Light-driven electron injection from a biotinylated triarylamine donor to [Ru(diimine)3]2+-labeled streptavidin

Article abstract of DOI:10.1039/c6ob01273f

Electron transfer from a biotinylated electron donor to photochemically generated Ru(iii) complexes covalently anchored to streptavidin is demonstrated by means of time-resolved laser spectroscopy. Through site-selective mutagenesis, a single cysteine res

Full text of DOI:10.1039/c6ob01273f

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Light-driven electron injection from a biotinylated
triarylamine donor to [Ru(diimine)3]2+-labeled
streptavidin†
Cite this: DOI: 10.1039/c6ob01273f
Received 13th June 2016,
Accepted 7th July 2016
Sascha G. Keller,a Andrea Pannwitz,b Fabian Schwizer,a Juliane Klehr,a
DOI: 10.1039/c6ob01273f
Oliver S. Wenger*b and Thomas R. Ward*a
www.rsc.org/obc
Electron transfer from a biotinylated electron donor to photochemi- genetic optimization can lead to catalytic performance which
cally generated Ru(III) complexes covalently anchored to streptavidin is superior to that observed for the related small metal catalyst
is demonstrated by means of time-resolved laser spectroscopy. outside the protected environment of the biotin binding
Through site-selective mutagenesis, a single cysteine residue was pockets of Sav, manifesting for example in unusually high
engineered at four different positions on streptavidin, and a Ru(II) enantioselectivity.4
tris-diimine complex was then bioconjugated to the exposed
In principle, such artificial metalloenzymes should also be
cysteines. A biotinylated triarylamine electron donor was added to amenable to the emerging field of redox photocatalysis,5 and
the Ru(II)-modified streptavidins to afford dyads localized within a for this purpose it would be desirable to equip the biotin–
streptavidin host. The resulting systems were subjected to electron streptavidin systems with redox photosensitizers. Complexes of
transfer studies. In some of the explored mutants, the photo- d6 metal ions such as ruthenium(II) and iridium(III) are good
triggered electron transfer between triarylamine and Ru(III) is com- candidates in this regard.
plete within 10 ns, thus highlighting the potential of such artificial
There have been several studies of the luminescence pro-
perties of biotinylated d6 metal complexes, including ruthe-
nium(II) polypyridines,6 rhenium(I) tricarbonyl diimines,7 and
cyclometalated iridium(III) complexes.8 In most cases, binding
to streptavidin leads to enhanced luminescence properties,
and this is important for example for cell imaging purposes.9
The above mentioned classes of metal complexes are well
suited for photosensitization of electron transfer reactions,10
but, to the best of our knowledge, this has not yet been rea-
lized within the context of the biotin–streptavidin techno-
logy.11 However, ruthenium(II) and rhenium(I) complexes have
been employed frequently for investigation of phototriggered
electron-transfer in other proteins (e.g., azurin, cytochrome c,
or plastocyanin), mostly for distance-dependence studies and
for elucidating electron tunneling pathways.12 The ruthenium(II)
and rhenium(I) metal centers were commonly ligated to histi-
dine ligands for this purpose, and the natural redox-active
groups of these proteins (e.g., blue copper centers or heme
groups) served as electron-transfer reaction partners.
Typical photoredox systems usually require a photosensi-
tizer combined with a distinct catalytic moiety. As the binding
of biotinylated probes to Sav is non-cooperative, introducing
two distinct dyads within a streptavidin tetramer remains an
unmet challenge.13 To assemble such dyads using a Sav
scaffold, we thus opted to bioconjugate the photosensitizer to
an engineered cysteine residue, thus leaving the four biotin-
binding sites of Sav free to introduce either a catalytic or an
electron-donor moiety.
metalloenzymes to perform photoredox catalysis.
Introduction
Streptavidin (hereafter Sav) is an exquisitely robust protein of
ca. 64 kDa comprised of four homologous sub-units, each of
which can bind a biotin molecule with an association constant of
∼1014 M−1 under physiological conditions.1 Sav can readily be
over-expressed and purified from the bacterium Escherichia coli.
Owing to this combination of favorable properties, biotin–strepta-
vidin systems have been exploited in various contexts where
host–guest recognition is desirable, ranging from targeted drug
delivery, to live cell imaging and new applications in catalysis.2
By biotinylation of small metal catalysts, novel catalytic
functions can be conferred to the resulting biotin–streptavidin
assemblies, resulting in artificial metalloenzymes.3 Their func-
tion can be tailored either chemically, i.e., through variation of
the biotin-linker-catalyst moiety, or genetically through site-
specific mutagenesis of Sav. In the best case, such chemo-
aDepartment of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 Basel,
Switzerland. E-mail: thomas.ward@unibas.ch
bDepartment of Chemistry, University of Basel, St. Johanns-Ring 19, CH-4056 Basel,
Switzerland. E-mail: oliver.wenger@unibas.ch
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c6ob01273f
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Fig. 2 Surface display representation of homotetrameric streptavidin,
highlighting the symmetry related positions selected for introducing
cysteine residues: K121 (orange), S112 (yellow), R84 (blue), T66 (green)
and biotin (red stick representation); (pdb code: 3PK2).15
Fig. 1 Chemical structures of the ruthenium(II)-label for streptavidin 1,
the Biot-TAA moiety 2 (biotin is displayed in blue) and the TAA-Ac refer-
ence molecule 3. See ESI† for syntheses.
Inspection of the X-ray structure of S112A Sav (pdb code:
3PK2)15 led us to select four positions for the introduction of a
cysteine residue by site-directed mutagenesis. The following
four mutants were produced using a previously published
method and purified by affinity chromatography16 and charac-
terized by ESI-MS: Sav T66C, R84C, S112C, and K121C (Fig. 2).
Subsequent bioconjugation was achieved by mixing the
mutant protein overnight with an eightfold excess of Ru-
complex 1 at 4 °C in phosphate buffer (50 mM, pH 7) in the
dark. The excess of the unbound Ru-complex 1 was removed
by dialysis and the resulting Ru4Sav bioconjugates (Ru4Sav
T66C, Ru4Sav R84C, Ru4Sav S112C, and Ru4Sav K121C respect-
ively) were characterized by ESI-MS, revealing a quantitative
conversion to the Ru-containing proteins (see ESI† for details).
The biotin-binding capacity of the Ru4Sav bioconjugates was
assessed relying on a displacement titration using HABA (2-(4-
hydroxyphenylazo)benzoic acid). Upon incorporation within
Sav, HABA displays an absorption at λmax 506 nm. Upon
addition of biotinylated probes, the HABA is displaced,
leading to a disappearance of the absorption at 506 nm.4a,17
This simple procedure revealed that all four biotin-binding
As Sav possesses no cysteine residue, site-directed muta-
genesis was used to engineer this highly nucleophilic residue
at selected positions. The resulting single-point mutants were
subsequently bioconjugated with a [Ru(bpy)2(phen)]2+-photo-
sensitizer 1 (Fig. 1, bpy = 2,2′-bipyridine, phen = 1,10-phenan-
throline). The triarylamine (TAA) unit was equipped with a
biotin group to yield Biot-TAA (see ESI† for details, 2 displayed
in Fig. 1). The resulting Biot-TAA/Ru(II)-streptavidin adducts
are donor–acceptor systems in which phototriggered electron
transfer can be investigated. The goal of this work was to
explore whether phototriggered electron injection from a bio-
tinylated donor to ruthenium(II)-labeled streptavidin is possible
and if so, what position on the surface of Sav is most suitable for
the covalent anchoring of the photosensitizer.
Results and discussion
Structural and synthetic aspects
The ruthenium(II) complex (rac-1 displayed in Fig. 1) was co- sites can accommodate the biotinylated cofactor Biot-TAA, and
valently tethered to streptavidin through nucleophilic attack by the affinity is similar to that of pure unmodified biotin (see
a cysteine residue with concomitant elimination of HBr.14 Fig. 3, more details in the ESI†). This confirms that the bulky
Single cysteine residues were introduced by site directed muta- Biot-TAA moieties bind to Ru4Sav isoforms, despite the pres-
genesis on each of the four homologous units of streptavidin, ence of the bulky Ru(bpy)2(phenNHCOCH2) moieties which,
leading to a fourfold Ru(II)-labeling of each streptavidin tetra- in the S112C and K121C mutants, lie in the proximity of the
mer. The biotinylated triarylamine unit Biot-TAA was syn- biotin-binding vestibule.
thesized as
a
binding partner. The acetyl-substituted
Since no crystal structure could be obtained so far, through-
triarylamine (TAA-Ac) 3 served as a reference compound space distances were estimated based on a previously pub-
(Fig. 1). Syntheses and mutagenesis procedures are presented lished crystal structure of an Ir-loaded streptavidin mutant
in the ESI.†
(pdb code: 3PK2).15 The arylated biotin moiety bearing a para-
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Fig. 3 HABA displacement titrations of biotin (black symbols) and the biotinylated triarylamine Biot-TAA (red symbols) for (a) WT streptavidin and
(b) K121C-Ru (see ESI† for experimental details).
sulfur atom was used for distance estimation. This latter sulfur
atom was selected as a surrogate for the nitrogen atom of the
Biot-TAA moiety (Fig. S16†). The shortest through-space dis-
tances from the sulfur atom to the α-carbon of the amino
acids targeted for mutagenesis ranges from 7.1 to 22.1 Å
(Table 1). As, by symmetry four cysteine residues result from a
single point mutation, the mean value of the two closest resi-
dues was also determined: it increases from K121 (7.9 Å) <
S112 (9.5 Å) < R84 (18.3 Å) < T66 (20.0 Å).
Electron transfer studies
To explore the electron transfer in the Biot-TAA/Ru(II)-streptavi-
din dyads a flash quench technique was applied (Fig. 4).18
Therefore, the ruthenium(II) photosensitizer was excited selec-
Fig. 4 Illustration of the flash-quench procedure used for studying
electron transfer in the Biot-TAA/Ru(II)-streptavidin adducts.12 MV2+
stands for methylviologen.
tively at 532 nm with laser pulses of ∼10 ns duration. In the
presence of 120 mM methylviologen dichloride (MV2+) added
3
to the aqueous solution, the luminescent MLCT excited state
buted unambiguously to MV•+.19,20 The oxidation product Ru(III)
of the photosensitizer *Ru(II) is quenched oxidatively with
manifests as a bleach at ∼450 nm. When adding 25 μM Biot-
diffusion-limited kinetics, i.e., initial excitation (“flash”) is fol-
TAA to the same solution, an additional band at ∼760 nm
lowed by oxidation of *Ru(II) to Ru(III) (“quench”) within the
becomes observable (black trace in Fig. 5a) in addition to the
∼10 ns duration of the laser pulse.
MV•+ bands. The band at 760 nm is typical for TAA+,21 and
In a sample containing 50 μM Ru(II)-streptavidin (K121C
mutant) and 120 mM MV2+ but no Biot-TAA in MilliQ water at
hence this is clear evidence for electron transfer from TAA to
Ru(III), which (based on the relevant redox potentials)21 is
25 °C, one observes the typical spectroscopic signatures of the
expected to be associated with a reaction free energy (ΔGET0) of
ca. −0.5 eV. In the reference experiment in which 50 μM Ru(II)-
streptavidin and 120 mM MV2+ were measured in the presence
of 25 μM TAA-Ac, the band at 760 nm does not appear (blue
trace in Fig. 5a), confirming that in the Ru(II)-streptavidin
(K121C mutant)/TAA-biotin system, the phototriggered elec-
tron transfer from TAA to Ru(III) does indeed rely on the tight
association between the streptavidin host and the Biot-TAA
guest.
MV•+ radical (green trace in Fig. 5a).19 Specifically, there is a
sharp and intense absorption at ∼400 nm and a broader, less
intense band with a maximum at ∼600 nm which can be attri-
Table 1 Estimated through-space distances between α-carbon atom of
the targeted mutagenesis positions and the sulfur atom of a biotinylated
cofactor based on the crystal structure of streptavidin (pdb code:
3PK2).15 The two closest Cα are listed as well as their mean values
(Fig. S16–S20 in the ESI for details)
The TAA+ signal at 760 nm rises with instrumentally limited
kinetics (black trace in Fig. 5b), indicating that phototriggered
electron transfer from TAA-biotin to Ru(III)-streptavidin (K121C
mutant) occurs with a rate constant kET ≥ 108 s−1. The signal
then decays on a timescale >10 μs, due to bimolecular electron
transfer between Biot-TAA+ and MV•+ (Fig. 5b). MV•+ has non-
Mutant
R1 [Å]
R2 [Å]
Mean [Å]
K121C
S112C
R84C
T66C
7.1
6.9
14.8
17.8
8.6
12.1
21.7
22.1
7.9
9.5
18.3
20.0
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Fig. 5 (a) Transient absorption spectra of K121C-Ru measured after excitation at 532 nm with laser pulses of ∼10 ns duration. The spectra were
time-integrated over 200 ns immediately after excitation. Sample concentrations were: 50 μM Ru(II)-streptavidin, 120 mM MV2+, 25 μM Biot-TAA or
TAA-Ac, where applicable. The solvent was MilliQ water at 25 °C. (b) Temporal evolution of the transient absorption signal at 760 nm for 3 of the
4 samples from (a).
negligible absorbance at 760 nm hence the observation of
small signals even in absence of Biot-TAA (blue and green
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biotinylated triarylamine guests. This findings correlate with
the increasing distances between the photosensitizer and the
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Summary and conclusions
Rapid phototriggered electron transfer from biotinylated
guests to ruthenium photosensitizers which are covalently
attached to cysteine residues of streptavidin is possible in care-
fully selected mutants. This is an important finding in view of
photoredox catalysis which makes use of catalysts which are
embedded in the protected environment of a biotin binding
pocket of streptavidin and photosensitizers which are bound
to the surface of streptavidin. Catalysts in the biotin binding
pocket can exhibit markedly better performance than outside
streptavidin,3a,4b and our study paves the way to photoredox
catalysis in specifically engineered artificial metalloenzymes.
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