Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group

Article abstract of DOI:10.1002/anie.201707212

Iron(III)-5,15-diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa, despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron-limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.

Full text of DOI:10.1002/anie.201707212

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: Construction and Crystal Structure Analysis of Heme Acquisition
Protein HasA Containing Iron(III)-5,15-Diphenylporphyrin and
Derivatives Thereof as an Artificial Prosthetic Group
Authors: Hiromu Uehara, Yuma Shisaka, Tsubasa Nishimura, Hiroshi
Sugimoto, Yoshitsugu Shiro, Yoshihiro Miyake, Hiroshi
Shinokubo, Yoshihito Watanabe, and Osami Shoji
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201707212
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10.1002/anie.201707212
Angewandte Chemie International Edition
DOI: 10.1002/anie.200((will be filled in by the editorial staff))
Artificial Hemoproteins
Construction and Crystal Structure Analysis of Heme Acquisition
Protein HasA Containing Iron(III)-5,15-Diphenylporphyrin and
Derivatives Thereof as an Artificial Prosthetic Group**
Hiromu Uehara†, Yuma Shisaka†, Tsubasa Nishimura, Hiroshi Sugimoto, Yoshitsugu Shiro, Yoshihiro
Miyake, Hiroshi Shinokubo, Yoshihito Watanabe, and Osami Shoji*
Abstract: Iron(III)-5,15-diphenylporphyrin (1) and its derivatives (2–
7) were accommodated by the heme acquisition protein HasA
secreted by Pseudomonas aeruginosa, despite possessing bulky
substituents at the meso-position of the porphyrin. Crystal structure
analysis revealed that the two phenyl groups at the meso-positions
of porphyrin extend outside HasA. It was shown that growth of P.
aeruginosa was inhibited in the presence of HasA coordinating the
synthetic porphyrins under iron-limiting conditions, and that the
structure of the synthetic porphyrins greatly affects the inhibition
efficiency.
Porphyrins are 18- electron aromatic macrocycles that are
composed of pyrrole subunits and found extensively in nature as
heme and chlorophyll. Metalloporphyrins are involved in a wide
variety of significant reactions in nature such as O2 activation
(cytochrome P450),[1] oxygen transport (hemoglobin),[2] and light-
energy harvesting (light-harvesting antennae complex).[3]
[*]
Dr. O. Shoji, H. Uehara†, Y. Shisaka†
Department of Chemistry, Graduate School of Science,
Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
Fax: (+81) 52-789-3557
E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp
Homepage: http://bioinorg.chem.nagoya-u.ac.jp/
Dr. O. Shoji, H. Sugimoto
Core Research for Evolutional Science and Technology,
Japan Science and Technology Agency, 5 Sanbancho,
Chiyoda-ku, Tokyo, 102-0075 (Japan)
Prof. Dr. Y. Shiro
Figure 1. (a) The structures of Fe-DPP and its derivatives used in
this study. (b) The heme acquisition system of Pseudomonas
aeruginosa. Under low-iron conditions, P. aeruginosa secrets apo-
HasA (PDB ID: 3MOK), which captures and transports heme (holo-
HasA, PDB ID: 3ELL) to the outer membrane receptor HasR.
Guraduate School of Life Science, University of Hyogo,
3-2-1 Kouto, Sayo, Hyogo 678-1297 (Japan)
Dr. H. Sugimoto
Given their biological importance, countless porphyrin
derivatives with various structures have been prepared so that
models of heme proteins can be constructed and bioinspired
catalysts may be developed.[4] The function of heme proteins is
dependent upon the properties of the heme molecule; thus, the
reconstitution of heme proteins with synthetic porphyrins, such
as protoporphyrin IX complexed with a metal other than iron, and
heme molecules that are modified at the propionate groups,
have been widely studied.[5] However, the incorporation of
synthetic metal porphyrins with structures that differ from heme
RIKEN SPring-8 Center
1-1-1 Kouto, Sayo, Hyogo 679-5148 (Japan)
Prof. Dr. H. Shinokubo, Dr. Y. Miyake, T. Nishimura
Department of Molecular and Macromolecular Chemistry,
Graduate School of Engineering, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8603 (Japan)
Prof. Dr. Y. Watanabe
Research Center for Materials Science, Nagoya University
Furo-cho, Chikusa-ku, Nagoya, 464-8602 (Japan)
[†] These authors contributed equally to this work.
remains
challenging,
because
most
heme
proteins
accommodate heme with high selectively.
[] This work was supported by Grant-in-Aid for Young
Scientists (A) to O.S. (26708018) from the Ministry of
Education, Culture, Sports, Science, and Technology
(Japan) and JST CREST Grant Number JPMJCR15P3,
Japan. This work was also supported by JSPS KAKENHI
Grant Number JP15H05806 in Precisely Designed
Catalysts with Customized Scaffolding to O. S.
5,15-diphenylporphyrin (DPP) is a simple porphyrin that can
be prepared by acid-catalyzed condensation of dipyrromethene
with benzaldehyde in relatively good yield (25–40%),[6] and
highly symmetric DPP derivatives with substitution at the meso
and/or meso-phenyl group can also be prepared in reasonable
yield.[7] The ease of access to DPP derivatives has prompted
their use as building blocks for the construction of multi-
Supporting information for this article is available on the
WWW under http://www.angewandte.org.
1
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10.1002/anie.201707212
Angewandte Chemie International Edition
porphyrin architectures[8] and catalysts.[9] However, neither DPP
nor any of its derivatives have yet been complexed with natural
heme proteins. Indeed, the incorporation of such compounds
into natural heme proteins is expected to be difficult, because of
the two bulky phenyl groups with perpendicular orientation to the
porphyrin plane. However, there are reports that de novo
designed peptides can accommodate DPP derivatives.[10] If it
was possible to incorporate DPP or its derivatives into natural
heme proteins, then these compounds could be used as
synthetic prosthetic groups. It may thereby be possible to tune
the biological function of heme proteins by changing the central
metal ion and substituent groups. Herein, we report the first
artificial metalloproteins incorporating Fe-DPP (1)[11] and its
derivatives 2–7 by using the extracellular heme acquisition
protein HasA secreted by Pseudomonas aeruginosa (Figure 1).
P. aeruginosa secretes HasA (apo-HasA)[12] under low-iron
conditions to acquire heme as an iron source.[13] HasA captures
heme and transports it to the specific receptor HasR, which is
expressed in the outer membrane of P. aeruginosa (Figure
1b).[14] The heme is then taken up into cells via HasR where it is
degraded for use as an iron source. The crystal structure of
heme-bound HasA (holo-HasA) showed that the heme is
coordinated by His32 and Tyr75, located in two independent
loops, which hold the heme molecule in a manner akin to a pair
of tweezers (Figure 1b).[15] The captured heme is highly exposed
to the solvent, with three of the four pyrrole rings of heme
accessible from outside the protein. This unique mode of heme
binding in HasA suggested that it may be possible for this protein
to accommodate DPP, including its two bulky phenyl groups at
the meso-position, and we investigated whether HasA could
indeed capture Fe-DPP (1). We also assessed the level of
incorporation of several Fe-DPP derivatives (2–6) and iron(III)-
5,15-diazaporphyrin (7).[16] Given that growth of P. aeruginosa
was shown to be inhibited in the presence of HasA coordinated
to iron phthalocyanine (Fe–Pc),[17] we also investigated how the
structure of the synthetic porphyrins captured by HasA affect the
growth of P. aeruginosa under iron-limiting conditions. We
envisaged that HasA containing suitably modified porphyrin
derivatives could also serve as growth inhibitors.
We started our investigation by examining the complex
formation of HasA with Fe-DPP (1). HasA without heme (apo-
HasA) was added to a solution of 1 in DMSO. After removal of
DMSO by dialysis, HasA with 1 was purified by anion-exchange
column chromatography. The UV-Vis spectrum of the resulting
brown solution showed absorption at 412 nm, which is
assignable to the Soret absorption band of 1 (Figure 2a). ESI-
TOF-MS analysis gave a peak with an m/z value corresponding
to that of HasA complexed with 1, and the ratio of peak intensity
of the complex to that of apo-HasA was the same as that for the
complex with heme-bound-HasA (Figures 2b and S1). Given that
the ratio of peak intensity of apo-HasA to HasA with synthetic
porphyrin reflects the stability of the complex,[18] HasA
complexed with 1 appears to be as stable as heme-bound HasA.
To further confirm the binding of 1 by HasA, a crystal structure of
the complex of HasA with 1 was obtained at 2.0 Å resolution
(Figure 2c). As was observed for heme-bound-HasA, the
structure showed clear electron density for 1 at the heme-binding
position, with both His32 and Tyr75 ligated to the iron of 1
(Figure 2d). The phenyl rings of 1 were accommodated in two
distinct sites, one formed by His32(C=O), Pro34,
Figure 2. (a) UV-Visible spectra of HasA with Fe-DPP (1; red) and without Fe-DPP (apo-HasA; gray) in PBS solution. Protein solutions of apo-
HasA and HasA with Fe-DPP (1) are shown in the inset. (b) ESI-TOF-MS spectrum of HasA with Fe-DPP (1) in 20 mM ammonium acetate
buffer. (c)–(f) X-ray crystal structure of HasA incorporating Fe-DPP (1) (PDB ID: 5XIB): (c) Overall structure of HasA coordinating Fe-DPP (1) is
indicated as pink sticks. (d) An enlarged view of the complex binding domain in HasA. The 2Fo-Fc electron density map of Fe-DPP (1) contoured
at the 1.0 level is shown in gray mesh. (e) Side views of Fe-DPP (1) and the surrounding amino acids of HasA. The two phenyl groups at the
meso-position of Fe-DPP (1) are shown in cyan. (f) Superimposition of HasA with Heme (PDB ID: 3ELL) and Fe-DPP (1). The angle of the
porphyrin of Fe-DPP and heme differs by approximately 45 rotation. In all structures, oxygen, nitrogen, and iron are indicated in red, blue, and
orange, respectively.
2
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10.1002/anie.201707212
Angewandte Chemie International Edition
Arg129, and Tyr138, and the second site composed of Thr43,
Gly44(C=O), Gly45, Phe46, Pro50(C=O), and Phe51 (Figure 2e).
same manner as holo-HasA and inhibit heme acquisition through
holo-HasA. In particular, we wished to establish whether
differences in the structure of the synthetic metal complexes 1–7
affect the inhibition of P. aeruginosa, as overall structures of
HasA containing synthetic porphyrins are identical irrespective of
the structure of synthetic porphyrins (1–4, and 7) examined in
this research (Figure S2). Initially, HasA with porphyrin
derivatives 1–7 were added to a culture solution of P. aeruginosa
to examine whether the organism could utilize the synthetic
porphyrins as a sole iron source. Under iron-limiting conditions P.
aeruginosa did not grow, showing that it is not able to use
synthetic porphyrins 1–7 as an iron source. Growth inhibition of
P. aeruginosa in the presence of HasA complexed with
Interestingly, the porphyrin ring of
1
was rotated by
approximately 45° with respect to the heme of holo-HasA (Figure
2f). This apparently serves to avoid steric crowding caused by
the bulky phenyl groups at the meso-position. As evident from
the crystal structure analysis, the overall structure of HasA
ligating 1 is essentially identical to that of heme-bound HasA
(root-mean-square deviation (RMSD) over 2–184 amino-acid
residues for C atoms: 0.18), which indicates that structural
perturbation upon binding of 1 is negligible.
Considering the stable complex formation between 1 and
HasA, we examined Fe-DPP derivatives 2–7, as shown in Figure
1a. ESI-TOF-MS analysis gave peaks corresponding to HasA
complexed with 2–7, although peaks for complexes of HasA with
3[19] and 6 could only be detected in low amounts, suggesting
that HasA does not form stable complexes with the latter
derivatives (Figure S1). It was possible to crystallize HasA with
2–7 under similar conditions to those used to crystallize HasA
with 1, and crystal structures of HasA complexed with 2, 3, 4,
and 7 were obtained (Figure 3). In all cases, clear electron
density of the DPP derivative was observed at the heme-binding
site, with the overall structures being identical to that of heme-
bound-HasA (Figures 3a, c, e, g, and S2). The angle of the
porphyrin rings and the location of two phenyl rings were the
same as for Fe-DPP (1) bound to HasA. The crystal structure of
the complex with
2 showed that its ethynyl group was
accommodated in the hydrophobic pocket, composed of Phe46,
Tyr56, Leu85, His134, Val137, and Met141, located deep within
the heme-binding site where, in the natural system, the vinyl
group of heme is accommodated (Figures 3b and S3). In
contrast to 2, the phenyl ring at the 10-position of 3 was
observed at the opposite side (outside of HasA) and exposed to
the solvent (Figure 3d); the electron density of this phenyl group
is partially disordered compared with the phenyl groups at the 5-
and 15-positions, suggesting that the 10-phenyl is not so rigidly
held, possibly due to the steric repulsion between the ring and
the two loops of HasA, and particularly residues Val37 and
Phe78 (Figure S4). This is consistent with the observation that
ESI-TOF-MS analysis gave a weaker signal for HasA complexed
with 3, indicative of unstable binding. These results showed that
the phenyl group at the 10-position of 3 is not accommodated in
the hydrophobic pocket located in the heme-binding site
because of its steric bulk. In fact, iron(III)-5,10,15,20-
tetraphenylporphyrin (TPP) was not accommodated in HasA.[17]
As evident from the crystal structure of HasA in complex with 4,
the ethynyl group can be located both inside and outside of the
heme-binding site (Figure 3f). Notably,
7
was also
accommodated by HasA even though the meso-carbon atoms
were replaced by nitrogen atoms (Figure 3g, h). Crystal structure
analysis revealed that HasA can accommodate Fe-DPP (1), 2, 3,
4, and 7 without any structural perturbation (Figure S2).
Finally, we evaluated the inhibition activity of HasA with
synthetic porphyrins 1–7. We previously reported that iron
phthalocyanine (Fe–Pc)–bound HasA markedly inhibits the
growth of P. aeruginosa under iron-limiting conditions, even in
the presence of heme-bound HasA (holo-HasA), which can
provide heme as an iron source.[17] We reasoned that Fe–Pc
bound HasA interacts strongly with its specific receptor, HasR, in
the outer membrane of P. aeruginosa, resulting in inhibition of
heme uptake via holo-HasA. Given that the overall structures of
HasA containing synthetic porphyrins 1–7 are identical to that of
holo-HasA, we expected that they could interact with HasR in the
Figure 3. X-ray crystal structures of HasA with Fe-DPP derivatives:
Fe-ethynylDPP (2; cyan; a, b; PDB ID: 5XIE); Fe-triPP (3; purple; c,
d; PDB ID: 5XIC); Fe-bis(ethynyl)DPP (4; orange; e, f; PDB ID:
5XKB); and Fe-diazaDPP (7; green; g, h; PDB ID: 5XA4). (a, c, e, g)
An enlarged view of complex binding domain in HasA. The 2Fo-Fc
electron density maps of each complex (2, 3, 4, and 7) contoured at
the 1.0 level are shown as a gray mesh. (b, d, f, h) Superimposition
of holo-HasA (white; PDB ID: 3ELL) and HasA coordinating Fe-DPP
derivatives 2, 3, 4, and 7. The upper side of these figures
corresponds to the outside, the opposite side of the hydrophobic
pocket (inside). In all structures, oxygen and nitrogen are shown in
red and blue, respectively.
3
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10.1002/anie.201707212
Angewandte Chemie International Edition
porphyrins 1–7 was then examined. Accordingly, P. aeruginosa
(PAO1) was first cultured in M9-based medium containing EDTA
as an iron scavenger, and subsequent growth of the organism
was monitored (absorption of the culture at 600 nm (OD600)) after
the simultaneous addition of 1 M of holo-HasA and 1 M of
HasA complexed with the synthetic porphyrins 1–7. Growth
inhibition of P. aeruginosa was observed for all HasA complexes.
The efficacy of growth inhibition depended upon the structure of
the porphyrin derivative employed (Figure 4a). Given that
complexes of HasA with either 1 or 6 elicited weaker growth
inhibition, further substitution at the meso-position of 1 with
bromine, ethynyl, or phenyl groups was thought to be critical for
efficient growth inhibition. Among the tested meso substituents,
bromo- (5) and ethynyl (2 and 4) groups were found to be more
suitable than a phenyl group at this position (3). Interestingly,
use of complex 7 with HasA led to intense inhibition of P.
aeruginosa growth. Given that we did not observe any clear
difference in the overall fold of HasA between the porphyrins 1–7
(Figure S2), interactions between HasA and HasR are assumed
to not differ significantly for this group. We assume that the
synthetic porphyrins 1–7 are transported from HasA to HasR,
whereupon they prevent the structural change of the receptor
HasR required for the release of apo-HasA. This blocking of the
receptor hinders heme transfer from holo-HasA to HasR,
resulting in growth inhibition (Figure 4b, c). Further studies are
necessary to elucidate the mechanism governing the observed
growth inhibition in more detail.
In conclusion, we have demonstrated that HasA secreted by
P. aeruginosa can accommodate Fe-DPP (1) and its derivatives
2–7 without any structural perturbation. Crystal structure analysis
revealed that two phenyl groups at the meso-position of the
porphyrins extend outside of HasA to avoid steric crowding and
are exposed to the solvent. To the best of our knowledge, this is
the first example of a natural protein that stably binds Fe-DPP
(1) and its derivatives 2–7. Furthermore, we discovered that
complexes of HasA with synthetic porphyrins inhibited the
growth of P. aeruginosa under iron-limiting conditions. The
structure of synthetic porphyrins greatly affected inhibition, with
either substitution at the meso-position or replacement of meso-
carbon atoms with nitrogen enhancing the inhibition efficiency.
We concluded that the critical step governing inhibition is not
interaction between HasA and HasR, but likely the interaction
between HasR and the synthetic porphyrins transported from
HasA to HasR. Although only a limited number of Fe-DPP
derivatives have been examined herein for complex formation
with HasA, further screening of synthetic porphyrins and/or
combination with amino-acid replacement (mutagenesis) to alter
the heme-binding site structure of HasA, based on the crystal
structure of HasA reported in this study, would expand the range
of synthetic porphyrins that can be accommodated by HasA. We
envisage that HasA could be used as a host protein to
accommodate various synthetic porphyrins to confer water
solubility as well as to adjust the characteristics of the molecules
such as electron potential and photophysical properties.
Although we reported herein on growth inhibition, we believe that
the combination of HasA with synthetic porphyrins can be used
as prospective biocatalysts by tailoring the active site via
mutagenesis for accommodation of substrates. Investigations
along these lines are under way in our research group.
Received: ((will be filled in by the editorial staff))
Published online on ((will be filled in by the editorial staff))
Keywords: synthetic porphyrins · heme proteins · artificial HasA ·
protein structures · growth inhibition
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Angewandte Chemie International Edition
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10.1002/anie.201707212
Angewandte Chemie International Edition
Entry for the Table of Contents
Artificial Hemoproteins
Iron(III)-5,15-diphenylporphyrin and its
derivatives were accommodated by
heme acquisition protein HasA secreted
by Pseudomonas aeruginosa despite
containing bulky groups at the meso-
position of the porphyrin. Crystal
structure analysis revealed that the two
phenyl groups at the meso-position of
the porphyrins extend outside of HasA.
__________ Page – Page
Construction and Crystal Structure
Analysis of Heme Acquisition Protein
HasA Containing Iron(III)-5,15-
Diphenylporphyrin and Derivatives
Thereof as an Artificial Prosthetic Group
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