PRINCIPLES OF TOXICOLOGY

66 BIOTRANSFORMATION: A BALANCE BETWEEN BIOACTIVATION AND DETOXIFICATION
Cytochrome P450 is a collective term for a group of related hemoproteins, all with a molecular
weight (MW) around 50,000 daltons, which as will be seen later, differ in their substrate selectivity
and in their ability to be induced and inhibited by drugs and chemicals (Table 3.4).
Cytochrome P450–catalyzed oxidations are categorized by the nature of the atom that is oxidized
(see Figure 3.8). Subsequent to the oxidation, the oxygen atom from molecular oxygen may be retained
within the major fragment of the chemical or it may be eliminated by molecular rearrangement (e.g.,
O and N dealkylations).
Whatever the atom oxidized, or the name given to the reaction, the cytochrome P450–mediated
oxidation involves the same cyclic three-step series (Figure 3.9).
Step 1. The xenobiotic [X] first binds to the cytochrome at a substrate binding site on the protein and
alters the conformation sufficiently to enable the efficient transfer of electrons to the heme from
NADPH via a nearby (see Figure 3.6) flavoprotein, NADPH cytochrome P450 reductase. (The
activity of this FAD- and FMN-containing flavoprotein is often determined experimentally using
exogenously added mitochondrial cytochrome c rather than microsomal cytochrome P450 as the
electron acceptor and so is often identified as NADPH cytochrome c reductase). The conformational
change can sometimes be seen in vitro (in the absence of electron transfer) as an alteration of the
heme from a low-spin to a high-spin state, which results in a blue shift in the absorbance maximum
of the hemoprotein. The gain at 390 nm and loss at 420 nm, when seen by difference spectroscopy,
is termed a type I binding spectrum (not to be confused with phase I metabolism).
Step 2. The reduction of the heme iron from its normal ferric state to the ferrous state allows a
molecule of oxygen (O–O) to bind (the binding of CO rather than oxygen to ferrous cytochrome
P450 in the in vitro situation provides a characteristic absorbance maximum around 450 nm, which
gives this cytochrome its name).
Step 3. The ternary complex of xenobiotic, cytochrome, and oxygen receives another electron, either
through the same flavoprotein as before or through an alternative path involving a different
flavoprotein in which the electron is first passed through cytochrome b5, another cytochrome
present in the endoplasmic reticulum (see Figure 3.6). This alternate pathway for the second electron
can also use NADH as the pyridine nucleotide electron donor. The addition of the second electron
to the ternary complex results in a eventual splitting of the molecular oxygen, one atom of which
oxidizes the chemical, the other atom picking up protons to form water, returning ferric cytochrome
P450 to repeat the cycle.
Flavoprotein-catalyzed oxidations differ from cytochrome P450–catalyzed oxidations in mechanism
and in substrate selectivity. For the flavoproteins (a 65,000-dalton protein containing only FAD), the
enzyme forms an activated oxygen complex (“cocked gun”) and the addition of a metabolizable
chemical discharges this, in the process of becoming oxidized. The electrophilic oxygenated species
attacks nucleophilic centers. A wide range of chemicals can thus be metabolized by this flavoprotein;
the important feature for metabolism being a heteroatom (nitrogen, sulfur) presenting a lone pair of
electrons (Table 3.5).
Some compounds are metabolized both by flavin-containing monooxygenases and cytochrome
P450 but to different products. An example is dimethylaniline, which is metabolized to the N-oxide
by the flavoprotein and is N-demethylated by cytochrome P450.
Nonmicrosomal Oxidations in other subcellular organelles can be catalyzed by flavoproteins (e.g.,
monoamine oxidase in mitochondria) or pyridine nucleotide linked dehydrogenases (e.g., alcohol and
aldehyde dehydrogenases in cytoplasm).
Dehydrogenase-catalyzed oxidations do not involve molecular oxygen. The oxidation of the
chemicals or drugs occurs through electron transfer to a pyridine nucleotide, usually NAD + . Most of
the dehydrogenases are cytoplasmic in location. The most noteworthy of this class of enzymes in
humans is the dehydrogenase responsible for the metabolism of ethanol. In contrast to the major