342
Z. Fu and J. Chen
alternatives. In fact, the existing knowledge of the catalytic cycle of P450 enzymes
has been complemented or supported by quantum chemical calculations.
17.1.2.2 Catalytic Cycle of P450 Enzymes and Related Common
Reaction Genres
P450 enzymes commonly act as monooxygenases that transform exogenous chemicals (e.g., alkanes, RH) into oxidative products and H 2 O using an O 2 molecule and
nicotinamide adenine dinucleotide phosphate (NADPH). The overall reaction can
be formulated as: RH + O 2 + NADPH + H
+
→ ROH + NADP
+
+ H 2 O. Through
more than half a century’s work, much of the mystery in P450 chemistry has been
unveiled, among which the catalytic cycle is the most eye-catching part [4, 8]. As seen
in Fig. 17.3, the catalytic cycle starts from a resting state (1), wherein the heme iron
is hexa-coordinated with H 2 O and cysteine in the axial direction and four porphyrin
nitrogen atoms in the radial direction. This trivalent iron atom is almost coplanar
with the porphyrin ring, rendering the whole system a low-spin doublet state. The
binding water molecule is then expelled as the substrate enters the active site, giving
a pentacoordinate Fe
III complex (2) that transports the iron from the heme plane to
a position below the porphyrin ring. Complex 2 is in a high-spin sextet state that
displays a strong electron-withdrawing capacity. Subsequently, this complex would
receive an electron from NADPH and synchronously bind with one O 2 molecule,
leading to singlet species Fe
III –O–O
− (3). 3 is still a good electron receptor and further
reduced to the anion peroxo Fe
III –O–O
2− species (4) by absorbing a second electron.
4 is a strong Lewis base that readily gets protonated to the Fe
III –O–OH
− species (5,
Compound 0) by capturing a H
+ from the active site. Compound 0 remains a Lewis
base that undergoes a coupling reaction (Coupling-I) via receiving another H
+ from
the enzyme, and produces a H 2 O molecule and high-valent iron-oxo (Fe
IV =O) complex, which is also called Compound I (6). Compound I is accepted as the terminal
oxidant that transforms substrates to oxidized products (SubO). The release of SubO
from the active site and rebinding of H 2 O accomplish the catalytic cycle and return
the system back to the resting state. Overall, P450 enzymes require two electrons,
two H
+ as well as one O 2 to fulfill the catalytic oxidative reactions, with one oxygen
atom incorporated into H 2 O and the other inserted into the substrate.
It deserves mentioning that the mechanism for protonation of Compound 0 is still
controversial. As shown in Fig. 17.3, when the protonation initiates on the distal O
atom, Compound I is yielded concomitant with O–O bond scission; otherwise, an
iron hydrogen peroxide Fe
III (O 2 H 2 ) complex (8) is produced when the protonation
takes place on the proximal O atom. This Fe
III (O 2 H 2 ) complex is susceptible to an
uncoupling reaction by losing H 2 O 2 and returning back to 2 in the case that H 2 O 2
binds loosely with the iron. In a similar way, the resting state 1 can be reactivated to 8
via a shunt pathway. When H 2 O 2 binds tightly with Fe in complex 8 (e.g., stabilized
by the surrounding residues via hydrogen bonding), the enzyme takes on another
coupling reaction scenario (Coupling-II) wherein one H 2 O is detached [9] and gives
rise to Compound I.
Z. Fu and J. Chen
alternatives. In fact, the existing knowledge of the catalytic cycle of P450 enzymes
has been complemented or supported by quantum chemical calculations.
17.1.2.2 Catalytic Cycle of P450 Enzymes and Related Common
Reaction Genres
P450 enzymes commonly act as monooxygenases that transform exogenous chemicals (e.g., alkanes, RH) into oxidative products and H 2 O using an O 2 molecule and
nicotinamide adenine dinucleotide phosphate (NADPH). The overall reaction can
be formulated as: RH + O 2 + NADPH + H
+
→ ROH + NADP
+
+ H 2 O. Through
more than half a century’s work, much of the mystery in P450 chemistry has been
unveiled, among which the catalytic cycle is the most eye-catching part [4, 8]. As seen
in Fig. 17.3, the catalytic cycle starts from a resting state (1), wherein the heme iron
is hexa-coordinated with H 2 O and cysteine in the axial direction and four porphyrin
nitrogen atoms in the radial direction. This trivalent iron atom is almost coplanar
with the porphyrin ring, rendering the whole system a low-spin doublet state. The
binding water molecule is then expelled as the substrate enters the active site, giving
a pentacoordinate Fe
III complex (2) that transports the iron from the heme plane to
a position below the porphyrin ring. Complex 2 is in a high-spin sextet state that
displays a strong electron-withdrawing capacity. Subsequently, this complex would
receive an electron from NADPH and synchronously bind with one O 2 molecule,
leading to singlet species Fe
III –O–O
− (3). 3 is still a good electron receptor and further
reduced to the anion peroxo Fe
III –O–O
2− species (4) by absorbing a second electron.
4 is a strong Lewis base that readily gets protonated to the Fe
III –O–OH
− species (5,
Compound 0) by capturing a H
+ from the active site. Compound 0 remains a Lewis
base that undergoes a coupling reaction (Coupling-I) via receiving another H
+ from
the enzyme, and produces a H 2 O molecule and high-valent iron-oxo (Fe
IV =O) complex, which is also called Compound I (6). Compound I is accepted as the terminal
oxidant that transforms substrates to oxidized products (SubO). The release of SubO
from the active site and rebinding of H 2 O accomplish the catalytic cycle and return
the system back to the resting state. Overall, P450 enzymes require two electrons,
two H
+ as well as one O 2 to fulfill the catalytic oxidative reactions, with one oxygen
atom incorporated into H 2 O and the other inserted into the substrate.
It deserves mentioning that the mechanism for protonation of Compound 0 is still
controversial. As shown in Fig. 17.3, when the protonation initiates on the distal O
atom, Compound I is yielded concomitant with O–O bond scission; otherwise, an
iron hydrogen peroxide Fe
III (O 2 H 2 ) complex (8) is produced when the protonation
takes place on the proximal O atom. This Fe
III (O 2 H 2 ) complex is susceptible to an
uncoupling reaction by losing H 2 O 2 and returning back to 2 in the case that H 2 O 2
binds loosely with the iron. In a similar way, the resting state 1 can be reactivated to 8
via a shunt pathway. When H 2 O 2 binds tightly with Fe in complex 8 (e.g., stabilized
by the surrounding residues via hydrogen bonding), the enzyme takes on another
coupling reaction scenario (Coupling-II) wherein one H 2 O is detached [9] and gives
rise to Compound I.