Nitroso-heme complex

In beef cubes thermally processed in water, weak radicals were detectable at room temperature, but were not strong enough to identify or quantitate. At 77K (liquid nitrogen temperature) signals from a nitroso>heme complex with two principal components appeared (Figure 1). The set of three sharp, narrow lines in the center of the spectra is typical of pentacoordinate Fe heme-NO complexes (13,14), while the broad downfield signal with moderate to no fine structure arises from electron interactions with tire heme apoprotein (14 -16). 

Figure 7.13. The spectroscopically detectable metabolic intermediate (Ml) heme complexes formed when some primary amines are oxidized by P450 enzymes involve oxidation of the nitrogen to a nitroso species that coordinates to the iron. The primary amine function can be unmasked by W-demethylation reactions, as is the case in the inhibition of CYP3A enzymes by troleandomycin (TAO) (AcO- in the structure represents CHjCOj). The arrow shows the nitrogen that is involved in the reaetion in TAO.

Figure 6 The structure of the potent quasi-irreversihle CYP3A4 inhibitor troleando-mycin (top panel), the metabolic steps required to convert troleandomycin into a nitroso metabolite that coordinates with the ferrous-heme of CYP3A4 (side panel), and the spectral changes associated with MI complex formation (bottom panel). Troleandomycin (50 pM) was incubated at 37°C with a human liver microsomal sample with high CYP3A4/5 activity (1 mg/mL) andNADPH (1 mM) for the times indicated. The reference cuvette contained the same components minus troleandomycin. Scans from 380 to 520 nm were recorded on a Varian Cary 100 BIO IJV/Vis dual beam spectrophotometer. Abbreviation MI, metabolic intermediate.

This is different when free amines are formed as a result of an N-demethylation reaction. Free amines bind rather strongly to the oxidized and reduced hemoprotein and thus may cause product inhibitionVery stable complexes of different nature with the reduced hemoprotein have been reported after incubation of certain N-alkyl-ated amines of the amphetamine type with liver microsomes Recent studies on the mechanism of formation of these complexes support a mechanism ofN-hydroxyl-ation leading to aliphatic nitroso compounds which form tight complexes with the ferrous heme-sulfur protein... 

Quasi-irreversible inhibition is observed when CYP metabohsm produces an intermediate that can form a stable metabolite-intermediate MI) complex. This is another example of mechanism-based inhibition. Erythromycin is one such quasi-irreversible CYP3A4 inhibitor. Upon didemethylation of its tertiary amine group and subsequent oxidation, the resulting nitroso species forms a tight complex with the Fe(II) atom of the CYP s heme unit. Unhke truly irreversible adducts, such complexes can be broken up, say by oxidation with potassium ferricyanide, but under normal physiological conditions this obviously doesn t happen. 

Time-dependent inhibition defined mainly by mechanism-based inhibition (MBI), which includes CYP suicide inactivation (irreversible inhibition, the more widely studied process) and metabolite-intermediate (MI) complex formation (quasi-irreversible inhibition), is responsible for most clinically significant DDIs (Silverman, 1995 Waley, 1980 Zhou et al., 2005). Suicide inactivation involves the formation of a reactive intermediate that irreversibly inactivates the CYP in the process of catalytic turnover. Quasi-irreversible inhibition occurs when the CYP produces a metabolite (e.g., nitroso intermediate) with the capacity to bind tightly to the CYP heme. TDI (time-dependent inhibition) can be characterized (1) to be dose dependent, (2) to be preincubation time dependent, (3) to have bioactivation of the inhibitor that is required for inactivation of the target enzyme, (4) to have de novo protein synthesis that is required to recover metabolic capacity, and (5) to have potentially slow onset of the effects but be more profound than reversible inhibition. If present, then TDI is the major component of overall enzyme inhibition and frequently leads to clinically relevant DDIs. Table 4.5 contains a list of inhibitors of TDI observed in vitro and in vivo. 

Unlike the MDP-ehcited MI complex discussed above, the alkyl/aryl amine -mediated P450 Ml-complex can be easily disrupted upon oxidation of the P450-ferrous heme to the ferric state with potassium ferricyarride, with consequent reversion of the P450 enzyme to its native resting state. This oxidation-dependent reversal serves as a reliable diagnostic test of both alkyl-arrd aiyl-rritroso MI complex es [231,240]. On the other harrd, in sharp contrast to the alkyl-nitroso... 

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