Corrole-Substituted Fluorescent Heme Proteins

Article abstract of DOI:10.1021/acs.inorgchem.0c03599

Although fluorescent proteins have been utilized for a variety of biological applications, they have several optical limitations, namely weak red and near-infrared emission and exceptionally broad (>200 nm) emission profiles. The photophysical properties of fluorescent proteins can be enhanced through the incorporation of novel cofactors with the desired properties into a stable protein scaffold. To this end, a fluorescent phosphorus corrole that is structurally similar to the native heme cofactor is incorporated into two exceptionally stable heme proteins: H-NOX from Caldanaerobacter subterraneus and heme acquisition system protein A (HasA) from Pseudomonas aeruginosa. These yellow-orange emitting protein conjugates are examined by steady-state and time-resolved optical spectroscopy. The HasA conjugate exhibits enhanced fluorescence, whereas emission from the H-NOX conjugate is quenched relative to the free corrole. Despite the low fluorescence quantum yields, these corrole-substituted proteins exhibit more intense fluorescence in a narrower spectral profile than traditional fluorescent proteins that emit in the same spectral window. This study demonstrates that fluorescent corrole complexes are readily incorporated into heme proteins and provides an inroad for the development of novel fluorescent proteins.

Full text of DOI:10.1021/acs.inorgchem.0c03599

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Corrole-Substituted Fluorescent Heme Proteins
Christopher M. Lemon* and Michael A. Marletta*
Cite This: Inorg. Chem. 2021, 60, 2716−2729
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ABSTRACT: Although fluorescent proteins have been utilized for
a variety of biological applications, they have several optical
limitations, namely weak red and near-infrared emission and
exceptionally broad (>200 nm) emission profiles. The photo-
physical properties of fluorescent proteins can be enhanced
through the incorporation of novel cofactors with the desired
properties into a stable protein scaffold. To this end, a fluorescent
phosphorus corrole that is structurally similar to the native heme
cofactor is incorporated into two exceptionally stable heme
proteins: H-NOX from Caldanaerobacter subterraneus and heme
acquisition system protein A (HasA) from Pseudomonas aeruginosa.
These yellow-orange emitting protein conjugates are examined by
steady-state and time-resolved optical spectroscopy. The HasA
conjugate exhibits enhanced fluorescence, whereas emission from the H-NOX conjugate is quenched relative to the free corrole.
Despite the low fluorescence quantum yields, these corrole-substituted proteins exhibit more intense fluorescence in a narrower
spectral profile than traditional fluorescent proteins that emit in the same spectral window. This study demonstrates that fluorescent
corrole complexes are readily incorporated into heme proteins and provides an inroad for the development of novel fluorescent
proteins.
One strategy to improve upon the optical properties of
traditional fluorescent proteins is to incorporate a highly
fluorescent cofactor into a stable protein scaffold. One example
of such a scaffold is the heme nitric oxide/oxygen binding (H-
NOX) protein from the thermophilic bacterium Caldanaer-
obacter subterraneus (Cs) (Figure 1a). H-NOX proteins are a
family of bacterial gas-sensing proteins that are homologous to
the heme domain of the mammalian nitric oxide receptor soluble
guanylate cyclase (sGC).9−15 They have a buried, hydrophobic
heme pocket comprising a heme-ligating histidine residue and a
conserved Y−S−R motif that hydrogen bonds with the
propionate chains of the heme cofactor. In oxygen-sensing H-
NOXs, such as Cs H-NOX, the protein also contains a distal
hydrogen-bonding network that stabilizes the O2 adduct. The
iron center of the heme cofactor binds diatomic gases, such as
NO or O2, resulting in a conformational change of the protein.
This triggers a signal cascade that culminates in a physiological
response to the gaseous signal, such as biofilm formation or
flagellar motion.15 The robust Cs H-NOX protein is readily
expressed in Escherichia coli, variants are easily made, and it
INTRODUCTION
■
Since the discovery of green fluorescent protein (GFP)1 and its
subsequent demonstration as a fluorescent tag for in vivo
labeling,2 the field of fluorescent proteins has greatly expanded.
Significant effort has been dedicated to tune the optical
properties of fluorescent proteins, spanning ultraviolet (UV),
visible, and near-infrared (NIR) wavelengths. Moreover,
fluorescent proteins have been utilized for a variety of
applications as sensitizers, labels, and sensors.3 Despite advances
to extend the emission of fluorescent proteins into the red and
NIR, the emission intensity of these proteins is quite weak,
exhibiting quantum yields (ϕ) < 20%.4 Since tissue is
transparent to red and NIR light in the 600−1100 nm range
(i.e., the “tissue transparency window”),5 the weak emission of
traditional fluorescent proteins in this spectral range greatly
limits in vivo utility. Indeed, the quantum yields of traditional red
fluorescent proteins are significantly lower than typical GFPs
and other fluorescent proteins that emit outside the tissue
transparency window. Another limitation of traditional fluo-
rescent proteins is their broad emission profiles, which can span
over 200 nm. This limits the utility of fluorescent proteins for
ratiometric sensing applications. In this sensing modality, two
fluorophores are incorporated into the sensor and the analyte is
quantified by measuring the emission intensity ratio of the two
components. This is a robust sensing methodology that is
independent of sensor concentration and is functional in
scattering media, such as the biological milieu.6−8
Received: December 9, 2020
Published: January 29, 2021
https://dx.doi.org/10.1021/acs.inorgchem.0c03599
© 2021 American Chemical Society
Inorg. Chem. 2021, 60, 2716−2729
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Chart 1. Comparison of the Native Heme Cofactor
Protoporphyrin IX and the Corrole Analog Utilized in This
Study.
porphyrin complexes exhibit ϕ = 4−5%,34 which is not an
improvement over extant fluorescent proteins. Corrole is a
closely related tetrapyrrole macrocycle with a contracted 23-
atom core due to the presence of a direct pyrrole−pyrrole
linkage.35 As a result, corrole is a trianionic ligand when fully
deprotonated, rather than dianionic like porphyrin, thereby
stabilizing different metal complexes and accessing different
molecular properties.36,37 While corroles have not been studied
as extensively as their porphyrin congeners, it has been
demonstrated that they exhibit optical properties superior to
related porphyrin complexes.38 In general, corroles have higher
fluorescent quantum yields than related porphyrin com-
plexes.39,40 Specifically, light main group corrole complexes of
gallium,39,41 phosphorus,42−44 aluminum,40,41,45,46 and sili-
con47,48 are highly fluorescent and have been utilized for
biological imaging applications.43,49−51 Therefore, main group
corrole complexes are ideal heme analogues for fluorescent
protein development.
Figure 1. (a) X-ray crystal structure of the Fe(II)−O2 complex of Cs H-
NOX (PDB ID: 3TF0), depicting the heme-ligating histidine (H102 in
cyan) and the hydrogen-bonding network that stabilizes the O2 adduct
(W9, Y140, and N74 in cyan). (b) X-ray crystal structure of Pa HasA
(PDB ID: 3ELL), illustrating the heme-ligating residues (Y75 and H32
in cyan).
To this end, the synthesis of a corrole analog that is
structurally similar to the native heme cofactor was targeted
(Chart 1) for ultimate reconstitution into the heme pocket of the
apoprotein to furnish a novel fluorescent protein. Previous
studies have examined corrole−protein interactions with a
variety of proteins, including serum albumin,52,53 viral capsids,54
and transferrin.55 Additionally, nanoparticle formulations have
been prepared with corrole conjugates of these proteins.56,57 In
all of these examples, the corroles are amphiphilic meso-aryl
corroles with β-sulfonate substituents. These conjugates, which
exploit nonspecific hydrophobic and charge interactions, have
been utilized for a variety of biomedical applications,51 including
drug delivery,54 in vitro cancer cell therapy,54 cell imaging,56 and
sonodynamic therapy.57 Conversely, only two studies have used
heme-like (meso-unsubstituted, β-octaalkyl) corrole complexes
to replace the native porphyrin cofactor of heme proteins: an
iron corrole in myoglobin and horseradish peroxidase (HRP) as
artificial peroxidases58 and a copper corrole in myoglobin.59
Here, this approach has been extended by developing
fluorescent H-NOX and HasA proteins using a phosphorus
corrole as the fluorescent cofactor.
readily binds unnatural heme cofactors. Together, these
properties render Cs H-NOX an ideal platform to construct
novel sensing and imaging agents.16 To this end, Cs H-NOX has
been tailored for a variety of applications, including NO
sensing,17 O2 sensing,18 magnetic resonance imaging,19 and
fluorescent labeling.20
Another stable protein scaffold is the heme acquisition system
protein A (HasA) from the pathogenic bacterium Pseudomonas
aeruginosa (Pa) (Figure 1b). Under iron-depleted stress
conditions, these bacteria secrete HasA to scavenge heme
from the host and subsequently import it through a membrane
receptor HasR.21−24 Unlike H-NOX proteins, HasA has a
surface-exposed heme-binding pocket. The secreted apoprotein
exhibits an extended conformation; two flexible loops sequester
the heme cofactor, leading to high-affinity binding (Ka = 5.3 ×
1010 M−1)25 and a large conformational change.26 The bound
heme is ligated by a tyrosine and a histidine residue, but it has
been demonstrated that either one of these two residues alone is
sufficient to bind heme.27,28 In addition to the native heme
cofactor, Pa HasA can also bind a variety of β-alkyl and meso-
aryl iron porphyrins, as well as related iron phthalocyanine and
salophen complexes.29,30 It has also been demonstrated that
HasA can bind gallium complexes of protoporphyrin IX31,32 and
phthalocyanine.33
EXPERIMENTAL SECTION
■
Materials for Chemical Synthesis and Characterization. The
following chemicals were used as received: dichloromethane (CH2Cl2),
ethyl acetate (EtOAc), hexanes, 4-(dimethylamino)pyridine, 3-nitro-2-
butanol (mixture of isomers), acetic anhydride (Ac2O), sodium
bicarbonate (NaHCO3), sodium sulfate (Na2SO4), ethyl isocyanoace-
tate, benzyl acetoacetate, methyl 4-acetyl-5-oxohexanoate, zinc powder
(Zn), triethylamine (NEt3), trimethyl orthoformate, L-histidine hydro-
chloride monohydrate, palladium on carbon with 10 wt % loading (Pd/
C), silica gel (60 Å pore size, 230−400 mesh particle size), potassium
Since both H-NOX and HasA bind artificial cofactors, a
strategy to incorporate fluorescent analogues of protoporphyrin
IX (Chart 1) was designed to serve as an alternative to
traditional fluorescent proteins. Although emissive in the red
region of the spectrum, free-base and metalloporphyrins are not
exceptionally bright fluorophores. For example, Zn(II)
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hydroxide (KOH), concentrated hydrobromic acid (HBr), pyridine,
phosphorus trichloride (PCl3), rhodamine 101, and Celite from Sigma-
Aldrich; trifluoroacetic acid (TFA), concentrated hydrochloric acid
(HCl), sodium hydroxide (NaOH), and tetrahydrofuran (THF) from
EMD Millipore; methanol (MeOH), chloroform (CHCl3), sodium
nitrite (NaNO2), and 85% phosphoric acid (H3PO4) from Fisher
Scientific; acetic acid (HOAc) from Macron; tetrachloro-1,4-
benzoquinone (p-chloranil) from TCI Chemicals; chloroform-d
(CDCl3) and dimethylsulfoxide-d6 (DMSO-d6) from Cambridge
Isotope Labs; hydrogen gas (H2) from Praxair; lead(IV) acetate
(Pb(OAc)4) from Alfa Aesar; and potassium carbonate (K2CO3) from
J. T. Baker Chemicals. Nitrogen gas (N2) from Praxair was passed over a
Drierite column prior to use. The phosphorus complex of 5,10,15-
tris(4-methoxyphenyl)-corrole was prepared as previously reported.42
Materials for Protein Expression, Purification, and Recon-
stitution. The following chemicals were used as received: potassium
phosphate dibasic trihydrate (K2HPO4), deoxyribonuclease I from
bovine pancreas (DNase), benzamidine, D-(+)-glucose, triethanol-
amine (TEA), 2-morpholinoethanesulfonic acid (MES), and antifoam
SE-15 from Sigma-Aldrich; Luria Broth (LB), Terrific Broth (TB),
ampicillin, and 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochlor-
ide (AEBSF) from Research Products International; agarose, DMSO,
BugBuster Master Mix, and Immobilon Classico Western HRP
substrate from EMD Millipore; hemin chloride from Frontier
Scientific; PrimeSTAR Max DNA polymerase and His60 Ni Superflow
resin from Takara Bio; mouse anti-His6 antibody and goat anti-mouse
HRP conjugate antibody from Life Technologies; Bacto Agar from BD;
glycerol from VWR; lysozyme (from egg white) from Amresco; TEA
hydrochloride (TEA·HCl) from Spectrum Chemical; imidazole from
Oakwood Chemical; sodium chloride (NaCl) from Macron; Gibson
Assembly Master Mix from New England BioLabs; nitrogen gas (N2)
from Praxair; isopropyl β-D-1-thiogalactopyranoside (IPTG) and
ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)
from Fisher Scientific; and PD-10 desalting columns (Sephadex G-25
medium) from GE Healthcare. Argon gas (Ar) from Praxair was passed
over an Oxiclear gas purifier prior to use. The DH5α and XL1-Blue E.
coli competent cells were obtained from the UC Berkeley Macrolab,
while the RP523(DE3) competent cells were obtained from lab-
generated stocks.13
8,12-Bis(2-methoxycarbonylethyl)-2,3,7,13,17,18-hexamethyl
Corrole (H3-2). In a 20 mL glass vial, 60 mg (83 μmol) of a,c-biladiene
1, which was prepared as described in the Supporting Information, was
suspended in 10 mL of MeOH. The mixture was briefly sonicated and
65 mg (264 μmol) of p-chloranil was added. The mixture was briefly
sonicated and then stirred at room temperature for 1.5 h. Two parallel
reactions were run and then combined for work-up and purification.
The reaction mixtures were transferred to a separatory funnel and
CH2Cl2 was added. The red-brown solution was washed with a
saturated solution of NaHCO3 and then with water. The organics were
dried over Na2SO4 and brought to dryness. The crude material was
loaded onto a silica gel column packed with CH2Cl2, and the product
was eluted with 4:1 CH2Cl2:EtOAc as a magenta solution. The solvent
was removed by rotary evaporation to afford 77 mg (83% yield for two
parallel reactions, or 120 mg of 1) of the title compound as a purple
solid. 1H NMR (400 MHz, CDCl3, 25 °C): δ 3.19 (t, J = 7.7 Hz, 4H),
3.35 (s, 6H), 3.45 (s, 6H), 3.53 (s, 6H), 3.71 (s, 6H), 4.23 (t, J = 7.7 Hz,
4H), and 9.19 (s, 1H), 9.35 (s, 2H). Anal. Calcd for (M + H)+ M =
C33H38N4O4, 555.2966. Found: ESI-MS: 555.2960. UV−vis (CHCl3),
λ in nm (ε in 103 M−1 cm−1): 398 (130.), 409 (97.8), 501 (7.0), 538
(16.3), 550 (16.6), and 593 (17.5).
PCl3. The product was extracted into CH2Cl2, and the organic layer was
washed with a saturated solution of NaCl. The organics were dried over
Na2SO4 and brought to dryness. The crude material was loaded onto a
silica gel column packed with EtOAc and the product was eluted with
5% MeOH in EtOAc as a pink-orange solution. The solvent was
removed by rotary evaporation to afford 18 mg (77% yield) of the title
compound as a red-orange solid. 1H NMR (400 MHz, CDCl3, 25 °C):
δ 3.25 (m, 4H), 3.57 (s, 6H), 3.63 (s, 6H), 3.70 (s, 6H), 3.77 (s, 6H),
4.38 (t, J = 7.8 Hz, 4H), 9.87 (s, 2H), and 9.96 (s, 1H). 31P NMR (162
MHz, CDCl3, 25 °C): δ −98.76. Anal. Calcd for (M + H)+ M =
C33H35N4O5P, 599.2418. Found: ESI-MS: 599.2410. UV−vis
(CHCl3), λ in nm (ε in 103 M−1 cm−1): 274 (16.9), 280 (16.2), 313
(12.3), 399 (215), 403 (238), 485 (3.4), 517 (11.6), 522 (11.2), and
558 (25.3).
8,12-Bis(2-carboxyethyl)-2,3,7,13,17,18-hexamethylcorrolato-
(oxo)phosphorus(V) (P-3). In a 50 mL round bottom flask, 29 mg (47
μmol) of corrole ester P-2 was dissolved in 10 mL of THF and 10 mL of
3 M NaOH was added. The biphasic mixture was stirred vigorously and
refluxed overnight, protected from light. After this reaction, the corrole
had transferred from the organic layer to the aqueous layer. The
reaction mixture was cooled in an ice bath and acidified by slowly
adding 1 M HCl to reach pH ∼0. The solution became cloudy,
indicating precipitation of the protonated carboxylic acid corrole. The
red-orange suspension was transferred to a 50 mL conical tube and was
centrifuged at 4300g for 20 min. The supernatant was discarded and the
pellet was resuspended in 20 mL of H2O, and the suspension was
centrifuged at 4300g for 20 min. This process was repeated until the
supernatant had a pH of ∼5. The pellet was resuspended in a minimal
amount of H2O and transferred to a round bottom flask. The solvent
was removed by rotary evaporation and then dried under vacuum for
several days to afford 20 mg (76% yield) of the title compound as a dark
red solid. 1H NMR (400 MHz, DMSO-d6, 25 °C): δ 3.13 (t, J = 7.6 Hz,
4H), 3.54 (s, 6H), 3.62 (s, 6H), 3.63 (s, 6H), 4.32 (m, 4H), 10.09 (s,
2H), 10.17 (s, 1H), and 12.39 (bs, 2H). 31P NMR (162 MHz, DMSO-
d6, 25 °C): δ −97.66. Anal. Calcd for (M + H)+ M = C31H31N4O5P,
571.2105. Found: ESI-MS: 571.2110.
Protein Expression and Purification. The protein expression
plasmids were prepared as described in the Supporting Information.
The plasmids were transformed into the RP523(DE3)13 strain of E. coli,
and then grown on an LB agar plate (1.5% (w/v) agar) supplemented
with ampicillin (100 μg/mL) and hemin (20 μg/mL). Ten colonies
were selected to test protein expression and were grown in 3 mL of TB
supplemented with ampicillin (100 μg/mL) and hemin (20 μg/mL) at
37 °C overnight. Glycerol stocks were prepared for each culture. The
cells were then subcultured (1:200 dilution) in 50 mL of TB
supplemented with ampicillin (100 μg/mL), hemin (20 μg/mL), and
0.2% (w/v) glucose and grown at 37 °C until the OD600 reached ∼0.6.
IPTG was added (100 μM final concentration) and the cultures were
then transferred to an 18 °C incubator and grown overnight. H-NOX
expression levels were determined by preparing cell density-matched
samples of each culture, as well as a pre-induction control. The cells
were pelleted (2300 g for 5 min), then lysed using BugBuster, and the
whole-cell lysate was analyzed by sodium dodecyl sulfate-polyacryla-
mide gel electrophoresis (SDS-PAGE) and Western blot to identify
His-tagged proteins. Western blotting was performed using a mouse
anti-His6 primary antibody, a goat anti-mouse−HRP conjugate
secondary antibody, and the Classico HRP substrate. Colonies with
the highest level of heme expression were then selected for large-scale
protein expression. For large-scale expression of the holoprotein, a 5 mL
overnight culture (prepared as described above) was inoculated directly
from a frozen glycerol stock and then grown at 37 °C overnight. This
culture was then used to inoculate 1 L of TB (containing 100 mg of
ampicillin, 20 mg of hemin, and 0.2% (w/v) glucose) and was grown at
37 °C until the OD600 reached ∼0.6. IPTG was added (100 μM final
concentration), and the cultures were then transferred to an 18 °C
incubator and grown for 24 h. Cells were harvested by spinning the
cultures at 4300 g for 20 min, and the resultant pellets were snap-frozen
in liquid nitrogen and stored at −80 °C.
8,12-Bis(2-methoxycarbonylethyl)-2,3,7,13,17,18-hexamethyl-
corrolato-(oxo)phosphorus(V) (P-2). In a 20 mL glass vial, 21 mg (38
μmol) of free-base corrole H3-2 was dissolved in 5 mL of dry pyridine
and 100 μL of PCl3 (0.16 g, 1.1 mmol) was added; the resultant mixture
was stirred at room temperature for 10 min. An additional 200 μL of
PCl3 (0.31 g, 2.3 mmol) was added and the mixture was stirred at room
temperature for 10 min. Then 200 μL of PCl3 (0.31 g, 2.3 mmol) was
added and the mixture was stirred at room temperature for 10 min. The
reaction mixture was dissolved in CH2Cl2 and transferred to a
separatory funnel; water was carefully added to react with the residual
For anaerobic protein expression to obtain the apoprotein, a starter
culture was prepared in a serum bottle containing 60 mL of TB,
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Scheme 1. Synthesis of Free-Base Corrole H3-2 and Subsequent Phosphorus Insertion to Yield P-2
supplemented with ampicillin (100 μg/mL) and 0.2% (w/v) glucose,
and then inoculated directly from a frozen RP523 glycerol stock. The
bottle was sealed with a rubber septum and purged with Ar for 1 h, and
then grown at 37 °C for at least 20 h. A freshly autoclaved 10 L
fermenter60 was charged with 7 L of hot, sterilized TB and 1 L of hot,
sterilized H2O (to account for the ∼1 L volume loss due to overnight
purging). After cooling to 37 °C, 2 mL of antifoam and 400 mg of
ampicillin were added, and the mixture was sparged with N2 overnight.
The following day, ampicillin (500 mg) and glucose (0.2% (w/v) final
concentration) were added to the media, which was then inoculated
with the 60 mL anaerobic starter culture. The resultant culture was
grown at 37 °C until the OD600 reached ∼0.45 and was then cooled to
18 °C prior to inducing protein expression. Once the OD600 reached
∼0.7 and the temperature reached 18 °C, IPTG was added (100 μM
final concentration) and the culture was grown at 18 °C for 24 h to
obtain the apoprotein. Cells were harvested by spinning the cultures at
4300 g for 20 min, and the resultant pellets were snap-frozen in liquid
nitrogen and stored at −80 °C.
Cell pellets of Cs H-NOX were thawed in a water bath at room
temperature, then resuspended in Buffer A (50 mM K2HPO4, 300 mM
NaCl, 20 mM imidazole, and 5% (v/v) glycerol at pH 8.0)
supplemented with 110 mM benzamidine, 0.4 mM AEBSF, 0.1 mg/
mL lysozyme, and 0.3 mg/mL DNase. Similarly, Pa HasA cell pellets
were resuspended in Buffer B (20 mM K2HPO4, 250 mM NaCl, and 30
mM imidazole at pH 7.4) supplemented with 110 mM benzamidine,
0.4 mM AEBSF, 0.1 mg/mL lysozyme, and 0.3 mg/mL DNase. For
both proteins, cells were lysed using a high-pressure homogenizer
(Avestin Emulsiflex-C5). In the case of Cs H-NOX, the lysate was
heated in a water bath at 60 °C for 1 h. The lysate was clarified by
spinning at 42,000 g, and the supernatant was loaded onto a column
containing His60 resin (3 mL for 1 L aerobic cultures or 5 mL for ∼7 L
anaerobic cultures) equilibrated with Buffer A for H-NOX or Buffer B
for HasA. The resin was then washed with 20 column volumes of Buffer
A for H-NOX or Buffer B for HasA. The protein was then eluted with
Buffer C for H-NOX (50 mM K2HPO4, 300 mM NaCl, 300 mM
imidazole, and 5% (v/v) glycerol at pH 8.0) or Buffer D for HasA (20
mM K2HPO4, 250 mM NaCl, and 300 mM imidazole at pH 7.4). The
eluent was collected in five, 2−3 mL fractions. The fractions were
analyzed by SDS-PAGE and UV−vis absorption spectroscopy.
Typically, the first fraction contained a significant amount of nucleic
acids. The main protein-containing fractions were the second and third
fractions. Later fractions contained little protein and typically exhibited
a shoulder on the red side of the 280 nm protein absorption peak.
Fractions with comparable purity were combined and then dialyzed
overnight into Buffer E (20 mM K2HPO4, 250 mM NaCl, and 1 mM
EDTA at pH 7.4) using snakeskin dialysis tubing (3500 MW cutoff).
The protein was then filtered with a 0.22 μm syringe filter and stored in
sterile tubes at 4 °C.
column (Sephadex G-25 medium), pre-equilibrated with Buffer F (20
mM TEA and 1 mM EDTA at pH 6.5). The pink eluent was collected in
1 mL fractions. In order to remove any traces of unbound P-3, the
protein-containing fractions were concentrated to <1 mL (using a 5000
MW cutoff filter), and a second PD-10 column was run using Buffer F.
Physical Measurements. NMR spectra were recorded on a Bruker
AVANCE 400 NMR spectrometer at the UC Berkeley College of
Chemistry NMR Facility. 1H NMR spectra were internally referenced
to the residual solvent signal (δ = 7.26 for CDCl3 or δ = 2.50 for DMSO-
d6),61 and 31P NMR were internally referenced to 85% H3PO4 (δ = 0)
using a capillary tube containing the standard. Mass spectra were
recorded on a Finnigan LTQ FT-ICR mass spectrometer (Thermo
Fisher Scientific) equipped with an electrospray ionization source in
positive ion mode at the QB3/Chemistry Mass Spectrometry Facility at
UC Berkeley. UV−vis absorption spectra were acquired using a Cary
300 spectrometer (Agilent) or a NanoDrop 2000C (Thermo
Scientific). Steady-state emission spectra were recorded on a Horiba
Scientific FluoroMax-4 spectrofluorometer. Time-resolved fluores-
cence was measured using a PicoQuant FluoTime 300 fluorometer
and a 520 nm PicoQuant pulsed diode laser with a PDL 820 driver.
Signal was collected at the emission maximum with a 2 nm bandwidth
and detected using a TimeHarp 260 time-correlated single-photon
counting board. Relative quantum yields of corroles in chloroform (η =
1.4459)62 or proteins in TEA buffer (η = 1.3329, interpolated from
published data)63 were calculated using Rhodamine 101 in basic EtOH
(η = 1.3611)62 as the reference (Φref = 0.96)64 according to the
following equation:
2
i
j
j
j
j
j
j
y
z
z
z
z
z
z
i
y
z
z
z
z
z
η
η
∇
j
sam
sam
j
= Φ j
refj
Φ
sam
j
∇
ref
(1)
k
{
ref
k
{
where ∇ is the slope of the plot of integrated fluorescence intensity
versus absorbance (constructed with 5 points) and η is the refractive
index of the solvent.
Computational Details. Density functional theory (DFT)
calculations were performed with the hybrid functional Becke-3
parameter exchange functional65−67 and the Lee−Yang−Parr nonlocal
correlation functional (B3LYP),68 as implemented in the Gaussian 16,
Revision A.03 software package.69 A polarized split-valence triple-ζ
basis set that includes p functions on hydrogen atoms and d functions
on other atoms (i.e., the 6-311G(d,p) or 6-311G** basis set) was used.
Calculations were performed with a polarizable continuum (PCM)
solvation model in chloroform using a polarizable conductor
calculation model (CPCM).70,71 Geometries were confirmed as local
minima structures by calculating the Hessian matrix and ensuring that
no imaginary eigenvalues were present. Excited state calculations were
performed using time-dependent DFT (TD-DFT)72−76 with the same
functionals, basis sets, and solvation details as the ground state but with
the inclusion of diffuse functions on all atoms (i.e., the 6-311++G(d,p)
or 6-311++G** basis set). Excited-state energies were computed for
the 20 lowest singlet and triplet excited states. Optimized geometries
were rendered in Gauss View 5. Simulated UV−vis spectra were
generated in the program Gauss View 5 by broadening transition lines
with Gaussian functions that have a half width of 0.03 eV.
Apoprotein Reconstitution. A 1 mg/mL stock solution of corrole
P-3 was prepared in DMSO. An aliquot of the H-NOX or HasA protein
(∼1 mg) was diluted to 1 mL with Buffer E in a 1.7 mL microcentrifuge
tube. Then, 100 μL of the P-3 corrole solution was slowly added
dropwise to the protein; the tube was gently inverted after each addition
of two drops of the corrole stock solution. The reaction mixture was
gently rocked at room temperature for 2 h, protected from ambient
light. After incubation, the reaction mixture was spun at 21,000 g for 1
min to pellet any solids. The solution was then applied to a PD-10
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The phosphorus center of P-2 and P-3 could exist as one of
three possible species: a six-coordinate dihydroxide, a five-
coordinate terminal oxo,82 or a five-coordinate cationic mono-
hydroxide83 (Chart 2). Mass spectrometry is consistent with the
RESULTS
■
Corrole Synthesis and Characterization. In order to
prepare a corrole analog with approximately the same size and
shape as the native heme cofactor, the synthesis of corrole H3-2
was targeted. In order to simplify the synthesis, vinyl groups
were substituted for more synthetically tractable methyl groups.
Although the 2,3,17,18-tetraethyl derivative of corrole H3-2 has
previously been incorporated into heme proteins,58,59 smaller
methyl groups would minimize the bulk of the corrole and
facilitate binding in the H-NOX heme-binding pocket. The free-
base corrole H3-2 was prepared via oxidative cyclization of the
linear tetrapyrrole a,c-biladiene 1 (see the Supporting
Information for synthesis), using p-chloranil as the oxidant
(Scheme 1).77 The scale of the cyclization is critical, and the
highest yields (>80%) were obtained when the reaction was run
on a 60 mg scale. For convenience, two parallel reactions were
performed and then combined for work-up and purification.
Metallation of the free-base corrole was attempted with a
variety of light main-group elements that are known to yield
highly fluorescent complexes. First, gallium coordination was
performed with GaCl3 in pyridine, following literature
procedures.39 A stark color change from the magenta free-base
to red-orange indicated the formation of the complex.
Purification of Ga-2 on silica resulted in the decomposition of
the molecule to the corresponding biliverdin. Presumably, the
gallium complex acted as a photosensitizer for the generation of
singlet oxygen, which subsequently reacted with the corrole
across the pyrrole−pyrrole linkage (Scheme S1). This reactivity
is consistent with previous reports of photochemical decom-
position of highly electron-rich β-octaalkyl corroles.78,79
Chart 2. Speciation of Phosphorus Corroles.
five-coordinate formulations. It should be noted that these two
scenarios are indistinguishable, as [PO] + H+ and [P−OH]+
have identical masses. The coordination number of the P(V)
center can be determined by 31P NMR. Chemical shifts in the
−90 to −100 ppm region correspond to five-coordinate species,
whereas six-coordinate complexes have resonances between
−180 and −200 ppm.37,42 P-2 and P-3 exhibit a single resonance
at −98.76 and −97.66 ppm, respectively, confirming that both
are five-coordinate species. These values are similar to the
chemical shift reported for P(V)O octaethylcorrole (OEC)
(−99.40 ppm).82 Finally, there is no evidence of axial −OH
1
signals in the H NMR spectrum, as evidenced by the lack of
signals at < −2 ppm. Together, these data indicate that P-2 and
P-3 are best formulated as five-coordinate PO complexes.
The electronic absorption spectra of H3-2, P-2 (Figure 2), and
P-3 (Figure S1) exhibit intense Soret (or B) bands in the near-
To circumvent this deleterious reactivity, the preparation of
complexes with the lighter main group elements, aluminum and
phosphorus, was targeted. Following published methods,40
aluminum coordination was performed using AlMe3 in toluene;
a drastic color change was consistent with complex formation.
While the primary method for purification of aluminum corroles
is recrystallization, attempts to purify Al-2 in this way resulted in
the decomposition of the corrole after ∼12 h. It is likely that this
complex undergoes photochemical degradation akin to the
gallium analog. Attempts to purify the aluminum corrole by
column chromatography were unsuccessful. The compound
stuck to silica, neutral alumina, and Florisil and could not be
eluted with polar solvent mixtures, including 100% methanol.
It should be noted that previously reported Ga39 and Al40
complexes utilize 5,10,15-tris(pentafluorophenyl)corrole
(TPFC). Since the corrole core is inherently more electron-
rich than porphyrin,80 the strongly electron-withdrawing
pentafluorophenyl substituents reduce the electron density of
the tetrapyrrole core. Consequently, H3TPFC is highly resistant
to decomposition and is one of the most stable corrole ligands.81
Conversely, H3-2 has electron-donating alkyl substituents,
which increase the density of the corrole core. This ligand and
other β-octaalkyl corroles are prone to oxidative degradation.78
Fortunately, the phosphorus complex of corrole 2 was readily
Figure 2. Absorption (solid lines) and emission (dashed lines) spectra
of H3-2 () and P-2 (red ) in CHCl3. For the emission spectra, the
absorbance of the samples was matched at the excitation wavelength:
A(525) = 0.1000 0.0003.
UV and weaker Q bands in the visible region of the spectrum
(Table 1). The free-base corrole (Cs symmetry) exhibits a split
Soret band, which arises from distinct x and y polarizations of
this transition.84 Out-of-plane coordination of the PO unit
renders P-2 and P-3 Cs symmetric. Consequently, it is expected
that the phosphorus complexes would also exhibit a split Soret
a
Table 1. Absorption of P Corroles and Conjugates
B1
B2
Q(1,0)
Q(0,0)
44
prepared via literature methods using PCl3 (Scheme 1), and
b
d
c
P-2
P-3
399 (sh)
517
380
403
404
403
520
519
523
558
558
561
559
the compound was obtained in 77% yield. In order to enhance
water solubility and enable hydrogen bonding with the Y−S−R
motif in Cs H-NOX, the methyl esters were hydrolyzed under
basic conditions. A biphasic mixture of the corrole ester P-2 in
THF and 3 M NaOH was refluxed overnight. Acidic workup
precipitated the carboxylic acid derivative P-3 in high yield.
d
P-3 H-NOX
P-3 HasA
399
d
a
b
Transition wavelengths are in units of nm. Measured in CHCl3.
c
d
Shoulder. Measured in TEA buffer at pH 6.5.
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Table 2. Summary of Emission Properties of Corroles and Protein Conjugates
a
b
b
Q(0,0) (nm)
Q(0,1) (nm)
τ (ns)
ϕf × 102
kr (s−1
)
k )
nr (s−1
c
H3-2
600
566
566
567
566
617
615
617
617
619
5.7
1.2
3.8
1.5
3.1
5.9
3.9
1.2
2.5
7.3
1.0 × 107
3.3 × 107
3.1 × 106
1.7 × 107
2.3 × 107
1.7 × 108
8.0 × 108
2.6 × 108
6.5 × 108
3.0 × 108
c
P-2
d
P-3
d
P-3 H-NOX
P-3 HasA
d
a
b
c
Fluorescence quantum yield measured relative to Rhodamine 101 and calculated using eq 1. Calculated using eq 2. Measured in CHCl3.
d
Measured in TEA buffer at pH 6.5.
band, consistent with previously reported symmetry-dependent
observations.44,85,86 However, the splitting is poorly resolved
and the spectrum is more similar to pseudo-octahedral
complexes with C2v symmetry85 and is consistent with
previously reported six-coordinate phosphorus corroles.87−89
The addition of pyridine to a CH2Cl2 solution of P-2 does not
change the absorption spectrum (Figure S2a). Similarly, the
absorption spectrum of a P-3 solution in TEA buffer does not
change upon the addition of histidine (Figure S2b). This result
suggests that, unlike meso-triaryl PO corroles (Figure S2c,d),
P-2 and P-3 do not bind nitrogenous bases and remain five-
coordinate complexes.
The emission spectra of H3-2, P-2 (Figure 2), and P-3 (Figure
S1) exhibit a sharp fluorescence transition with weaker
vibrational overtones at longer wavelengths. Figure 2 illustrates
the emission intensity for absorbance-matched samples of H3-2
and P-2, indicating that the emission from the free-base corrole
is more intense than the phosphorus complex. Indeed, the
fluorescence quantum yields for H3-2 and P-2 are 5.9 and 3.9%,
respectively (Table 2). Emission from P-3 is extremely weak in
an aqueous buffer, exhibiting a quantum yield of only 1.2%.
Time-resolved measurements (Figure S3) indicate that the
excited-state lifetime for P-2 is extremely short (1.2 ns), whereas
the lifetime of H3-2 is considerably longer (5.7 ns), and this
value is consistent with previously reported data for free-base
corroles.90 Despite the low quantum yield, P-3 exhibits a longer
lifetime of 3.8 ns when fit to a monoexponential decay. We note
that a biexponential decay function nominally improves the fit of
the data (τ1 = 1.5 ns, 84%; τ2 = 4.9 ns, 16%), but the physical
significance of this two-component fit is unclear. Using the
lifetime and quantum yield, the radiative (kr) and nonradiative
(knr) rate constants can be determined using the following
equation:
reported corrole structures determined by X-ray crystallog-
raphy.44,83,91,92
Since it appears that P-2 does not bind pyridine (Figure S2),
calculations were also performed for a six-coordinate complex of
P-2 with an axial imidazole ligand (P(O)(Im)-2). This structure
would mimic the histidine−corrole interaction in the H-NOX
and HasA heme-binding pockets. As a starting point for the
geometry optimization, one of the axial hydroxide ligands of
P(OH)2-2 was replaced with an imidazole ligand, while the
other was converted to an oxo. Interestingly, the imidazole
ligand dissociates from the phosphorus center in the optimized
structure (Figure S7 and Tables S4 and S5), resulting in a long
P−N distance of 4.43 Å. The presence of imidazole destabilizes
the structure by 7.2 kcal/mol (relative to the five-coordinate
complex and imidazole): ΔGrxn = +7.2 kcal/mol for P-2 +
imidazole → P(O)(Im)-2. This supports the experimental
observation that P-2 does not bind nitrogenous bases,
demonstrating that the phosphorus center prefers to remain 5-
coordinate.
The four frontier molecular orbitals (highest occupied
molecular orbital (HOMO), HOMO−1, lowest unoccupied
molecular orbital (LUMO), and LUMO+1) are energetically
well-separated from the rest of the orbital manifold (Figure S8
and Table S6). Based on the Gouterman four orbital model for
porphyrins,93−95 which has also been shown to hold for
corroles,84,96,97 it is expected that the Soret and Q bands arise
due to transitions between these four frontier orbitals. To more
rigorously characterize the optical properties of these corroles,
TD-DFT calculations were performed using the same methods
as for the geometry optimization, but with the inclusion of
diffuse functions on all atoms (i.e., the 6-311++G** basis set).
Single-point excited-state energy calculations were performed
for the 20 lowest singlet and triplet states (Tables S7−S12) of
H3-2, P-2, and P(OH)2-2. The four lowest-energy singlet states
(S1−S4) give rise to two Q-like and two Soret-like transitions.
These states exclusively involve the four frontier orbitals, with
the exception of a minor (10%) contribution to S3 of H3-2 that
involves HOMO−2. The deviation between the calculated and
experimental transitions ranges from 0.01 to 0.18 eV, although
S1 for H3-2 deviates by 0.24 eV. Nevertheless, these differences
are substantially smaller than the absolute mean error of 0.3−0.5
eV determined from TD-DFT benchmark studies on test sets of
organic molecules.98−100
kr
ϕ = krτ =
kr + knr
(2)
where ϕ is the fluorescence quantum yield and τ is the excited-
state lifetime. While kr is similar for H3-2 and P-2, this value is an
order of magnitude slower for P-3. Conversely, knr is similar for
both H3-2 and P-3, but this rate constant is nearly 4 times higher
for P-2.
DFT Calculations. In order to gain insight into the
electronic structure of the corrole complexes, DFT calculations
were performed using the B3LYP functional. Ground-state
geometry optimization calculations were performed for H3-2
and P-2, both as a terminal oxo and a dihydroxide (P(OH)2-2),
using the 6-311G** basis set and a CPCM solvation model in
chloroform. The structures were verified as local minima by
performing frequency calculations and ensuring that there were
no imaginary frequencies. The optimized structures (Figures
S4−S6 and Tables S1−S3) are qualitatively similar to previously
Although the predicted transitions do not precisely align with
the data, the calculated spectra (Figures S9−S11) qualitatively
reflect experimental observations. The two Q bands and the two
Soret bands are orthogonally polarized; this data is summarized
in Tables S13−S15, which list the electronic dipole moment
vectors for states S1−S4. This demonstrates that both the Q and
B bands are split into x and y polarizations due to the low
symmetry of the corrole, consistent with previous reports.84
Calculations indicate that H3-2 has a substantial energy
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difference between the Soret transitions (202 cm−1), which
results in clear separation of bands (676 cm−1) in the
experimental spectrum. It should be noted that these TD-
DFT calculations have underestimated the experimental split in
the Soret transitions. Given the out-of-plane coordination of the
PO unit in P-2, the calculated splitting of the Bx and By
transitions is 519 cm−1, which is larger than the experimental
splitting of 249 cm−1. Conversely, calculations for P(OH)2-2
show a very small 28 cm−1 separation between the Bx and By
transitions. The experimental absorption spectrum of P-2
exhibits a poor resolution of the Soret transitions, which is
more similar to the calculated UV−vis spectrum of P(OH)2-2
rather than the terminal oxo P-2.
Protein Expression. Expression-based methods were
initially explored to incorporate the carboxylic acid derivative
P-3 into Cs H-NOX, as this has been a successful methodology
to replace the native cofactor of heme proteins with a variety of
unnatural porphyrin complexes.60 This approach requires the
RP523(DE3) strain of E. coli, which has a disrupted gene in the
heme biosynthesis pathway and also harbors a mutation that
renders the cell well permeable to heme and heme-like
porphyrins (e.g., mesoporphyrin IX).101 High levels of artificial
porphyrin incorporation are achieved by supplementing the
media with the cofactor under anaerobic conditions. Given the
structural similarity of the corrole analog to the native heme
cofactor, an extension of this approach to prepare corrole-
substituted heme proteins was reasonable.
Small-scale (1 L) anaerobic protein expression of Cs H-NOX
was performed in a three-neck bottle sealed with silicone septa
and the media was supplemented with 7 mg of P-3. Surprisingly,
the bacterial cell pellet was not pink, as would be expected if the
corrole diffused across the bacterial cell wall. Although the
purified protein appeared colorless, a minimal amount of P-3
was incorporated into the H-NOX, as evidenced by a small Soret
band in the absorption spectrum (Figure S12). It is possible that
the speciation of P-3 is different in water than it is in organic
solvents; additional charge (e.g., [P−OH]+) may prevent the
molecule from crossing the bacterial cell wall. The absorption
spectrum of P-3 in the buffer is pH-dependent, which likely
reflects changes in the protonation state of the molecule (Figure
S13); such events are more likely for a hydroxide ligand rather
than an oxo.
Encouraged by the successful heme reconstitution of an sGC
from Chlamydomonas reinhardtii to yield a functional enzyme,102
we then turned to reconstitution methods. Both Pa HasA and Cs
H-NOX were expressed using the RP523(DE3) strain of E. coli.
When RP523 cells are grown anaerobically, the bacteria can
grow in the absence of heme, enabling apoprotein isolation. This
methodology circumvents the harsh, denaturing conditions
necessary to remove the heme.19,29 The apoprotein was isolated
in moderate yield: 2 mg/L for Cs H-NOX and 5 mg/L for Pa
HasA. While Pa HasA is stable for over 1 year in imidazole-free
buffer at 4 °C, Cs H-NOX undergoes complete degradation over
the course of several weeks at 4 °C (or several hours at room
temperature), as evidenced by SDS-PAGE. If the H-NOX
protein is instead stored at 4 °C in buffer containing 300 mM
imidazole, then the protein remains intact for over 1 year. This
observation is consistent with a contaminating metalloprotease;
imidazole coordination to the active-site Zn center inhibits
activity. Leveraging the thermal stability of Cs H-NOX, heat
denaturation can be utilized to inactivate any protease
contaminants. Although there are several thermostable (>49
°C) E. coli proteins, none are proteases.103 After heating the
crude Cs H-NOX lysate at 60 °C for 1 h, the IMAC-purified
protein is stable for at least 4 months in an imidazole-free buffer
when stored at 4 °C. It was determined that 60 °C was the
optimal temperature because the majority of E. coli proteins
denatured at this temperature while preserving the apo H-NOX,
which significantly degrades above 66 °C (Figure S14).
Conjugate Preparation and Characterization. The
conditions used for protein reconstitution were surveyed using
apo H-NOX. A DMSO solution of P-3 was added dropwise to a
solution of the protein, and the resultant mixture was gently
rocked at room temperature for 2 h. The reconstituted protein
was separated from excess corrole on a PD-10 G-25 desalting
column. The eluent was collected in 1 mL fractions and analyzed
by UV−vis absorption spectroscopy (Figure S15). The first
fraction exhibited a split Soret band (∼400 nm) that is
significantly red shifted relative to the free corrole (∼380 nm),
indicating incorporation in the heme-binding pocket. Later
fractions have a Soret band comparable to the corrole in buffer,
suggestive of contamination from the unbound corrole. While
this spectral feature does not decrease after dialyzing the sample
overnight, it is readily removed by passage through a second PD-
10 desalting column (Figure S16). Perhaps the excess corrole
can nonspecifically bind to the H-NOX surface, if given a
sufficient amount of time.
In order to optimize P-3 incorporation in H-NOX, the effects
of buffer identity and salt concentration were surveyed for the
buffer used for both the reconstitution reaction and the desalting
column (Figure S17). The buffer (phosphate at pH 7.4 vs TEA
at pH 7.5) used for the reconstitution reaction had a nominal
effect on P-3 incorporation. The inclusion of 250 mM NaCl in
the desalting buffer eliminated nonspecific binding to the
protein surface, but drastically decreased the level of corrole
incorporation. Next, the pH dependence of the TEA desalting
buffer was examined over the 6.5−8.5 range, and it was found
that P-3 incorporation decreased monotonically with increasing
pH (Figure S18). A less dramatic effect was observed for the pH
dependence of the reconstitution reaction, although pH 7.5 was
optimal (Figure S19). Additional experiments were performed
using TEA (pKa = 7.8) (Figure S20), TEA·HCl at pH 4.8
(Figure S21), and MES buffer (pKa = 6.1) (Figure S22), but
these conditions did not improve P-3 incorporation.
P-3 incorporation in Cs H-NOX is maximized when the
reconstitution reaction is performed in phosphate buffer at pH
7.5 and the PD-10 desalting column is run using TEA buffer at
pH 6.5 (Figure S23a). These optimized conditions were then
utilized to incorporate P-3 into Pa HasA and led to high levels of
corrole incorporation (Figure S23b). Importantly, these samples
are quite stable when stored at 4 °C protected from light,
resulting in nominal corrole loss after nearly 2 months (Figure
S24). In the case of P-3 H-NOX, a 13% loss of corrole was
observed after 56 days, while a 5% loss was observed for P-3
HasA after 44 days.
Figure 3a,b compares the absorption spectra of P-3 with the
H-NOX and HasA conjugates. A significant red shift of the Soret
band is observed upon protein binding and the spectrum is
comparable to P-2 in CHCl3 (Figure 2, Table 1). Another
distinction in the absorption spectra of free and protein-bound
P-3 is the width of the Q(0,0) transition ∼560 nm. While the full
width at half maximum (fwhm) of this feature is 25 nm for P-3,
the values are 13 and 14 nm for the H-NOX and HasA
conjugates, respectively, which is similar to the 15 nm fwhm for
P-2 in CHCl3. These results suggest that P-2 in CHCl3 is a better
standard for comparison than P-3 in an aqueous buffer. Indeed,
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times weaker than the emission from the HasA conjugate
(Figure 3c). This is more clearly illustrated in Figure 4, which
compares the emission of absorbance-matched samples of P-2,
P-3, as well as the protein conjugates.
Figure 4. Emission spectra of absorbance-matched samples (A(520) =
0.0575 0.0005) of P-3-substituted H-NOX (), HasA (red ), and
P-3 (blue ) in TEA buffer at pH 6.5, as well as P-2 (green ) in
CH2Cl2.
It was our expectation that conjugate formation would
enhance the fluorescence intensity (and quantum yield) relative
to the free corrole because binding in the heme pocket would
restrict the conformational flexibility of the molecule, decreasing
nonradiative deactivation of the excited state. We also expected
that the fluorescence enhancement would be greater for H-NOX
because hydrogen bonding of the propionate chains to the Y−
S−R motif would greatly restrict the conformational flexibility of
the corrole, whereas the carboxylate groups of the propionate
chains are solvent exposed in HasA. Thus, we predicted that
ϕ(P-2) < ϕ(P-3 HasA) < ϕ(P-3 H-NOX). It should be noted
that P-2 is a better comparison for the conjugates than P-3 (vide
supra). However, we find that ϕ(P-3 H-NOX) = 2.5% < ϕ(P-2)
= 3.9% < ϕ(P-3 HasA) = 7.3% (Table 2), and this is
corroborated by the emission spectra of Figure 4. Time-resolved
measurements (Figure S25) indicate that the excited-state
lifetime for the H-NOX conjugate is 1.5 ns, which is similar to
that of P-2 (1.2 ns). Conversely, the lifetime of the HasA
conjugate is longer (3.1 ns) and quite similar to that of P-3 (3.8
ns). As in the case of P-3, a fit of the HasA data to a biexponential
decay function nominally improves the fit (τ1 = 1.9 ns, 89%; τ2 =
5.0 ns, 11%), but the physical significance of this two-
component fit is unclear. Using this data and eq 2, we find
that the nonradiative rate constant knr decreases upon protein
conjugation: 1.2-fold decrease for H-NOX and 2.7-fold decrease
for HasA (relative to P-2). While this result confirms our
hypothesis, it is counter to the expectation that H-NOX would
restrict the conformation of the corrole more than HasA and
result in a lower knr. It should be noted that the relative intensity
of the Q(0,1) vibrational shoulder (∼620 nm) decreases upon
protein binding and this feature is highly suppressed for the H-
NOX conjugate. The Q(0,0) to Q(0,1) intensity ratios are as
follows: 4.61 for P-3 < 5.27 for P-2 < 5.49 for HasA <7.41 for H-
NOX. This result suggests that the corrole conformation is more
restricted in H-NOX than in HasA. Since the Q(0,0) to Q(0,1)
intensity ratio does not align with the observed values of knr,
there are likely additional factors that dictate the photophysics of
these conjugates.
Figure 3. (a) Absorption (solid lines) and emission (dashed lines)
spectra of P-3 () and the H-NOX conjugate (red ) in TEA buffer
at pH 6.5. (b) Absorption (solid lines) and emission (dashed lines)
spectra of P-3 () and the HasA conjugate (blue ) in TEA buffer at
pH 6.5. (c) Comparison of the absorption (solid lines) and emission
(dashed lines) spectra of the H-NOX (red ) and HasA (blue )
conjugates of P-3. For the emission spectra, the absorbance of the
samples was matched at the excitation wavelength: A(520) = 0.0735
0.0005.
the extinction coefficients for P-2 were measured in CHCl3
because this solvent has a similar dielectric constant (ε =
4.806)62 to the hydrophobic interior of proteins (ε = 6−7).104
For the H-NOX conjugate (Figure 3a), the observed Soret to
protein (280 nm) ratio is 4.88. Based on the extinction
coefficients of P-2 and H-NOX (and accounting for corrole
absorbance at 280 nm), the expected ratio is 5.06, indicating that
>96% of the protein is in the holo form. Similarly, the observed
Soret to 280 nm ratio for the HasA conjugate (Figure 3b) is 5.33,
which is similar to the expected ratio of 5.18. These nominal
deviations from the expected value could reflect differences in
the extinction coefficient in the protein environment versus
organic solvent. Nevertheless, the observed ratios indicate a high
extent of P-3 incorporation in both proteins.
Figure 3c compares the absorption and emission spectra of
the H-NOX and HasA conjugates. The H-NOX conjugate
displays a split Soret band, while the HasA conjugate displays a
single transition. This result suggests that there are distinct
corrole−protein interactions in HasA and H-NOX, which could
account for the difference between the observed and expected
Soret to 280 nm ratios. Unexpectedly, the emission intensity of
the H-NOX conjugate is weak, exhibiting a signal that is 4.7
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P(OH)2 complex of TPFC,44 whereas ϕf = 13% for the free-
base.90 In the case of corrole 2, the free-base has a higher ϕf than
the phosphorus complex: 5.9% versus 3.9% (Table 1). It is
unlikely that the difference in substitution pattern (meso vs β)
accounts for this observation. Indeed, the fluorescence lifetime
of H3-2 is comparable to free-base meso-triaryl corroles (4−6
ns),90 and the 2−4 fold decrease in kr likely reflects the
molecular motions of the propionate chains. Since the excited-
state dynamics of H3-2 is consistent with free-base meso-triaryl
corroles, it is expected that P-2 should behave similarly to
phosphorus meso-triaryl corroles. The weak fluorescence and
short 1.2 ns lifetime of P-2 may be due to solvent interactions
that result in the formation of nonradiative decay channels. Since
the excited singlet state of P(V) triaryl corroles is a potent
oxidant,106 charge-transfer to solvent could occur, thereby
enhancing nonradiative deactivation of the excided state. If this
were the case, it would be expected that this is a general
phenomenon for all P(V) corroles. However, six-coordinate P
corroles and related analogues exhibit longer singlet excited state
lifetimes (3−4 ns).106−109 Alternatively, the weak fluorescence
of P-2 likely reflects differences in phosphorus speciation. Five-
coordinate meso-triaryl corroles can be obtained by treating the
six-coordinate complex with TFA, and it was shown that this
five-coordinate species exhibits a lower fluorescence quantum
yield (27%) and shorter excited-state lifetime (1.61 ns) than the
analogous six-coordinate complex (ϕ = 44%, τ = 3.01 ns).91 The
short lifetime (1.2 ns) and low quantum yield of five-coordinate
P-2 are consistent with these observations. While the speciation
accounts for the short lifetime, the underlying chemical
explanation for this observation remains unknown.
The short lifetime (1−2 ns) of the phosphorous corrole is
consistent for P-2, P-3, and the protein conjugates. Although the
excited state of P-3 and the HasA conjugate exhibits
biexponental kinetics, the major contribution to the fit
(∼85%) falls in this range. Despite the similarity in lifetime,
the fluorescence quantum yields are quite variable, reflecting
differences in the nonradiative decay of the excited state. The
protein environment surrounding the corrole likely dictates the
photophysical properties of P-3. Both Cs H-NOX and Pa HasA
have a tyrosine residue at the heme-binding pocket; the phenol
side chain is redox active, albeit at high potentials (1.46 V vs
NHE).110 However, since P(V) corroles are potent photo-
oxidants (∼1.2 V vs Ag/AgCl or 1.4 V vs NHE),106 tyrosine
oxidation could contribute to the nonradiative deactivation of P-
3 in the protein conjugates. Using electrochemical and
spectroscopic data, the reduction potential of the singlet excited
state can be estimated. Given the structural and spectral
similarity of P-2 and the P(V)O complex of OEC,82 it is
expected that the electrochemical properties will also be similar.
Using the one-electron reduction potential of PO(OEC) (−1.58
vs SCE or −1.34 V vs NHE)82 and the energy of the Q(0,0)
transition of P-2 (2.19 V), the estimated excited-state reduction
potential is 0.85 V versus NHE. Consequently, these five-
coordinate phosphorus corroles are considerably weaker photo-
oxidants than six-coordinate derivatives by 0.55 V. While
proton-coupled electron transfer lowers the barrier for tyrosyl
radical generation,110 it seems unlikely that this process is
operative for P-3. A more likely contribution to nonradiative
decay of P-3 is histidine residues in the heme-binding pocket.
Amines are well-known fluorescence quenchers. Since P-3 does
not bind histidine, the nitrogen lone-pair of the imidazole side
chain can quench corrole fluorescence through photo-induced
electron transfer. Both Cs H-NOX and Pa HasA have heme-
DISCUSSION
■
Phosphorus meso-triaryl corroles exhibit red-orange emission
profiles (λmax > 590 nm) with high fluorescence quantum yields
(ϕf = 20−50%).42,44,105 We hoped to translate these favorable
optical properties into a protein environment with P-3, thus
generating fluorescent proteins with optical properties that are
superior to traditional fluorescent proteins. However, P-2 and P-
3 exhibit yellow-orange emission with a maximum at 566 nm,
which is significantly blue-shifted relative to phosphorus meso-
triaryl corroles. This observation can readily be rationalized with
a qualitative molecular orbital diagram (Figure 5). The b1
Figure 5. Qualitative molecular orbital diagram illustrating the
perturbation of the four frontier orbitals for an unsubstituted corrole
core upon meso-aryl substitution and β-alkyl substitution.
HOMO is significantly more destabilized by an electron-
donating meso-substituent than a β-substituent because this
orbital has a greater density at the meso positions relative to the β
positions. As a result, the HOMO−LUMO gap (ΔE) for meso-
substitution is smaller than β-substitution (ΔEmeso < ΔEβ),
which manifests in lower energy (longer wavelength) emission
for meso-triaryl corroles relative to β-octaalkyl corroles.
While the majority of P(V) meso-triaryl corroles are six-
coordinate species, P-2, P-3, and other β-octaalkyl corroles are
five-coordinate. It seems that the coordination number
correlates with the nature of the peripheral substituents.
Phosphorus triaryl corroles exhibit an equilibrium between
five- and six-coordinate species, as observed by solution 31P
NMR. Electron-donating substituents give rise to a 1:1 mixture
of five- and six-coordinate complexes, whereas electron-
withdrawing groups (e.g., 4-NO2-phenyl, 4-CN-phenyl) yield
primarily six-coordinate species.42 Similarly, P corroles with
fluorinated aryl groups are exclusively isolated as six coordinate
complexes.44,87 Perhaps the reduced electron density of the
macrocycle increases the effective positive charge on the P
center. This leads to an increase in the ionic character of the
bonding situation, which could readily be offset with two anionic
ligands. Conversely, β-substituted corroles with electron-
donating alkyl substituents are exclusively isolated as five-
coordinate species.82,83 Perhaps this higher electron density at
the P center increases the covalent character of the bonds; this
may be efficiently utilized for π-bonding to result in the isolation
of terminal oxo (PO) derivatives.
Although phosphorus complexes of β-octaalkyl corroles have
been previously characterized,82,83 the emission properties of
these molecules have not yet been reported. The fluorescence
quantum yield of P-2 is unexpectedly low. This can partially be
explained by the conformational flexibility of the propionate
chains, which would engender molecular motions that readily
deactivate the excited state. In general, six-coordinate P(OH)2
corrole complexes exhibit higher fluorescence quantum yields
than the corresponding free-base. For example, ϕf = 31% for the
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ligating histidine residues, which are not participating in a
bonding interaction with P-3, that are sufficiently close to the
corrole cofactor to quench fluorescence. These hypotheses can
be tested through site-directed mutagenesis, but such experi-
ments are beyond the scope of this study.
The absorption spectrum of P-3 in buffer is significantly
different from that of the protein-bound corrole (Figure 3).
Additional evidence of protein-induced changes includes a
decrease in the fwhm of the Soret and Q bands, as well as the
suppression of the Q(0,1) emission band. One interesting
observation is the splitting of the Soret band for the H-NOX
protein. Protein binding sharpens the Soret band to enable
resolution of the x and y polarizations of this transition. DFT
calculations demonstrate the presence of two orthogonally
polarized transitions (vide supra), but they are rarely resolved
experimentally.84 It is likely that hydrogen bonding of the
propionate chains to the Y−S−R motif in H-NOX helps to
differentiate the dipole moments along the x and y axes of the
corrole, enabling the resolution of the polarized Soret
transitions. Conversely, HasA lacks this Y−S−R motif and the
propionate chains are solvent exposed; the lack of a hydrogen-
bonding partner does not enable sufficient differentiation of the
x and y dipole moments, resulting in the experimental
observation of a single Soret transition.
Despite the low fluorescence intensity, the emission profile is
concentrated in a relatively sharp band (fwhm = 13−14 nm).
This is in contrast to traditional fluorescent proteins,111 which
exhibit broad emission profiles that can span over 200 nm, as in
the case of mHoneydew, mBanana, and mTangerine (Figures 6a
and S26). Of the examples illustrated in Figures 6a and S26,
mKO (Kusabira Orange) and mKO2 have the narrowest peak
for the main emission transition (fwhm = 27−30 nm), but this is
nearly twice as broad as the H-NOX and HasA conjugates. As a
result, the protein conjugates of P-3 are better suited for FRET-
based and ratiometric applications because the emission
intensity is concentrated in a significantly smaller spectral
window. This attribute results in enhanced emission intensity,
despite their low fluorescence quantum yields. Figure 6b shows
the emission profiles of the fluorescent proteins in Figure 6a that
have been scaled to the quantum yield of the protein. In this way,
the emission intensity of the proteins is directly comparable.
Although the HasA conjugate exhibits an 8.5-fold lower
fluorescence quantum yield than mKO2, the corrole conjugate
only exhibits a 5-fold decrease in relative fluorescence intensity
at the emission maximum. Similarly, the HasA conjugate
exhibits only a 1.7-fold decrease in emission intensity relative
to mTangerine, despite having a 4-fold lower fluorescence
quantum yield. The most striking example is a comparison of the
HasA conjugate and mBanana; while the quantum yield is nearly
10 times less, the emission intensity of HasA is only 4-fold lower
than mBanana. Finally, the H-NOX conjugate and mHoneydew
display equal emission intensity, but the HasA conjugate has a 5-
fold lower quantum yield. These comparisons illustrate that,
despite their low fluorescence quantum yield, the P-3 conjugates
of H-NOX and HasA offer several advantages over traditional
fluorescent proteins.
Figure 6. (a) Normalized emission spectra of the P-3 conjugates and
traditional fluorescent proteins that emit in a similar spectral window:
P-3 H-NOX (), P-3 HasA (red ), mBanana (yellow ),
mHoneydew (green ), and mKO (blue ). Spectral data for the
fluorescent proteins was obtained from the online fluorescent protein
database FPbase (fpbase.org).111 (b) Emission spectra of fluorescent
proteins that is scaled by quantum yield: P-3 H-NOX ϕ = 2.5% (), P-
3 HasA ϕ = 7.3% (red ), mBanana ϕ = 70% (yellow ),
mHoneydew ϕ = 12% (green ), mKO ϕ = 60% (blue ), mKO2 ϕ =
62% (violet ), and mTangerine ϕ = 30% (orange ). Quantum yield
data was obtained from FPbase (fpbase.org).111
enhanced in the HasA conjugate, while it is quenched in H-
NOX, relative to the free molecule. This observation is counter
to the expectation that corrole emission would be most
enhanced in H-NOX because of the buried heme pocket and
hydrogen bonding with the Y−S−R motif, which would limit the
conformational flexibility of the corrole. Although vibrational
motions are more restricted in H-NOX relative to HasA (i.e.,
suppression of the Q(0,1) transition), the nonradiative rate
constant is slower in HasA than H-HOX. This disparity suggests
that environmental factors in the heme-binding pocket are
playing a significant role in dictating the photophysical
properties of these conjugates.
We are currently working to improve the optical properties of
the conjugates by modifying both the corrole and the heme-
binding pocket of the proteins. Conversion of five-coordinate P-
2 to a six-coordinate species (as a difluoride (PF2),44 dialkyl
(PR2),82 or dialkoxide (P[OR]2)42,89) should enhance the
fluorescence of the corrole. However, it is unclear if or how such
a six-coordinate species would bind to the H-NOX and HasA
proteins. Alternatively, modification of the corrole by β-
fluorination or β-trifluoromethylation would reduce the electron
density of the macrocycle and could enable the isolation of stable
Al and Ga complexes. These derivatives are expected to exhibit
more red-shifted emission profiles and higher fluorescence
quantum yields than the corresponding P complex. So far,
attempts to adapt the synthesis of 1 using 3,4-difluoropyrrole
instead of 3,4-dimethylpyrrole have been unsuccessful. Addi-
tionally, it is unclear exactly how the corrole complex is
CONCLUSIONS
■
We have presented a proof-of-principle study demonstrating
that fluorescent phosphorus corroles can be incorporated into
the protein scaffolds of Cs H-NOX and Pa HasA. Distinctive
changes in the absorption and emission spectra confirm corrole
binding in the heme pocket of the proteins. Corrole emission is
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c03599.
■
sı
NMR spectra, results of DFT and TD-DFT calculations,
as well as additional experimental details and results
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Characterization of Ratiometric, pH Sensing Nanoparticles with
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AUTHOR INFORMATION
Corresponding Authors
■
Christopher M. Lemon − Department of Molecular and Cell
Biology, California Institute for Quantitative Biosciences
(QB3), and Miller Institute for Basic Research in Science,
University of California, Berkeley, Berkeley, California 94720,
United States; orcid.org/0000-0001-9493-5488;
Email: clemon@berkeley.edu
Michael A. Marletta − Department of Molecular and Cell
Biology, California Institute for Quantitative Biosciences
(QB3), and Department of Chemistry, University of
California, Berkeley, Berkeley, California 94720, United
States; orcid.org/0000-0001-8715-4253;
(10) Boon, E. M.; Huang, S. H.; Marletta, M. A. A Molecular Basis for
NO Selectivity in Soluble Guanylate Cyclase. Nat. Chem. Biol. 2005, 1,
53−59.
(11) Erbil, W. K.; Price, M. S.; Wemmer, D. E.; Marletta, M. A. A
Structural Basis for H-NOX Signaling in Shewanella oneidensis by
Trapping a Histidine Kinase Inhibitory Conformation. Proc. Natl. Acad.
Sci. U. S. A. 2009, 106, 19753−19760.
(12) Weinert, E. E.; Plate, L.; Whited, C. A.; Olea, C.; Marletta, M. A.
Determinants of Ligand Affinity and Heme Reactivity in H-NOX
Domains. Angew. Chem., Int. Ed. 2010, 49, 720−723.
(13) Herzik, M. A.; Jonnalagadda, R.; Kuriyan, J.; Marletta, M. A.
Structural Insights into the Role of Iron-Histidine Bond Cleavage in
Nitric Oxide-Induced Activation of H-NOX Gas Sensor Proteins. Proc.
Natl. Acad. Sci. U. S. A. 2014, 111, E4156−E4164.
(14) Horst, B. G.; Marletta, M. A. Physiological Activation and
Deactivation of Soluble Guanylate Cyclase. Nitric Oxide 2018, 77, 65−
74.
(15) Guo, Y.; Marletta, M. A. Structural Insight into H-NOX Gas
Sensing and Cognate Signaling Protein Regulation. ChemBioChem
2019, 20, 7−19.
(16) Senge, M. O.; MacGowan, S. A.; O’Brien, J. M. Conformational
Control of Cofactors in Nature - The Influence of Protein-Induced
Macrocycle Distortion on the Biological Function of Tetrapyrroles.
Chem. Commun. 2015, 51, 17031−17063.
(17) Boon, E. M.; Marletta, M. A. Sensitive and Selective Detection of
Nitric Oxide Using an H−NOX Domain. J. Am. Chem. Soc. 2006, 128,
10022−10023.
(18) Winter, M. B.; McLaurin, E. J.; Reece, S. Y.; Olea, C.; Nocera, D.
G.; Marletta, M. A. Ru-Porphyrin Protein Scaffolds for Sensing O2. J.
Am. Chem. Soc. 2010, 132, 5582−5583.
(19) Winter, M. B.; Klemm, P. J.; Phillips-Piro, C. M.; Raymond, K.
N.; Marletta, M. A. Porphyrin-Substituted H-NOX Proteins as High-
relaxivity MRI Contrast Agents. Inorg. Chem. 2013, 52, 2277−2279.
(20) Nierth, A.; Marletta, M. A. Direct meso-Alkynylation of
Metalloporphyrins Through Gold Catalysis for Hemoprotein Engineer-
ing. Angew. Chem., Int. Ed. 2014, 53, 2611−2614.
(21) Binet, R.; Létoffé, S.; Ghigo, J. M.; Delepelaire, P.; Wandersman,
C. Protein Secretion by Gram-Negative Bacterial ABC Exporters - A
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Email: marletta@berkeley.edu
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c03599
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
C.M.L. acknowledges the Miller Institute at UC Berkeley for a
Postdoctoral Fellowship. This research was supported by a grant
from the National Institutes of Health (NIH): R01GM127854
(to M.A.M.). Calculations were performed using the Molecular
Graphics and Computation Facility at UC Berkeley, which is
partially supported by NIH S10OD023532. We are deeply
indebted to Dr. Benjamin Guthrie for his guidance with cloning
and protein expression. We thank Dr. Sho Takatori for providing
a sample of Pseudomonas aeruginosa PAO1 and Sarah Chen for
assisting with the expression of HasA. We thank Trevor Roberts
for acquiring time-resolved emission data as well as Professor
Naomi Ginsberg for allowing us to access the instrument. The
members of the Marletta lab are graciously thanked for critically
evaluating this manuscript.
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