An efficient light-driven P450 BM3 biocatalyst

Article abstract of DOI:10.1021/ja409337v

P450s are heme thiolate enzymes that catalyze the regio-and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

Full text of DOI:10.1021/ja409337v

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An Efficient Light-Driven P450 BM3 Biocatalyst
Ngoc-Han Tran, Daniel Nguyen, Sudharsan Dwaraknath, Sruthi Mahadevan, Garrett Chavez,
Angelina Nguyen, Thanh Dao, Sarah Mullen, Thien-Anh Nguyen, and Lionel E. Cheruzel*
́ ́
Department of Chemistry, San Jose State University, One Washington Square, San Jose, California 95192-0101, United States
S
* Supporting Information
Current strategies to overcome these limitations include the
use of NAD(P)H recycling systems,8 surrogate oxygen atom
donors,9 as well as chemical,10 electrochemical,11 enzymatic,12
and light-activated systems,13 to deliver the reducing equiv-
alents to the heme domain. The most successful examples have
achieved high P450 catalytic activity using cofactor regeneration
systems, fusion P450 systems, and electrochemistry. The vast
majority of these approaches are, however, still dependent on
native or nonphysiological redox partners for high activity. This
dependency is limiting their broader application due to the
specificity of each reductase.12 On the other hand, very few
alternative approaches that use only the P450 heme domain,10
including those using terminal oxidants,9b,11e,f result in efficient
biocatalysts despite successful heme reduction achieved in
certain cases.11a
ABSTRACT: P450s are heme thiolate enzymes that
catalyze the regio- and stereoselective functionalization of
unactivated C−H bonds using molecular dioxygen and
two electrons delivered by the reductase. We have
developed hybrid P450 BM3 heme domains containing a
covalently attached Ru(II) photosensitizer in order to
circumvent the dependency on the reductase and perform
P450 reactions upon visible light irradiation. A highly
active hybrid enzyme with improved stability and a
modified Ru(II) photosensitizer is able to catalyze the
light-driven hydroxylation of lauric acid with total turnover
numbers of 935 and initial reaction rate of 125 mol
product/(mol enzyme/min).
Our laboratory has focused on utilizing the photochemical
properties of Ru(II)−diimine complexes14 covalently attached
to P450 BM3 heme domain mutants to perform selective light-
driven hydroxylation reactions (Figure 1).15 A light-activated
he field of direct C−H functionalization has seen great
Tadvances in recent decades with diverse emerging
approaches,1 including the use of photoredox catalysts,2 to
activate rather inert bonds toward the construction of complex
molecular scaffolds. Nevertheless, selective functionalization of
unactivated C−H bonds using molecular dioxygen as the sole
oxidant still remains one of the hallmarks of catalysis.3 While
few synthetic examples of both homogeneous and heteroge-
neous catalysts are emerging, enzymatic processes continue to
be of considerable importance. In particular, the Cytochrome
P450 superfamily of heme-thiolate enzymes, which catalyze a
myriad of chemical reactions often with high regio- and
stereoselectivity, has received great attention.4 Activation of
molecular dioxygen at the heme center generates a high-valent
Fe(IV)-oxo porphyrin radical species, namely, Compound I,
capable of performing the desired C−H bond functionaliza-
tion.5 Among the large and diverse family of P450 enzymes, the
fatty acid hydroxylase CYP102A1 or P450 BM3 from Bacillus
megaterium shows the highest catalytic hydroxylation rate ever
measured for P450 enzymes.6 Its high catalytic activity is
attributed to the efficient coupling between the delivery of
electrons from the reductase and the activation of oxygen at the
heme center. The singularity of P450 BM3 is that the NADPH-
dependent reductase and the heme domain are fused together
in a single peptide chain. The fused reductase in conjunction
with the high catalytic activity have rendered P450 BM3 an
effective model to study P450 monooxygenase and an
important design platform for biocatalysis.6 However, in
order for P450 BM3 and P450s in general to become valuable
biocatalysts, certain challenges such as the requirement of the
expensive NAD(P)H cofactor and the dependency on redox
partners must be addressed.7
Figure 1. (Top) Schematic representation of the hybrid enzyme
approach vs the natural pathway involving the redox partner (CPR)
and NAD(P)H. (Bottom) Close up of the active site where the Ru(II)
photosensitizer (X = H (1) or OMe (2)) is covalently attached to a
non-native single cysteine (L407C) residue of P450 BM3 heme
domain. Under flash quench reducing conditions (DTC: sodium
diethyldithiocarbamate), the photogenerated Ru(I) species is able to
provide electrons to the heme center in order to activate molecular
dioxygen and carry out the hydroxylation of lauric acid, yielding three
monohydroxylated products.
Received: April 22, 2013
© XXXX American Chemical Society
A
dx.doi.org/10.1021/ja409337v | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

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protein engineering have led to a highly active P450 hybrid
enzyme.
Table 1. Mutations and Half Denaturation Temperatures of
the Heme Domains Used in This Study
The nature of the Ru(II) photosensitizer was modified to
increase the driving force of the electron transfer steps. The
4,4′-dimethoxy-2,2′-bipyridine ligand ((OMe)2bpy) containing
the electron-donating methoxy groups was used instead of the
bipyridine (used in 1). This substitution led to a 80 mV
cathodic shift in the first reduction potential of Ru-
((OMe)2bpy)2Phen (E2+/1+= −1.27 V vs Ag/AgCl) compared
to −1.19 V vs Ag/AgCl for Ru(bpy)2Phen (Figure S3). The
labeled enzymes containing Ru((OMe)2bpy)2PhenA (2) also
show a red-shifted metal-to-ligand charge transfer (MLCT)
absorption and emission18 compared to their counterparts
having the initial photosensitizer (1) as illustrated in Figure 2A.
So far, P450 BM3 heme domain mutants with a single non-
native cysteine residue were engineered in order to control the
position of attachment of the Ru(II) photosensitizer.19 The
library of triple mutants was originally generated where the two
native cysteine residues, C62 and C156, were mutated to
alanine and serine, respectively, and a non-native single cysteine
residue was inserted by site-directed mutagenesis.19 Inves-
tigation into the thermostability of the enzymes revealed that
the triply mutated heme domain, tL407C, has lower thermal
stability (T50 = 52 °C) compared to the wild-type P450 BM3
heme domain (hBM3, T50 = 58 °C) (Table 1).20 This
prompted us to reintroduce the two original cysteine residues
in the L407C mutant in order to regain some of the stability
and catalytic activity of the wild-type heme domain. The C62
residue appears to be involved in hydrogen bonding
interactions with neighboring residues K391 and N395 (Figure
S10) and likely plays a role in the enzyme stability. The
resulting singly mutated sL407C has a T50 of 56 °C, which is
closer to the T50 observed for the hBM3 (Table 1).
a
b
enzymes
mutations
T50 (°C)
hBM3
Wild-type (C62, C156)
L407C
58.1 0.2
56.4 0.1
51.8 0.2
sL407C
tL407C
C62A, C156S, L407C
a
Residues in bold indicate position of photosensitizer covalent
attachment. T50 values are the mean SD of triplicate measurements.
b
system capable of harvesting the synthetic potential of P450
enzymes is of particular interest considering the current efforts
dedicated to the utilization of light energy to drive chemical
reactions. Herein, we report an optimized hybrid enzyme able
to achieve, upon light activation, total turnover numbers of over
900 with initial reaction rate of 125 mol product/(mol enzyme/
min), among the highest values reported for alternative electron
delivery approaches in the hydroxylation of lauric acid.9b,11c,d,13a
Besides the use of light activation to drive P450 reactions, the
hybrid enzyme approach circumvents the use of reductase and
NAD(P)H cofactor (Figure 1).
Briefly, the hybrid enzyme15 can be assembled by attaching
Ru(II)−diimine complexes of general formula Ru(LL)2PhenA,
(LL = bipyridine (bpy) and phenA = 5-acetamido-1,10-
phenanthroline) to a strategically positioned non-native single
cysteine residue (e.g., L407C) of P450 BM3 mutant heme
domains. Under the photocatalytic cycle highlighted in Figure
1,16 the photogenerated Ru(I) is able to provide the necessary
electrons to the heme domain in successive electron transfer
steps and sustain photocatalytic activity. A series of hybrid P450
BM3 enzymes15 was generated by varying the nature and
position of the Ru(II)−diimine photosensitizer to optimize the
electron transfer rate according to the semiclassical Marcus
theory.16,17 The most active tL407C-1 mutant having the
Ru(bpy)2PhenA complex (1) covalently attached to the
tL407C heme domain triple mutant (Table 1) showed modest
total turnover numbers of 140. This L407C label attachment
site has been the focus of our studies since, and changes in the
nature of the photosensitizer as well as optimizations through
The hBM3 and sL407C enzymes were then submitted to the
same labeling reaction conditions. The hBM3 could be singly
labeled in low yield (<10% after extended reaction time) at the
more reactive C156 residue,21 whereas the sL407C is singly
labeled at the preferred L407C position in a much higher
Figure 2. Photophysical properties and catalytic activities of the different hybrid enzymes. (A) UV−vis and luminescence spectra of the different
hybrid enzymes (hBM3, wild-type P450 BM3 heme domain, triply (tL407C) and singly (sL407C) mutated P450 BM3 heme domains; 1,
Ru(bpy)2PhenA; 2, Ru((OMe)2bpy)2PhenA) and model complexes (Ru(bpy)2Phen and Ru((OMe)2bpy)2Phen) at the same concentration. (B)
Plot showing the total turnover numbers (TTN) as mole of hydroxylated products/mole of enzymes for the various hybrid enzymes in the light-
activated hydroxylation of lauric acid. From left to right, the TTN range from 8 and 9 1 for the hBM3-1 and -2, to 140 5 (*, for previously
reported tL407C-1), 438 15 and 548 22 for tL407C-1 and -2; and 856 23, 935 27 for sL407C-1 and -2. Error bars indicate standard
deviations.
B
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Table 2. Summary of the Current Data (TTN and Reaction Rates) On the Alternative P450 BM3 Strategies To Perform the
Selective Hydroxylation of Lauric Acid
a
entry
system
enzyme
total turnover numbers (TTN) initial reaction rate (mol product/(mol enzyme/min))
reference
b
1
2
NADPH
holoBM3
sL407C-2
holoBM3
holoBM3
21B3
n/a
81.1 2.7 - 4100 150
125
6, 22
this work
11c
hν/Ru(II) photosensitizer
935 27
4
3
Co(III) sepulchrate/electrode
835
698
280
224
76
7
37.8 0.3
1.95
c
4
hν/deazaflavin/EDTA
13a
5
H2O2
50
9
6
Cobaltocene/electrode
Co(III) sepulchrate/electrode
H2O2
holoBM3
hBM3
7
16.4 0.6
2.2 0.1
10
11c
7
7
7
11c
8
hBM3
70
9b
9
Cobaltocene/electrode
Pt electrode
hBM3
58
1.8 0.5
110
11c
b
10
holoBM3
n/a
11d
a
b
c
Full-length wild-type enzyme (holoBM3), heme domain (hBM3) and 21B3 heme domain variant. n/a: not available. EDTA:
ethylenediaminetetraacetic acid.
labeling yield (>95%, similar to that observed with the tL407C
mutant). The labeling of the heme domain mutants is verified
by ESI-MS, UV−vis, and luminescence measurements (Figure
2A and Supporting Information). The position of attachment of
the photosensitizer was confirmed by Chymotrypsin digestion
of the hybrid enzymes (Figure S7).
the sL407C-2, the kcat is 130 min−1 and the Km is 199 μM. The
Km values are consistent with other values determined for the
full-length wild-type enzyme with lauric acid.6
P450 BM3 is regarded as the benchmark P450 enzyme with
its high catalytic rates and coupling efficiency largely due to its
fused reductase. A number of alternative approaches have been
developed to mimic such efficient system and artificially drive
P450 BM3 and various other P450s.8−13 As shown in Table 2,
the light activated sL407C-2 hybrid enzyme (entry 2) compares
favorably in terms of TTN and reaction rate with the current
P450 BM3 alternative approaches to hydroxylate lauric acid.
NAD(P)H recycling systems8 and studies using other model
substrates10,11a,b have been omitted from the table for the sake
of direct comparison. The entry 1 of the table is the full-length
wild-type enzyme (holoBM3) with NADPH as source of
reducing equivalents, showing initial reaction rates ranging from
81 to 4100 min−1. Such large discrepancies in the reaction rates
have already been noted elsewhere.6 The other entries (3−10),
listed in decreasing TTN order, include the electrochemical
delivery of electrons via mediators (entries 3, 6, 7, and 9), the
light-activated deazaflavin (entry 4), and the peroxide shunt
(entries 5, 8). In general, low activity is usually observed using
only the heme domain (entries 5, 7−9), and significant activity
improvement is obtained using the full-length enzyme
(holoBM3) with the fused reductase. The peroxide shunt
(entry 8) typically leads to rapid protein degradation, which
had been minimized through protein engineering in the more
stable 21B3 heme domain. To the best of our knowledge, the
observed total turnover numbers (935) and initial reaction rate
(125 min−1) for the sL407C-2 are the highest among the
existing alternative electron delivery approaches, even those
taking advantage of the linked reductase.
In comparison with other alternative P450 systems, the
hybrid enzyme approach offers the advantage of achieving high
catalytic activity upon light activation using only the heme
domain, circumventing the need for redox partners and
NAD(P)H cofactors. This approach has therefore the potential
to be more generally applicable to other P450 isoforms,
especially those lacking a fused reductase. In the hybrid
enzyme, the high initial reaction rates and TTN indicate that
oxygen activation is coupled to electron transfer from Ru(II)
photosensitizers strategically positioned at the proximal heme
side where the reductase is thought to interact (Figure S11).25
In conclusion, improvement in the stability of the hybrid
enzymes with a more native-like structure and a change in
nature of the Ru(II) photosensitizer have resulted in a sL407C-
Significant differences in total turnover numbers (TTN) are
observed (Figure 2B) when exposing a solution of the purified
hybrid enzymes containing excess substrate and reductive
quencher to visible light irradiation (with UV- and IR-cutoff
filters). The TTN are determined as mole of hydroxylated
products per mole of enzymes after 2 h, after which no further
significant turnover was observed. The monohydroxylated
products at subterminal positions (ω1, ω2, ω3) are identified
and quantified, after derivatization, by gas chromatography−
mass spectrometry (GC−MS) analysis. The ratio of hydroxy-
lated products in the photocatalytic reaction is identical to that
observed with the wild-type heme domain and values reported
for NADPH/holoenzyme.6,15 No catalytic turnover is observed
in the absence of visible light irradiation or reductive
quencher.15
Under optimal reaction conditions (1.5 μM hybrid enzymes,
1.5 mM lauric acid, and 100 mM sodium diethyldithiocarba-
mate), the labeled sL407C enzymes display a 100-fold increase
in TTN (Figure 2B) in the photocatalytic hydroxylation of
lauric acid compared to the labeled hBM3 enzymes (856 vs 8
and 935 vs 9 with labels 1 and 2, respectively), emphasizing the
importance of the site of label attachment. The sL407C shows
7-fold higher TTN compared to the previously reported value
for tL407C, which resulted from optimization of the protein
expression conditions23 with a higher heme incorporation (Rz
ratio,24 A418/A280 > 1.5) and greater thermostability of the
heme domain achieved by reinserting the two native cysteines.
In addition, ligand substitution in the photosensitizer resulted
consistently in a 10−25% increase in TTN.
Initial reaction rates were measured as turnover numbers
obtained after 1 min of visible light irradiation and range from
62 min−1 for the tL407C-2 to 125 min−1 for the sL407C-2 at
1.5 mM lauric acid. These catalytic rates are significantly
improved relative to the apparent specific activity reported
previously for the tL407C-1 (∼1 min−1).15b The hybrid
enzymes follow apparent Michaelis−Menten kinetics under
the optimal flash−quench reductive conditions. The kcat and Km
parameters were determined by measuring initial reaction rates
at various substrate concentrations (Figure S9). The kcat for the
tL407C-2 hybrid enzyme is 79 min−1 with a Km of 357 μM. For
C
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V.; Kuznetsova, G. P.; Samenkova, N. F.; Archakov, A. I. J. Inorg.
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2 system able to efficiently catalyze the light-driven
hydroxylation of lauric acid. The use of Ru(II) photosensitizer
in the hybrid enzymes is becoming a valuable approach to
perform P450 reactions upon light activation, expanding the
scope of photocatalytic reactions carried out by enzymatic
systems containing polypyridyl Ru(II) complexes.26
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ASSOCIATED CONTENT
* Supporting Information
■
S
(14) Balzani, V.; Bergamini, G.; Campagna, S.; Puntoriero, F. Top.
Curr. Chem. 2007, 280, 1.
Experimental section; photosensitizers and hybrid enzyme
characterizations (1H NMR, ESI-MS, Chymotrypsin digest,
GC−MS, cyclic voltammetry). This material is available free of
charge via the Internet at http://pubs.acs.org.
(15) (a) Tran, N. H.; Huynh, N.; Bui, T.; Nguyen, Y.; Huynh, P.;
Cooper, M. E.; Cheruzel, L. E. Chem. Commun. 2011, 47, 11936.
(b) Tran, N. H.; Huynh, N.; Chavez, G.; Nguyen, A.; Dwaraknath, S.;
Nguyen, T. A.; Nguyen, M.; Cheruzel, L. J. Inorg. Biochem. 2012, 115,
50.
AUTHOR INFORMATION
Corresponding Author
lionel.cheruzel@sjsu.edu
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was financially supported by the National Institute of
Health (GM095415), the Research Corporation for Science
Advancement. L.E.C. thanks the National Science Foundation
(MRI grant 0923573) and San Jose
of mass spectrometry facilities.
́
State University for the use
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