C O M M U N I C A T I O N S
mechanism.20,21 Consistent with this mechanism, there was no
kinetic deuterium isotope effect (kapp(H)/kapp(D) ) 1.0) when deu-
terated substrate (p-ClC6D4OH) was employed (Figure S4).
The oxygenation of p-cresol (21 mM) by hemocyanin (2.2 ×
10-3 mM) also proceeded catalytically when the reaction was
carried out under aerobic conditions in the same borate buffer
solution (pH 9.0) containing NH2OH (7.0 mM) as an electron donor
at 25 °C.22 The yield of catechol product was 77% based on the
initial O2 concentration (0.25 mM).
In summary, we have found that the oxygen carrier protein
hemocyanin can get the monooxygenase activity when it is treated
with a high concentration of urea (8 M). Oxy-hemocyanin is stable
enough to be examined in the single-turnover reaction under
anaerobic conditions. Thus, the oxygenation reaction of phenols
by oxy-hemocyanin can be followed directly by using an ordinary
UV-vis spectroscopic method (Figure 1). Preliminary kinetic
studies on the single-turnover reaction have suggested that the
reaction mechanism of the phenol-monooxygenation reaction by
oxy-hemocyanin is the same as that of phenolase reaction of
tyrosinase. Effects of urea on the stability and the reactivity of
hemocyanin are now under investigation.
Figure 1. (A) Spectral change observed upon addition of p-cresol (16 mM)
to Octopus hemocyanin (0.17 mM) in 0.5 M borate buffer (pH 9.0)
containing 10% MeOH and 8 M urea at 25 °C under Ar. Inset: Time course
of the absorption change at 348 nm. (B) Plot of Vapp versus [p-cresol].
Table 1. Apparent First-Order Rate Constants for the
Oxygenation of Phenols (p-RC6H4OH) by Oxy-Hemocyanin
1
p-substituent (R)
k
app (s-
)
-OCH3
-CH3
-F
9.0 × 10-4
1.9 × 10-4
3.5 × 10-5
3.8 × 10-5
2.2 × 10-5
4.2 × 10-6
1.1 × 10-6
-Cl
Acknowledgment. This work was financially supported in part
by Grants-in-Aid for Scientific Research from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
-Br
-COOCH3
-CN
Supporting Information Available: Details of the experimental
procedures and the spectral and kinetic data (Figures S1-S5). This
catechol (4-methyl-1,2-dihydroxylbenzene) was obtained in a 74%
yield based on the initial concentration of oxy-hemocyanin (the
product yield of single-turnover reaction under anaerobic condi-
tions). In the absence of urea, the spectral change was much slower,
as shown in Figure S2. These results clearly indicate that the
addition of urea causes not only the dissociation of the subunits
from the supramolecular assembly but also a partial conformational
change of the protein to open a space for the substrate binding as
suggested previously.3,14 Nonetheless, the active site structure of
the (µ-η2:η2-peroxo)dicopper(II) core of oxy-hemocyanin is main-
tained to react with the substrate. This is the first spectroscopic
detection of the monooxygenation reaction of the (µ-η2:η2-peroxo)-
dicopper(II) species in the biological systems.
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corresponding o-quinones. In this system, NH2OH had to be used as an
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The linear dependence of Vapp against the substrate concentration
in the single-turnover reaction (Figure 1B) may indicate that the
binding of the substrate to oxy-hemocyanin is weak. This is
reasonable since hemocyanin is essentially an oxygen carrier
protein, but not an enzyme, thus having no proper substrate-binding
pocket.
To get insight into the oxygenation mechanism of phenols by
oxy-hemocyanin, electronic effects of the phenol substituents (R)
on the reaction rate were examined, as shown in Figure S3. The
plot of log(kapp) against the Hammett σ+ gave a linear correlation,
from which a Hammett F constant was obtained as -2.0. It should
be noted that the F value of the present reaction is very close to
that of the phenolase reaction of mushroom tyrosinase (F ) -2.4).20
This result strongly suggests that the oxygenation of phenols by
oxy-hemocyanin involves the same mechanism as the phenolase
reaction of tyrosinase, that is, an electrophilic aromatic substitution
JA061631H
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J. AM. CHEM. SOC. VOL. 128, NO. 21, 2006 6789