MECHANISMS OF CHOSEN ENZYME REACTIONS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 103

observe the active oxidizing species of cytochrome P450cam, the reaction of superoxide
with the enzyme ferrous form was monitored by employing the stop-flow spectral
technique (Kobayashi et al., 1994). The intermediate spectrum was found to be quite
different from charcteristic spectra of compound I of horseradish peroxidase, or
intermediate products of reaction of ferric cytochrome P450cam with peracetic acid or
iodobenzene.
The enzyme species of at temperature 100 K with the dioxygen, substrate
and second electron were produced by x-ray radiolysis of water, which is one of the
triggering methods in crystallographic enzymekinetics, (Schlichtich and Goody 997).

The X-ray analysis of the radiolysis product suggested that O-O bond cleavage had
occurred, leaving a single atom on the heme iron (Schlichtich et al., 2000). This
conversion is not complete. Nevertheless, the electron density in the species was found
to be similar to that observed by the time-resolved x-ray diffraction studies of the
compound I intermediate in cytochrome c peroxidase and catalase (Groves and
Subramanian, 1984). Other changes which may be important for the enzyme catalytic
mechanism are the move of the camphor molecule by about 0.2 Å towards the heme iron
and the appearance of a new water molecule close to the oxyferryl oxygen which might
be leaving water molecules produced after the O-O bond scission. After warming the
radiolitically treated crystal, its electron density was found to be consistent with that for
the product complex 5-exo-hydroxycamphor (Poulos et al., 1985).
The EPR and ENDOR spectroscopy was used for studies of catalytic intermediates in
native and mutant cytochrome P450cam in cryogenic temperatures (6 and 77K)
(Davydov et al., 2001). The ternary complex of camphor, dioxygen, and ferrous-enzyme
was irradiated with to inject the second electron. This process showed that the
primary product upon reduction of the complex is the end -on intermediate. This species
converts even at cryogenic temperatures to the hydroperoxo-ferriheme form and after
brief annealing at a temperature around 200 K, causes camphor to convert to the product.
In spite of conclusions derived from x-ray analysis (Schlichtich et al., 2000) no
spectroscopic evidence for the buildup of a high-valance oxyferryl/porphyrin
radical intermediate during the entire catalytic circle has been obtained.
Freeze-quenching technique in combination with ESR and Mossbauer spectroscopy
was used for monitoring intermediates in the reaction of substrate free with
peroxy acetic acid (Schünemann et al., 2000). In such a condition, the oxidant oxidized
the enzyme active site iron (III) to iron (VI) and Tyr 96 into tyrosine radical, 90% and
10% from the starting material, respectively. Thus the tyrosine residue may be involved
in the catalytic process.
The kinetic methods and analysis of products can provide valuable information about
mechanisms of the cytochrome P450 reactions. According to the pioneering works of the
Groves group (Groves and McGlusky, 1976; Groves, 1985 and references therein) the
observed kinetic isotope effect (KIE) is large: for benzylic and aliphatic
hydroxylation. This observation was confirmed in kinetics studies of various systems. In
one instance a large intramolecular KIE was observed for flour derivative of camphor
(Sono et al., 1986; and references therein). The experimental KIF was attributed to the
Groves rebound mechanism in which the iron-oxo species abstracts an H atom from
substrate to give an iron-hydroxo species and an alkyl radical, followed by

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