Heme contained in hemoglobin is involved in delivering O2 to tissues, and also the heme contained in variety of heme enzymes catalyzes oxidative reactions and electron transfer processes involved in generating Og and NO in cells. Heme can carry out these numerous physiological functions only when it is associated with various proteins. Free heme or protein unbound heme is toxic and it dose not occur in normal cells; it is deposited in tissues only under pathological conditions. Heme is a tetrapyrrole porphyrin molecule joined with Fe++ and this conjugation gives the specific color and fluorescence of various iron-porphyrins. Porphyrin molecule by itself does not bind O2 and does not participate in oxidative reactions or carry out electron transfer processes; these properties are endowed only when the porphyrin is bound with divalent transition metals (i.e., Fe, Mg, Cu, Zn, Sn, and Co) (46). Pro-toheme is ferro-protoporphyrin (Fe++-state) and it is readily oxidized to ferri-protoporphyrin (Fe+++-state), which then undergoes reduction back to protoheme in the presence of O2 and reducing agents. This generates Og in aerobic cells. Because the free heme not bound to proteins can participate in Fenton or Haber-Weiss reactions and produces highly reactive hydroxyl radical (HO*), it is highly toxic by altering protein structures and by causing lipid peroxidation. To protect from this toxicity of free heme, aerobic organisms have developed highly efficient means to compartmentalize and regulate heme synthesis, to sequester heme into proteins, to store and to transport as well as to degrade.
Heme molecule can be released from heme proteins by the ROS-driven structural alteration, fragmentation, and enhanced proteolytic digestion (47). Released heme undergoes oxidative degradation by heme oxygenases (HOs) contained in endoplasmic reticulum membranes. Heme is the natural substrate of HO. In the course of HO catalyzed heme oxidation reaction, NADPH is utilized both for activation of O2 and reduction of heme-iron substrate from the HO-undegradable Fe+++ form to the HO-degradable Fe++-state (48). Thus, initial input of an electron from NADPH starts the multistep process of HO reaction, oxidatively cleaving the methene-carbon bridges in protoporphyrin tetrapyrrole substrate, which is holding the Fe++. After an initial release of CO and Fe++, biliverdin is formed. Thus, the microsomal HO enzyme system requires concerted activity of microsomal NADPH-P450 reductase, which transfers an electron from NADPH to the heme substrate and also to reduce the O2 for utilization in oxidative cleavage of heme that has entered heme pocket in the HO enzyme protein (see below). For each molecule of heme oxidized, three molecules of O2 and three molecules of NADPH are utilized. Also, for each mole of O2 used in the HO reaction, one mole of H2O is produced and thus, HO is a microsomal-mixed function oxidase (46).
There are at least three distinct isoforms of HO, the (heme-substrate) inducible HO-1 and the constitutively expressed noninducible HO-2 and HO-3 (49). These HO iso-forms are the products of different genes and they have wide differences in amino acid composition (50-52). Despite major differences between HO-1 and HO-2, there is a conserved 24 amino acid segment both in HO-1 and HO-2 (53). This conserved 24 amino acid segment is hydrophobic and forms a pocket that binds the hydrophobic heme substrate, thus constituting the heme pocket (54). This heme pocket does not recognize the metal portion of metalloporphyrins, but has specificity for the side chain of porphyrin ring. Thus, some of the nonphysiological metalloporphyrins like Zn- or Sn-protoporphyrins, which have the same porphyrin side chain as the Fe-protoporphyrin (heme), can compete with heme binding to the pocket and can inhibit the HO activity (55). Both HO-1 and HO-2 proteins are anchored to endoplasmic reticulum by another hydrophobic amino acid sequences present at the carboxyl terminal of these HO proteins and this hydrophobic region is not involved in the catalytic activity (56).
In recent years, physiological role of HO has changed from a simple task of heme degradation to the biological effects of its activity, namely, biliverdin (bilirubin) and CO. During the late 1980s, Ames and coworkers demonstrated that both biliverdin and bilirubin have strong antioxidant properties detoxifying ROS and RNS (42). Also, in the early 1990s, Marks et al. (57) and Snyder and coworkers (58) have suggested that CO mediates vasorelaxation and neurotransmission, respectively. Therefore, all products of HO activity are biologically active; CO is a signal molecule activating gua-nyl cyclase (GC) to enhance the generation of cGMP (59,60), Fe++ downregulates the expression of many genes including that of iNOS (61), and bilirubin (biliverdin) is a potent antioxidant (42,43,62,63). Thus, transgenic mice overexpressing HO-1 and HO-2 were found to have lower activity of lipid peroxidation (64), perhaps due to enhanced degradation of heme and also to the overproduction of bilirubin, the antiox-idant. In addition, these transgenic animals were found to have altered behavioral patterns, perhaps due to abundant production of CO, a retrograde neurotransmitter in brain (65).
Aside from these functions of the products of HO activity, HO system plays a key role in maintaining cellular redox homeostasis by eliminating the toxic-free heme. Thus, HO system protects cells from deleterious effects of free-heme molecule, which is known to be the most effective promoter causing formation of reactive OH* and lipid peroxidation (66). Furthermore, HO is involved not only in the catabolic pathway of disposing toxic heme, but also in the anabolic pathway of producing bioactive molecules like bilirubin pigments and CO, which bring, respectively, the antioxidant and the cGMP-elevating physiologic effects. Thereafter, it was also learned that HO activity could be enhanced not only by heme, the native substrate, but also by various other non-heme agents like endotoxin (LPS), heavy metals, cytokines, mitogens, and hormones (67-74). Indeed, studies have revealed that following induction of HO-1 expression in vascular tissues, there is an increased production of CO and cGMP (75,76). It has also been shown that upregulation of HO-1 expression and consequent overproduction of intracellu-lar bilirubin is associated with protection against the peroxy-nitrite (ONOO)-mediated apoptosis (77), oxidant-dependent microvascular leukocyte adhesion (78), and postischemic myocardial dysfunction (79). In support of this, cells treated with hemin (oxidized heme) had elevated HO-1 expression and bilirubin production and these hemin-treated cells were found to be highly resistant to the cytotoxicity caused by stronger or additional oxidants. High resistance to cytotoxicity was observed only when the cells are producing the bile pigment actively. This strongly implicated that activation or induction of HO-1 pathway overproducing bilirubin provides cytoprotec-tion against the toxicity caused by oxidative stress (62,80). Thereafter, this increase of HO activity was found to be due to de novo synthesis of HO-1 protein and subsequently, the list of HO-1 inducers has been expanded to include many of the heme and nonheme compounds as well as stressful nonspecific physical stimuli. Intense efforts have been made to define the unifying mechanism to explain the induction of HO-1, which is caused by chemically and structurally unrelated diverse agents.
In 1991, Tyrrell and coworkers (81) noted that many known inducers of HO-1 could promote cellular oxidative stress and that induction of HO-1 represented a general cellular response to oxidative stress. Cellular oxidative stress, resulting either from increased production of ROS or from decreased levels of intracellular reductants, appeared to be the common effector mechanism for various inducers of
HO-1 expression. They concluded that induction of HO-1 "reflects a powerful mechanism by which the pro-oxidant state of cells can be transiently reduced in order to avoid cellular damage during a sustained oxidative stress'' (82). In support of this, Nath et al. (83) demonstrated that prior induction of HO-1 protected rats from renal failure and mortality resulting from glycerol-induced oxidative stress called rhabdomyolysis; and the opposite effect was observed upon inhibition of HO-1 activity. Subsequently, many investigators have confirmed the protective function of HO-1 induction in preventing the oxidative injury caused by heme or nonheme insults (62,70,84-86).
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Our internal organs, the colon, liver and intestines, help our bodies eliminate toxic and harmful matter from our bloodstreams and tissues. Often, our systems become overloaded with waste. The very air we breathe, and all of its pollutants, build up in our bodies. Today’s over processed foods and environmental pollutants can easily overwhelm our delicate systems and cause toxic matter to build up in our bodies.