Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters

Intermediate filaments (IFs) represent the largest cytoskeletal gene family comprising ~70 genes expressed in tissue specific manner. In addition to scaffolding function, they form complex signaling platforms and interact with various kinases, adaptor, and apoptotic proteins. IFs are established cytoprotectants and IF variants are associated with >30 human diseases. Furthermore, IF-containing inclusion bodies are characteristic features of several neurodegenerative, muscular, and other disorders. Acidic (type I) and basic keratins (type II) build obligatory type I and type II heteropolymers and are expressed in epithelial cells. Adult hepatocytes contain K8 and K18 as their only cytoplasmic IF pair, whereas cholangiocytes express K7 and K19 in addition. K8/K18-deficient animals exhibit a marked susceptibility to various toxic agents and Fas-induced apoptosis. In humans, K8/K18 variants predispose to development of end-stage liver disease and acute liver failure (ALF). K8/K18 variants also associate with development of liver fibrosis in patients with chronic hepatitis C. Mallory-Denk bodies (MDBs) are protein aggregates consisting of ubiquitinated K8/K18, chaperones and sequestosome1/p62 (p62) as their major constituents. MDBs are found in various liver diseases including alcoholic and non-alcoholic steatohepatitis and can be formed in mice by feeding hepatotoxic substances griseofulvin and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDBs also arise in cell culture after transfection with K8/K18, ubiquitin, and p62. Major factors that determine MDB formation in vivo are the type of stress (with oxidative stress as a major player), the extent of stress-induced protein misfolding and resulting chaperone, proteasome and autophagy overload, keratin 8 excess, transglutaminase activation with transamidation of keratin 8 and p62 upregulation.

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Intermediate filament cytoskeleton of the liver in health and disease

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine. Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown.

Toxicological significance of mechanism-based inactivation of cytochrome p450 enzymes by drugs.

Differential apoprotein loss of rat liver cytochromes P450 after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine: a case for distinct proteolytic mechanisms?

Occurrence and biological properties of N-methyl protoporphyrin.

The ferrochelatase-reducing activity and cytochrome P-450- and heme-destructive effects of a variety of analogues of 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine (DDC) were studied in chick embryo liver cells. A group of DDC analogues was found in which an inability to reduce ferrochelatase activity corresponded with an inability to cause cytochrome P-450 and heme destruction. In a second group of DDC analogues, the ability to reduce ferrochelatase activity corresponded with the ability to cause cytochrome P-450 and heme destruction. These observations support the idea that the protoporphyrin IX moiety of N-alkylprotoporphyrin IX originates from the heme moiety of cytochrome P-450. A third group of DDC analogues caused cytochrome P-450 and heme destruction despite an inability to reduce ferrochelatase activity. With this third group of DDC analogues, the heme moiety of cytochrome P-450 is likely degraded to products other than N-alkylporphyrins. The inability of several lipophilic DDC analogues [4-benzyl, 4-isopropyl, 4-cyclohexyl, 4-(3-cyclohexenyl)] to reduce hepatic ferrochelatase activity may explain their low porphyrinogenicity. The pattern of porphyrin accumulation produced in response to two DDC analogues that did not inhibit ferrochelatase was investigated using high performance liquid chromatography. Coproporphyrin was the major porphyrin to accumulate in response to the 4-isopropyl analogue and uro- and heptacarboxylic acid porphyrins in response to the 4-benzyl analogue. These patterns of porphyrin accumulation are consistent with the inability of these analogues to inhibit ferrochelatase.

Suicidal destruction of cytochrome P-450 and reduction of ferrochelatase activity by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine and its analogues in chick embryo liver cells.

K Nakatsu

Papers

Suicidal destruction of cytochrome P-450 and reduction of ferrochelatase activity by 3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-trimethylpyridine and its analogues in chick embryo liver cells.