Nanohybrids composed of quantum dots and cytochrome P450 as photocatalysts

Article abstract of DOI:10.1002/anie.200503084

(Chemical Equation Presented) Synthesis with light: CdS quantum dots (QDs) generate superoxide and hydroxyl radicals upon UV irradiation in aqueous solution. The radicals are used for activating P450_BSβ enzymes attached at the QD surface, effecting the catalytic transformation of myristic acid into α- and β-hydroxymyristic acid (see picture, R = (CH ₂)₁₀CH₃).

Full text of DOI:10.1002/anie.200503084

Communications
Photocatalysis
DOI: 10.1002/anie.200503084
Nanohybrids Composed of Quantum Dots and
Cytochrome P450 as Photocatalysts**
Binil Itty Ipe and Christof M. Niemeyer*
Semiconductor nanomaterials, such as quantum dots (QDs),
quantum rods (QRs), and nanotubes, have potentials in a
[
1]
wide variety of fields ranging from nanoelectronics and
[
2–7]
nanophotonics to the broad area of nanosensing
bioimaging.
and
[
8,9]
Moreover, the unusual physical and chemical
properties of nanomaterials resulting from the spatial con-
finement of electrons have made them attractive candidates
[
10]
for the fabrication of new devices in energy technology. For
example, the absorption of light and the occurrence of
electron-transfer reactions at the semiconductor/liquid inter-
face have been investigated in detail for the design of solar
[
11]
cells. In addition, the photocatalytic activity of semicon-
ductor QDs has been a subject of intense research ever since
[
12]
their initial description, and organic reactions carried out
[
13–17]
with QDs as photosensitizers have also been reported.
[
*] Dr. B. I. Ipe, Prof. Dr. C. M. Niemeyer
Biologisch-Chemische Mikrostrukturtechnik
Fachbereich Chemie, Universität Dortmund
Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
and
ISAS – Institute for Analytical Sciences
Bunsen-Kirchhoff-Strasse 11, 44139 Dortmund (Germany)
Fax: (+49)231-755-7082
E-mail: christof.niemeyer@uni-dortmund.de
[
**] This work was supported bythe Deutsche Forschungsgemeinschaft
and the Alexander von Humboldt Foundation (research fellowship
for B.I.I.). We thank Dr. Isamu Matsunaga for the generous donation
of the plasmid pQE-30tBSb for overexpression of recombinant
P450, Andreas Arndt for help with the overexpression, and Dr.
Ljiljana Fruk for help setting up the HPLC assay.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Currently, nanotechnology and biotechnology are being
joined to develop novel materials and devices, leading to the
measured by means of FRET-quenching studies (FRET=
fluorescence resonance energy transfer) between CdS
(donor) and the heme enzyme (acceptor). Defined volumes
of 6 ” -His-tagged P450BSb were titrated against a stock
solution of CdS QDs, and the change in fluorescence intensity
at 550 nm was monitored. A similar method was recently used
to establish the interactions between CdSe QDs and cyto-
[
18]
establishment of the new field of nanobiotechnology. In this
context, we report here on the photoactivated organic
transformation catalyzed by hybrid devices composed of
semiconductor nanoparticles and the enzyme cytochrome
P450BSb. P450BSb belongs to the broad class of monooxygenase
enzymes which are well known to catalyze a range of
stereospecific and regioselective oxygen-insertion reactions
[
27]
chrome c. The concentration of P450BSb was determined by
[
28]
means of CO differential absorption studies, whereas the
concentration of CdS QDs was calculated from inductively
[
19]
into organic compounds. In particular, P450BSb catalyzes the
[
24]
hydroxylation of long-chain fatty acids at the a and b
coupled plasma mass spectrometry. Figure 2A shows the
[
20]
positions using hydrogen peroxide as an oxidant.
The choice of this hybrid system was based, on the one
hand, on the knowledge that radical species are involved in
the catalytic cycle of P450-mediated oxygenation reac-
[
21–23]
tions
and, on the other hand, on the results of previous
studies showing that the irradiation of CdS QDs leads to the
À
+
formation of excitons (e and h ), which generate superoxide
À
[24–26]
(
O C ) and hydroxyl (OHC) radicals in aqueous solutions.
2
We speculated that the superoxide and hydroxyl radicals
could activate the enzymes adsorbed at the QD surface,
thereby inducing specific monooxygenation of a suitable
substrate. Such a photoactivation of enzymes could have
significant advantages over conventional activation of P450BSb
enzymes using H O , because the light-induced reaction
2
2
would offer a much higher degree of control for the on/off
switching of chemical reactivity than that possible in chemi-
cally initiated reactions.
To prepare the QD/P450BSb nanohybrids, recombinant
[
21]
P450BSb enzyme containing a 6 ” His tag at its C-terminus,
was overexpressed in E. coli and purified by nickel nitrilo-
acetate (Ni-NTA) chromatography. CdS nanoparticles
approximately 3 nm in diameter capped with a layer of
mercaptoacetic acid were prepared in reverse-micellar
Figure 2. A) Photoluminescence quenching of CdS in the presence of
[
24]
medium, as previously described.
The P450BSb enzyme
different concentrations of the 6”His-P450BSb enzyme. [CdS]/[P450BSb
ratio: a) 1:0, b) 1:1, c) 1:2, d) 1:3, e) 1:4, f) 1:5, g) 1:6, h) 1:7, i) 1:8.
B) Luminescence quenching at 540 nm upon bioconjugate formation
at increasing P450BSb/CdS ratios (data normalized against unconju-
gated QDs).
]
was attached to the CdS QDs by means of the electrostatic
interaction between the positively charged hexahistidine tail
of the enzyme and the negatively charged mercaptoacetic acid
ligand shell of the CdS QDs (Figure 1).
For the preparation of the QD/P450BSb hybrid materials,
the number of P450BSb molecules attached per QD was
fluorescence spectra of QD/P450BSb nanohybrids. Each spec-
trum represents QDs mixed with variable molar equivalents
of P450BSb. The decrease in fluorescence emission of CdS QDs
at 550 nm upon titration with P450BSb is shown in Figure 2B. It
is evident that the continuous quenching of QD fluorescence
ceases at approximately six molar equivalents of the P450BSb
per QD. This surface coverage correlates well with theoretical
considerations based on the size of the QDS and the enzyme
(see the Supporting Information). The slight decrease in
fluorescence observed on further increase of P450BSb concen-
tration might be attributed to solution-based quenching
[
29]
effects.
In an initial demonstration of the QD/P450BSb nano-
hybrid-mediated photocatalysis we chose the well-established
[
21]
hydroxylation of myristic acid as a model reaction, and we
investigated whether hydroxymyristic acid products are
formed by using HPLC and ESI-LCMS analysis. To this
end, myristic acid (8 mm) in EtOH was added to the P450BSb
/
Figure 1. Monooxygenation of myristic acid (R=(CH ) CH ) using
2
10
3
P450BSb/QD nanohybrids.
QD hybrid solution (20 mm), and the mixture was irradiated
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Communications
for 20 minutes at room temperature. The reaction mixture
was extracted with chloroform, and the products were
analyzed by LC/ESI-MS. This analysis revealed the character-
+
istic mass peak of hydroxymyristic acid ([MÀH ] = 243.09,
calculated M = 244.37, see the Supporting Information). In
contrast, no product peaks were observed in control experi-
ments with: 1) pure QDs in the absence of P450BSb; 2) with
pure P450BSb in the absence of QDs; and 3) without irradi-
ation.
We could also observe subtle changes in the optical
absorption of P450 in the presence of the myristic acid
substrate. An approximately 5-nm blue shift of the 417-nm
absorption of the P450BSb attached to the CdS QD was
observed (Figure S5B in the Supporting Information), and
P450BSb alone also showed an ꢀ 3-nm blue shift in the
presence of myristic acid (Figure S5A). As reported earlier,
this blue shift is indicative of the binding of the substrate to
[
22,30]
the active site of P450.
Although substrate binding is not
[
31]
a prerequisite for electron transfer,
the spectroscopic
results together with the nonformation of hydroxymyristic
acid products in the absence of P450BSb provides evidence that
the myristic acid is bound to the active site of the P450BSb in
the CdS/P450BSb nanohybrids.
To further elucidate this system, the product yields of
similar reactions were determined systematically using a
standardized assay which included esterification of the fatty
acids and their subsequent HPLC analysis using hydroxylau-
[
21]
ric acid as an internal standard for quantification (For a
detailed protocol, see the Supporting Information.) Various
experiments were carried out under different irradiation
times, as well as different surface coverages of the CdS QDs
with P450BSb. The results indicated that both a- and b-
hydroxymyristic acid isomers are formed in approximately
equal amounts in the P450BSb/CdS photocatalysis, in analogy
to the hydrogen peroxide initiated reaction. As expected,
the amount of hydroxylation products increased with increas-
ing amounts of P450 per CdS (Figure 3A) as well as
increasing irradiation time (Figure 3B).
To further investigate the P450 enzyme activity, controls
were carried out with a binary system composed of CdS QDs
and P450BSb without the 6 ” -His tag. Under standardized
conditions, the system produced similar amounts of the
hydroxylation products, as determined by the HPLC assay
Figure 3. Identification of monooxygenation products by HPLC. Elution
profile of myristic acid and its metabolites produced from QD/P450BSb
hybrids: A) with different CdS/P450BSb ratios: a) 1:1, b) 1:2, c) 1:3,
d) 1:4. e) 1:5; B) variation of irradiation times: a) 1 min, b) 2 min,
c) 5 min, d) 10 min. The insets show complete chromatograms. The
[
20]
left part of the split product peak (t =11.5 min) corresponds to the b
R
isomer and the right part (t =11.9 min) to the a isomer of hydroxyl-
R
myristic acid. The peak at t =13.6 min corresponds to myristic acid.
R
The broad shoulder (t =10.8 min) is an HPLC artifact resulting from
R
large injection volumes (25 mL), see also Figure S6.
the enzyme molecules might occur during the adsorption and
purification steps. Nonetheless, despite this reduction in
turnover, the P450BSb/CdS nanohybrids were capable of
producing similar amounts of hydroxylation products as the
native enzyme. Therefore, these results suggest that the close
(
see Figure S7). Hence, it appeared questionable whether the
close proximity of the two components in the P450BSb/CdS
nanohybrids is, indeed, beneficial for the enzymatic activity of
this system. To estimate proximity effects, we compared the
turnover of the P450BSb/CdS nanohybrids with that of the
native enzyme activated with H O . The turnover of the
proximity between the enzymes and the QDs in the P450BSb
/
CdS nanohybrids improves the overall catalytic power over
that of the system containing the two components in
separated form.
To further investigate the P450BSb/CdS photocatalysis,
hydroxylation experiments were carried out in the presence
of superoxide dismutase (SOD) enzyme. SOD effectively
2
2
À1
P450BSb/CdS system was found to be 61 min , while that of
P450 with H O was 122 min . The value is even about six
À1
2
2
times less than that reported for a similar P450
catalysis
BSb
À1
(
363 min ), and therefore our results indicate that the
activity of the P450BSb in the nanohybrids is significantly less
than that of the native enzyme. This might be a consequence
of the negative effects of the attached nanoparticles on the
enzymeꢀs catalytic performance, such as the deceleration of
diffusion processes or the restriction of conformational
degrees of freedom. Alternatively, (partial) denaturation of
decomposes superoxide radicals, and we had previously used
À
this activity to prove the identity of O C species produced by
2
[
24]
CdS QDs. With the P450BSb/CdS system investigated here,
we observed a decrease of the hydroxymyristic acid products
formed to approximately 20% chemical yields in the presence
of SOD (see Figure S8 in the Supporting Information).
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Angewandte
Chemie
Similar experiments carried out with the two-component
system, that is, the mixture composed of CdS QDs and
P450BSb without the His tag, revealed a decrease of the
hydroxymyristic acid products to approximately 10% chem-
ical yields. These results, again, hint at beneficial proximity
effects in the P450BSb/CdS nanohybrids. Apparently, the close
distance between the QD and the P450 reduces the SOD-
mediated degradation of superoxide radicals. Nonetheless,
the results also suggest that the diffusion of radicals and their
decomposition by SOD occurs at faster rates than the uptake
and conversion of the radicals by the P450 enzyme.
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In conclusion, we here report the design of quantum dot/
enzyme nanohybrids that are capable of catalyzing an organic
transformation through photoactivation of the QDs. The
À
+
À
[
[
[
photogenerated excitons (e and h ) produce O C and/or OHC
2
radicals, which in turn activate the P450 enzymes to catalyze
monooxygenation of fatty acid substrates. In our example
myristic acid is hydroxylated to give a- and b-hydroxymyristic
acid. Although we could clearly prove the chemical compo-
sition and the catalytic activity of the novel P450BSb/CdS
nanohybrids, the characterization of their detailed kinetic
properties will require more sophisticated studies. Hence,
further work will focus on potential proximity effects that
might arise owing to the close distance between the enzyme
molecules and the surface of the radical-generating nano-
particle. In general, the light-induced triggering of enzyme
activity confers explicit advantages over chemical initiation
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[
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In
[
[
addition to the investigation of other semiconductor QDs for
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currently under way, we are also aiming towards applications
as photocatalysts in synthetic organic chemistry and as
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irradiation for 20 minutes led to neither precipitation nor to a
decrease in enzymatic turnover. However, our efforts of reusing
the nanohybrids for an additional round of catalysis have been
unsuccessful so far, probably as a result of enzyme denaturation
during the workup and extraction procedures.
Received: August 30, 2005
Revised: October 14, 2005
Published online: December 19, 2005
Keywords: nanostructures · photochemistry· quantum dots ·
.
radicals
[
[
[
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Angew. Chem. Int. Ed. 2006, 45, 504 –507
ꢀ 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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507