the well-documented green chloramphenicol, an antibiotic that is hydroxylated, in pigments that result from N-alkylation of the iron porpart, to form an oxamyl chloride intermediate that acylates phyrin apparently by reactive radical or cation radical lysine residue(s) (7, 8). This modification seems to inmetabolites of the substrate (for review see ref 4). In the terfere with the electron transport from NADPH-cytocase of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,4-di- chrome P-450 reductase to the cytochrome (9). Other hydropyridine (DDEP), it is thought that this process examples include the suicide inactivation of cytochrome occurs by a one-electron oxidation of the nitrogen of DDEP P-450b, the major phenobarbital-inducible form, by Nby cytochrome P-450 to yield a radical cation intermediate methylcarbazole (10) and lauric acid hydroxylase by an that subsequently aromatizes and releases an ethyl radical acetylenic fatty acid (11). (4-6), which ethylates the pyrrole nitrogen of the prosthetic More recently, a third mechanism (Scheme I) was disheme moiety ( 5 ) . covered, which involves the irreversible binding of the The second pathway (Scheme I) involves the covalent prosthetic heme to the protein presumably involving a modification of the protein moiety by a reactive metabolite. covalent bond (12). This pathway occurs with a variety
6-
Of
This article not subject to US. Copyright. Published 1989 by the American Chemical Society
132 Chem. Res. Toxicol., Vol. 2, No. 3, 1989
Osawa and Pohl
Table I. Destruction of Cytochrome P-450and Irreversible Binding to Protein of [SH]Heme Radiolabel i n Rat Liver Microsomes 1 h after CCla Treatmenta irreversible binding of 3H label to protein, % loss of % of total microsomal treatment cytochromes P-450 3H label 4 control 0 CCll 64 28 "Rats were pretreated with phenobarbital (80 mg/kg) for 4 days and then administered NaH"C03 and [3,5-3H]ALAto radiolabel microsomal protein and heme, respectively (21). Two rats received CCl, (26 mmol/kg, ip in 50% sesame oil) and two rats sesame oil as control. The radioactivity (dpm/mg of protein; average of two values) of the microsomal suspensions prepared from these rats was as follows: control group, 3H label 280000, "C label 35000; CCl, group, 3H label 213 000, 14C label 32 000. Microsomes were precipitated with 5 volumes of acetone containing 0.5 M HCl and washed with the same solution. The numbers represent the average of single determinations (22).
of structurally diverse compounds, including those that cause N-alkylation of the prosthetic heme. In this perspective, we will discuss the following: (1) describe how this pathway was first discovered with the hepatotoxic agent CCl,; (2) illustrate that the formation of heme-protein adducts can be mediated by a variety of other xenobiotics, as well as endogenous compounds such as hydrogen peroxide and linoleic hydroperoxide; (3) describe how this pathway can occur with other hemoproteins; (4) propose possible mechanisms for the formation of heme-protein adducts; ( 5 ) speculate on the physiological and pathological consequences of such an alteration of the heme prosthetic group of hemoproteins. It will be illustrated that heme-protein adduct formation does not necessarily produce an inactive protein, but instead may result in the formation of an activated protein, a process we define as "suicide activation".
Covalent Bonding of the Prosthetic Heme to P-450 Cytochromes Carbon Tetrachloride. This hepatotoxic agent is one of the most extensively studied xenobiotics and is a classic example of a compound that causes free-radical-mediated tissue injury (13, 14). The initial event involved in its hepatotoxicity is the metabolism to the trichloromethyl radical by P-450 cytochromes (14-16). One consequence of this reaction is the inactivation of the P-450 and formation of uncharacterized heme breakdown products (14, 17-20). The mechanism for the CC1,-induced metabolism of heme is independent of the heme oxygenase pathway of heme catabolism, since CO is not formed from the heme during this process (18). Our interest in the metabolism and toxicity of various halogenated hydrocarbons led to the observation that, after incubation of liver microsomal cytochrome P-450 preparations with CCl,, a reddish brown chromophore persisted in the protein fraction after precipitation of the reaction mixture with acidic acetone. This finding indicated that heme products were irreversibly bound to protein. The definitive experiments to demonstrate that heme was metabolized to protein-bound adducts were carried out with cytochrome P-450 containing radiolabeled heme. This was accomplished by administering radiolabeled &aminolevulinic acid (ALA), a precursor to heme, to phenobarbital-pretreated rats (21). The P-450 apoprotein was also labeled in vivo by NaH14C03so that simultaneous measurements of radiolabeled protein and heme could be made (21). Administration of CCl, to these prelabeled rats caused an extensive loss of P-450 as measured by its fer-
Migration
-
Dve Front GEL FRACTION NUMBER
Figure 1. SDS-PAGE radioelectrophoretogramsof 40-pg samples of liver microsomes from control and CCL-treated rats (22).Rats were administered [3H]ALA to radiolabel microsomal heme and given CCl, as described in Table I.
rous-carbonyl complex (Table I) (22). Precipitation and extensive washing of the liver microsomal protein from these rats with acetoneHC1, which removes noncovalently bound heme from hemoproteins, revealed that 28% of the total heme was irreversibly bound to the protein fraction (Table I) (22). This indicated that approximately half of the destroyed P-450 could be accounted for by covalent modification of heme to protein. Analysis of the labeled microsomal proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and radioisotope counting of gel slices revealed that the proteinbound heme-derived material was confined to a region corresponding to molecular masses of 47-56 kDa, the range where P-450 cytochromes dominate (Figure 1) (22). Interestingly, a small amount of heme-derived material was found to be irreversibly bound even in untreated animals (Figure 1,control), suggesting an endogenous level of these adducts. The target of the irreversible binding of heme was confirmed to be cytochrome P-450 in immunoprecipitation studies. Immunoprecipitation of P-450b with specific antibody followed by SDS-PAGE and radioisotope counting revealed that heme was bound to the P-450b protein in liver microsomes of both control (Figure 2, panels A and B) and CC14-treatedrats (Figure 2, panels C and D)(12). The amount of irreversibly bound [3H]heme label, however, was at a much higher level in the livers of the CC1,-treated rats; i n this case 39% of the total heme irreversibly bound to microsomal protein was bound to this isozyme (12). Moreover, it appears that the activation of heme and its subsequent irreversible binding occurs before the heme dissociates from the apoprotein. This was shown by using a reconstituted system containing purified cytochrome P-450b, which contained a [3H]heme prosthetic group, NADPH-cytochrome P-450 reductase, NADPH, and CClk After incubation for 5 min, the reaction mixture was separated by SDS-PAGE and gel slices were analyzed for radioactivity (Figure 3). Under these conditions, approximately 90% of the cytochrome P-450 was lost, as measured by the absorbance of the ferrous-CO complex, and 44% of the total heme was irreversibly bound to P-450;
Chem. Res. Toxicol., Vol. 2, No. 3, 1989 133
Perspective
Table 11. Destruction of Cytochrome P-450 Heme, Irreversible Binding to Protein of [ “CIHeme Radiolabel, and Malondialdehyde Formation in Incubations of Rat Liver Microsomes” irreversible binding
z
Pi, d
incubation
U
conditions
A w
u
aerobic -NADPH +NADPH
E
F
+CC14
n 75
+CCl,
I-
o k Migrabon
+
of “C label to protein, % loss of cytochrome % of initial “C dpm MDA,b P-450 hemeb added to incubationsc pM
0 10 3 69
2 6 2 31
0.03 0.40
