Is CuII Coordinated to Patellamides inside Prochloron Cells?

Article abstract of DOI:10.1002/chem.201700895

Dinuclear Cu^II–patellamide complexes (patellamides are naturally occurring cyclic pseudo-octapeptides) are known to be efficient catalysts for hydrolysis reactions of biological importance, for example, those of phosphatase, carbonic anhydrase, and glycosidase. However, the biological role of patellamides is still unknown. Patellamides were originally extracted from the sea squirt Lissoclinum patella, but are now known to be ribosomally expressed by the blue-green algae Prochloron that live in symbiosis with L. patella. In a further step to unravel the metabolic significance of the patellamide complexes, the question as to whether these are also formed inside Prochloron cells is addressed. In this study, a biocompatible patellamide–fluorescent dye conjugate has been introduced into living Prochloron cells and, by means of flow cytometry and confocal microscopy, it is shown that Cu^II ions are coordinated to patellamides in vivo.

Full text of DOI:10.1002/chem.201700895

A Journal of
Accepted Article
Title: Is CuII coordinated to patellamides inside Prochloron cells?
Authors: Peter Comba, Annika Eisenschmidt, Lawrence R. Gahan,
Dirk-Peter Herten, Geoffrey Nette, Gerhard Schenk, and
Martin Seefeld
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201700895
Link to VoR: http://dx.doi.org/10.1002/chem.201700895
Supported by

Chemistry - A European Journal
10.1002/chem.201700895
II
Is Cu coordinated to patellamides inside Prochloron cells?
*
[
a]
[a]
[b]
[c]
[d]
Peter Comba* , Annika Eisenschmidt , Lawrence R. Gahan , Dirk-Peter Herten , Geoffrey Nette ,
[
b]
[c,e]
Gerhard Schenk and Martin Seefeld
[
a]
Universität Heidelberg, Anorganisch-Chemisches Institut and Interdisciplinary Center for Scientific Computing (IWR),
Im Neuenheimer Feld 270, 69120 Heidelberg, Germany. Email: peter.comba@aci.uni-heidelberg.de
[
b]
School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
[
c]
CellNetworks Cluster und Physikalisch-Chemisches Institut, Universität Heidelberg, Heidelberg, Germany.
Independent Marine Biochemistry Research, (IMBCR Pty. Ltd.) Point Lookout, Queensland 4183, Australia.
Biochemistry Center (BZH) Universität Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany.
[
d]
[
e]
*
Part of this article is reproduced from the PhD thesis of A.E.1
1
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
Abstract
II
Dinuclear Cu patellamide complexes (patellamides are naturally occurring cyclic pseudo-octapeptides)
are known to be efficient catalysts for hydrolysis reactions of biological importance, e.g. phosphatase,
carbonic anhydrase and glycosidase. However, the biological role of patellamides is still not known.
Patellamides were originally extracted from the sea squirt L. patella but are now known to be
ribosomally expressed by the blue-green algae Prochloron that live in symbiosis with L. patella. In a
further step to unravel the metabolic significance of the patellamide complexes, the question as to
whether these are also formed inside Prochloron cells is addressed. In this study a bio-compatible
patellamide-fluorescent-dye conjugate has been introduced in living Prochloron cells, and with flow
II
cytometry and confocal microscopy it is shown that Cu ions are coordinated to patellamides in vivo.
2
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
Introduction
Small cyclic octapeptides extracted from the blue-green algae Prochloron have been the subject of
intensive investigations regarding their putative biological role.1-3 They are commonly referred to as
"
patellamides", as they were initially isolated from the sea squirt Lissoclinum patella, the host of
4
II
Prochloron. Despite its biotoxicity, the concentration of Cu is significantly increased in the sea squirt
5
L. patella when compared to the surrounding sea water, and this has led to a detailed investigation of
the metal ion binding behavior of naturally occurring cyclic peptides like ascidiacyclamide and
6
patellamides A-D (Scheme 1). It was shown that the amide nitrogen atoms as well as the azole
II
nitrogens serve as binding sites for Cu complexation, with two azole-amide-azole binding sites per
peptide. To enable the investigation of in vitro coordination chemistry and also to make patellamide
derivatives available for structure-activity relationships, synthetic routes have been developed to
produce cyclic pseudo-peptides, delivering various model peptides as an alternative to the biosynthesis
and isolation from natural sources (Scheme 1).2, 7 Detailed investigations on the dinuclear CuII
complexes were carried out in combination with binding affinity measurements demonstrating that
the model ligands exhibit similar affinities as reported for the natural patellamides.2, 8-10
1
2
II
Scheme 1. From left to right: Patellamide D, model patellamides H
4
pat and H pat , dinuclear Cu complex of
4
2
-
H
4
pat ; X=substrate / H O / OH .
2
To shed light on the biological role of the patellamides, hydrolase studies were conducted showing
II
that the dinuclear Cu complexes of the synthetic peptides act as potent phosphatase and carbonic
anhydrase mimics at pH 7-8.11, 12 At pH 10-11, α- and β-glycosidase as well as β-lactamase activity was
1
observed, giving rise to the idea that metal complexes of the cyclic peptides might act as pH-
dependent catalysts. This is underpinned by a study which showed that the pH in L. patella varies
13
greatly from pH 7.5 in darkness to almost 11 in the light. Thus, all hydrolase activities observed to-
II
date might be equally metabolically important for Prochloron, and the dinuclear Cu complexes might
switch their activity from carbon-assimilation to carbohydrate degradation depending on the level of
irradiation. This pH-dependent function is known for metalloproteins as exemplified by the
3
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
organophosphate-degrading hydrolase from Agrobacterium radiobacter14 and purple acid
phosphatases.15
II
As a first step towards in vivo testing of the activities of Cu patellamide complexes with the ultimate
goal being the establishment of the true biological function(s) of the cyclic peptides, we now present
II
our results that answer the question asked in the title: is Cu coordinated to these ligands in cells?
II
Therefore, the fluorescence and fluorescence quenching by Cu coordination to a model patellamide-
dye conjugate, H pat-Atto550, introduced into living Prochloron cells, was analyzed by confocal
4
microscopy and flow cytometry.
Results
1
Ligand synthesis and structure of the putative copper complexes
The synthesis of the cyclic octapeptide H pat-Atto550 was accomplished analogously to the modular
4
approach used for the synthesis of other cyclic peptides prepared for our earlier studies (Scheme 2).7
The α-Boc ԑ-Cbz protected lysine was activated by treatment with isobutyl-chloroformate and coupled
to the keto ester 2 to give the resulting amidoketone 3, which was reacted with methylamine in the
presence of acetic acid under azeotropic removal of water from refluxing xylenes, delivering the
monomeric building block 4. The removal of the Boc protecting group was accomplished by addition
of TFA in DCM, which afforded 5. Analogous to the strategy reported for 4, the respective valine-
imidazole monomer was produced, which afforded after hydrolysis upon addition of NaOH in
dioxane/MeOH compound 6. The coupling of 5 and 6 was accomplished through COMU activation and
addition of EDIPA in anhydrous DMF and yielded the pseudo-dipeptide 7 in 55 % yield. 7 was
subsequently deprotected at the N-terminus, and coupled with 9, which was obtained analogously to
the described route to 7, yielding the linear pseudo-tetrapeptide 10 in 48 % yield. Deprotection of the
N- as well as the C-terminus produced 11, which was then cyclized in a high-dilution reaction to give
2
7 % of the Cbz-protected cyclic peptide 12. This was followed by deprotection of the ԑ-N-terminus
upon treatment with H and 5 % palladium on carbon producing 13, which could then be reacted with
the NHS activated ester of the reporter group Atto550. The fluorescence-tagged cyclic pseudo-
octapeptide H pat-Atto550, 14, was purified by HPLC and characterized by UHPLC-MS as well as by
2
4
UV/vis spectroscopy.
The choice of the right fluorescent dye was crucial, since Prochloron is one of the few prokaryotic
oxygenic autotrophs containing chlorophyll b in addition to chlorophyll a. This means that the range
for observation, often referred to as "green gap" between 520 nm and 600 nm, is relatively narrow.16
4
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
Therefore, as a photophysically sensitive group Atto550 was chosen with an excitation maximum of
exc=554 nm and an emission maximum of λem=574 nm.
λ
Scheme 2. Synthesis of H
accomplished analogously as described in .
4
pat-Atto550, 14; the syntheses of 6 and 9 are omitted for clarity and are
7
Figure 1. DFT optimized models of dinuclear [Cu
2
H
pat-FT] complexes (triplet ground state); left: CuII
2
II
coordinated by three water molecules and a hydroxido ligand, C
4
-chain outside the coordination site of the Cu
II
ions (⫠); right: Cu coordinated by two water molecules and a bridging hydroxido coligand, C
4
-chain inside the
II
coordination site of the Cu ions (ǁ), FT=fluorescent tag.
5
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
From structure optimization of a conjugate with a rhodamine derivative (the chemical structure of
II
Atto550 is not available) it became clear that the distance between the Cu centers and the fluorescent
tag (C
1
, Figure 1) is between 13.5 and 25.4 Å (Cu
1
and Cu ), depending on where the side chain is
2
II
II
II
located relative to the Cu binding sites, i.e. parallel or perpendicular to the Cu ∙∙∙Cu vector. For the
perpendicular arrangement (Figure 1, left) distances between 13.5–21.7 Å are expected, whereas the
C -Cu
1
n
distance is approx. 18.5–25.4 Å for the parallel orientation (Figure 1, right; see Supporting
Information for details).
2
2
Fluorescence and in vivo experiments
.1 Titration
II
Initially, the sensitivity of the fluorophore-labeled patellamide H pat-Atto550 towards Cu (1.8 μM
4
17
patellamide-dye conjugate in 100 mM aqueous TRIS buffer, pH=8.2 as observed for sea water) was
II
determined in vitro by titration with Cu (0.125–10 eq). A strong fluorescence quenching was observed
II
upon addition of Cu (Figure 2).
A
B
1
1
8
6
2
2
1
1
500000
000000
500000
000000
I
f(0)
14
12
Titration of H pat-Atto550
4
from 0 to 10 eq of Cu(II)
2+
Cu
H pat-Atto550
4
1
0
8
6
4
2
0
5
00000
0
buffer
Atto550-NHS
5
60
580
600
[nm]
620
640
0
5
10
15
20
25
30

2+
Cu [M]
II
Figure 2. Titration of H
4
pat-Atto550 with Cu , measured by fluorescence spectroscopy (aqueous solution of
H
4
pat-Atto550 in 100 mM TRIS buffer, pH=8.2, T=293 K; concentrations: [H
4
pat-Atto550]=1.8 μM [Atto550-
II
NHS]=2.3 μM). A Full emission spectrum and B Stern-Volmer plot (H
to titrations of the conjugate H
4
pat-Atto550 + Cu , up triangles) compared
II
4
pat-Atto550 + buffer (diamonds) and Atto550-NHS + Cu (down triangles),
The control experiments, i.e. titrations of H pat-Atto550 with equivalent volumes of buffer (instead of
4
II
II
Cu ) and of Atto550-NHS with Cu , led to significantly smaller fluorescence intensity decreases (Figure
II
2
B). The quenching behavior of the fluorescence-dye-conjugate is biphasic as the Cu concentration
II
increases. The initial linear increase in fluorescence quenching gradually levels off at excess Cu . Static
II
quenching is expected due to Cu complexation of the patellamide ligand in the starting phase.
Fluorescence lifetime measurements, as shown in Figure 3, confirm this assumption as no significant
changes of the fluorescence lifetime of Atto550 are observed upon addition of CuII.
6
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
1
1
1
0
0
.016
.008
.000
.992
.984
1
1
1
1
0
0
0
.024
.016
.008
.000
.992
.984
.976
H pat-Atto550 titration with Cu(II)
4
Atto550 titration with Cu(II)
=3.63 ns
0.976
=3.61 ns
0
1
2
3
4
5
6
7
8
9
10
11
0
1
2
3
4
5
6
7
8
9
10
11
eq Cu(II)
eq Cu(II)
II
Figure 3. Ratio of fluorescence lifetimes τ
0
/τ dependent on the amount of added Cu (100 mM TRIS buffer,
pH=8.2, RT; concentrations: [H pat-Atto550]=1.8 μM, [Atto550-NHS]=2.3 μM).
4
The second phase of the quenching might be due to a saturation effect or could result from the
formation of a dinuclear species while the first phase then would be the formation of the
II
corresponding mononuclear complex. However, binding of Cu to patellamide derivatives has been
found to show cooperativity, i.e. it is not likely that formation of dinuclear complexes only occurs after
addition of 10 eq of CuII.2, 9 In first approximation, the model for static quenching is applied to the initial
18
constant slope of the Stern-Volmer plot to the equivalent point (Figure 2B, eqn. 1). Here, I is the
f
measured fluorescence intensity at the metal ion concentration of each titration step. A linear fit yields
II
the Stern-Volmer constant K
s
of the Cu patellamide complex.
퐼푓0
퐼퐼
=
1 + 퐾 [퐶푢 ]
(1)
푠
퐼푓
6
-1
The fit estimates K to be 1.35(2) x 10 M , and this value is very similar to the stability constants for
s
II
1
6
-1
the dinuclear Cu complexes of the model patellamides (Scheme 1) H
4
pat (K
s
= 1.7 x 10 M ) and
2
4
-1
19
H
4
pat (K
s
= 4.0 x 10 M ), determined by isothermal titration calorimetry in a methanolic solution.
II
pat2
Interestingly, the complex of H
4
pat-Atto550 with Cu seems to be more stable than the related H
4
complex as both have the same backbone and only differ in one side chain. However, the model used
for the determination of the Stern-Vollmer constant does not describe the whole set of experimental
data and the conditions used for the different experiments (ITC, fluorescence spectroscopy) are not
II
identical. Therefore, further investigations of the binding of Cu to H
4
pat-Atto550, e.g. by ITC, are
needed to validate the binding affinity.
7
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
2
.2 Uptake of H
4
pat-Atto550 by Prochloron
In order to find the best method to stimulate the uptake of H
4
pat-Atto550 by Prochloron, different
protocols were investigated. As not many reports are published on the introduction of small peptides
into prokaryotes, standard techniques for the stimulation of biomolecule uptake, as established for
20
Escherichia coli, were applied, i.e. heat shock treatment at 42°C for up to 40 s and incubation at room
temperature for up to 30 min. After the treatment, the cells were washed twice with buffer, and
subsequently, pulsed amplitude-modulated photosynthesis measurements (PAM) were used to
ascertain that the Prochloron cells were still photosynthetically active. The amount of patellamide
taken up by the cells was determined by flow cytometry; here the excitation wavelengths λexc=535 nm
(
excitation of H pat-Atto550) and λexc=640 nm (excitation of chlorophyll b) were used (Figure 4). The
4
fluorescence of chlorophyll b is used as an internal standard. It is apparent that the heat shock
treatment (40 s at 42°C) leads to the same ratio of fluorescence at 575nm and 675nm (FL575/FL675,
i.e. the ratio of fluorescence by H pat-Atto550 and chlorophyll b, respectively) of about 0.016, as
4
observed for incubation for 30 min at room temperature. This indicates that the transport of
patellamides into Prochloron occurs by a passive mechanism. The setup of the experiment leads, after
-
8
dilution with cyanobacteria and buffer, to the final concentration in the sample of 5.8 x 10 M for
pat-Atto550. With the expected passive mechanism, this concentration consequently is the
H
4
maximum expected concentration inside the cells; however, this cannot be independently validated
and is also dependent on the corresponding equilibrium constant. Since the emission of chlorophyll b
is very intense, it was also verified that the observed fluorescence intensity does not solely originate
from the fluorescence of chlorophyll b. The corresponding experimental data from fluorescence
spectroscopy and confocal microscopy are shown in Figure 4.
2
.3 In vivo fluorescence quenching
Since it could be shown that the fluorescence intensity of H
pat-Atto550 is dependent on the CuII
4
concentration in vitro (see Figure 3) and that the conjugate is taken up by the cells, in a third step the
II
Cu sensitivity of H
4
pat-Atto550 was investigated in vivo. Two different techniques were applied, single
cell confocal microscopy and flow cytometry (FCM). Both techniques allow the determination of optical
parameters of single complete cells instead of giving average values for the whole population.
Additionally, flow cytometry is a technique that quantifies various optical parameters of complete cells
simultaneously with a high throughput rate. For these reasons it is a quick and reliable technique,
especially when compared to the time consuming preparation and data acquisition required for
8
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
confocal microscopy experiments.21 The advantage of confocal microscopy is that it provides
information on the spatial organization of each single cell, which is why both techniques were
exploited.
B
A
8
7
6
5
4
3
2
1
0
1.2
1.0
H pat-Atto550 + Prochloron
4
Prochloron
H pat-Atto550 + Prochloron
4
Prochloron
0
0
.8
.6
H pat-Atto550
4
0.4
0
0
.2
.0
Chlorophyll b
540 570 600 630 660 690 720
570
600
630
[nm]
660
690
720

[nm]

D
C
0
0
0
0
0
0
.018
0.018
0.016
Incubation of Prochloron at RT
with H pat-Atto550
heat shock treatment
of Prochloron
4
.016
.014
.012
.010
.008
with H pat-Atto550
4
0
0
0
0
.014
.012
.010
.008
0
5min
10min
30min
1
0s
20s
30s
40s
Figure 4. A: Fluorescence intensity (measured with a fluorescence spectrometer) of a Prochloron sample
treated with H pat-Atto550 for 40 s at 42°C (λexc=500 nm). B: Fluorescence intensity from confocal microscopy;
Prochloron treated with H pat-Atto550 for 40 s at 42°C (for raw data see Supporting Information); C and D:
4
4
FL575/FL675, i.e. ratio of the fluorescence intensity of H
chlorophyll b (FL675).
4
pat-Atto550 (FL575) to the fluorescence intensity from
9
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
A
B
0
0
0
0
0
0
.6
.5
.4
.3
.2
.1
n.s.
n.s.
0
0
.03
.02
***
H pat-Atto550
**
H pat-Atto550
4
H pat-Atto550
4
4
H pat-Atto550
4
+
Cu(II) + cyclam
+
Cu(II) + cyclam
H pat-Atto550
4
+
Cu(II)
H pat-Atto550
4
+
Cu(II)
0.01
0
.00
(
a)
(b)
(c)
(
a)
(b)
(c)
Figure 5. Mean and SEM of FL575/FL675, determined by FCM, for A Prochlorothrix hollandica (n=12,000) and B
II
Prochloron (n=4,000), treated (a) with H
4
pat-Atto550, (b) with H
4
pat-Atto550 and Cu , and (c) with H
4
pat-
II
Atto550 and Cu and subsequently with cyclam (10 eq) [p ≥ 0.05 – not significant (n.s.), p ≤ 0.05 – significant
(*), p ≤ 0.01 – highly significant (**), p ≤ 0.001 extremely significant (***)].
II
For the establishment of a valid protocol to investigate the Cu binding behavior of the fluorescent
dye-patellamide conjugate by means of flow cytometry and confocal microscopy, Prochlorothrix
hollandica (P.H.) was studied. P.H. was chosen as it is the taxonomically closest cyanobacterium and
readily available from a cyanobacteria database, in contrast to Prochloron, which can only be handled
for about 1-2 weeks in symbiosis with the host in a fresh water aquarium after sampling from tropical
waters. H pat-Atto550 was added to the cyanobacteria, which were then heat shocked for 40 s at 42°C
4
and subsequently chilled on ice for 1 min (a). Alternatively, samples (b) and (c) were incubated with
II
H
4
pat-Atto550 and 5 eq of a Cu salt. After rinsing the cells, sample (c) was additionally treated with
an excess of cyclam (10 eq, cyclam=1,4,8,11-tetraazacyclotetradecane), applied as a strong competitor
II
II
for Cu coordination to reinstall the fluorescence upon removing Cu from H
were analyzed by FCM, and the mean ratio (and its SEM) of fluorescence observed at 575 nm
corresponding to Atto550) compared to fluorescence at 675 nm (corresponding to chlorophyll b, used
4
pat-Atto550. All samples
(
here as an internal standard) is plotted in Figure 5. For Prochlorothrix hollandica samples (a) and (c)
are not significantly different, whereas sample (b) shows a large (p<0.001) difference compared to (a)
II
and (c). This lends strong support for the hypothesis that Cu is binding to H
4
pat-Atto550 in
II
Prochlorothrix hollandica. In addition, the binding constant of the Cu patellamide complex seems to
II
be significantly smaller than that of the corresponding Cu -cyclam complex, which is in accordance
with expectations, as the stability constant for [Cu (cyclam)]2+ is logK = 14.7 (pH 8.2),22 orders of
II
magnitude higher than what was observed from photophysical measurements for the H pat-Atto550
4
ligand studied here (vide supra). Consequently, the established protocol was also applied to Prochloron
cells. The results are summarized in Figure 5B. It emerges that sample (a) (0.01874 ± 0.0015) and (c)
1
0
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
(0.02026 ± 0.0010) are not significantly different, but sample (b) is significantly different from (a) and
II
(
c), exactly as observed for Prochlorothrix hollandica. This implies that a Cu -patellamide complex is
stable in the Prochloron cells and could possibly act as a catalyst, as observed in vitro. It should be
noted here that in spite of Prochlorothrix hollandica being the taxonomically closest cyanobacterium
it contains significantly less chlorophyll b than Prochloron, which leads to larger values for the observed
ratios FL575/FL675 in the range of 0.2-0.4 compared to the values observed for Prochloron (0.013-
0
.020).
Samples prepared identically as described for FCM were also investigated concerning their relative
brightness at 574 nm by confocal microscopy, and the results are depicted in Figure 6. In order to
validate that the observed brightness originates solely from the fluorescent-dye-patellamide
conjugate, the relative brightness of Prochloron at λem=574 nm was plotted as the first bar in Figure 6.
2
1
1
1
1
1
000
800
600
400
200
000
*
*
*
*
H pat-Atto550
4
+ Cu(II) + cyclam
H pat-Atto550
4
H pat-Atto550
+ Cu(II)
4
8
6
4
2
00
00
00
00
0
Prochloron
(
a)
(b)
(c)
Figure 6. Left: Confocal microscopy pictures of Prochloron cells and right: mean and SEM of FL574 for
II
Prochloron (n=600) treated (a) with H
and Cu and subsequently with cyclam.
4
pat-Atto550, (b) with H
4
pat-Atto550 and Cu , and (c) with H
4
pat-Atto550
II
Sample (b) shows a significantly lower brightness compared to samples (a) and (c) but at the same time
II
sample (c) is significantly brighter than sample (a). This implies that Cu is binding to the patellamide-
II
fluorescent-dye conjugate. Upon addition of an excess of cyclam the Cu that was added as well as
II
endogenous Cu ions are removed by cyclam. Although FCM does not show a significant difference for
samples (a) and (c) of Prochloron (Figure 5), it is worth noting that the mean of (c) is higher than the
mean observed for (a) although the difference is not significant. Moreover, the sample size for FCM is
~12,000 per population, whereas the populations investigated with confocal microscopy consist of
~600 Prochloron cells. Importantly, unlike the FCM data (normalized to background chlorophyll), the
data from confocal microscopy are not normalized.
1
1
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
To exclude the occurrence of an 'inner filter effect', UV-vis-NIR absorption measurements of a sample
II
of Prochlorothrix hollandica lysate, treated identically (same population density and Cu concentration)
to the samples prepared for FCM and confocal microscopy, were carried out (see Figure 7). These
II
experiments were aimed to elucidate whether putative Cu complexes with intracellularly abundant
chelators like amino acids absorb strongly at 570-620 nm, causing the fluorescence intensity of
Atto550 to appear attenuated.
0
0
0
0
0
.12
.09
.06
.03
.00
P.H. lysate +TRIS
II
P.H. lysate + Cu
400
500
600
700

[nm]
II
II
-7
Figure 7. Absorption spectrum of a Prochlorothrix hollandica lysate solution treated with Cu , [Cu ]=2.9x10 M,
and a solution of P.H. lysate solution treated with the equivalent volume of TRIS buffer.
II
From these measurements it appears that the treatment of the cells with Cu does not lead to any
change in the absorption spectrum of the corresponding cell lysate. Consequently, the potential
II
formation of Cu complexes with abundant chelators does not alter the absorption behavior of the
cells significantly.
2
.4 Copper ions in living cells
Knowledge of the concentration of copper ions in living cells and their oxidation state is of significance
for in vivo studies and, unfortunately, this is not well studied for cyanobacteria. It is known that
mammalian (eukaryotic) cells exhibit a reductive interior condition, and the free copper cations are
I 23
thus present as Cu . The copper ion homeostasis is regulated by a copper ion importing and exporting
23
system and leads to a copper ion concentration in the cell in the femtomolar range. Similar transport
systems were also found in the prokaryotes Enterobacteria.24 However, here, interior copper ion
-
8
concentrations in the micromolar range were reported, as compared to the nanomolar range (10 M)
24
in the surrounding medium. From a proteomic study of the cyanobacterium Synechocystis sp. it has
emerged that the export mechanism is the main regulator of the copper cation concentration.25
However, exact copper cation concentrations for cyanobacterial cells are not published so far. In 2001
1
2
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
II
it was reported that after acidic lysis of L. patella with Prochloron cells, the Cu concentration in the
whole organism corresponded to a 10 times enrichment compared to the surrounding sea water.26,5
From these results a maximum intracellular concentration of 1.6 µM can be estimated for L. patella
with Prochloron (including the copper cations that are potentially bound to storage proteins).5
The question about the reductive/oxidative milieu in the cells remains unanswered. It is known that,
depending on the level of irradiance, the pH is fluctuating and the same is true for the level of oxygen
saturation.17, 27 As a consequence of the oscillation during day/night cycles, the oxidative environment
in the cells is expected to be correlated to the level of oxygen in the cells. If this is the case, one can
expect an alkaline environment (pH 11) as well as dioxygen super saturation upon solar irradiation17
4
II
and consequently copper cations to be present as Cu . In the dark, on the other hand, an interior milieu
17
I
of pH 7.0, in combination with an anoxic environment is expected, which would favor Cu . To date,
I
the coordination chemistry of Cu to patellamides has not been studied in detail. Research in this field
I
II
would improve the understanding of relative stabilities of the putative Cu versus Cu complexes. This
II
II
leaves us to propose that the maximum internal concentration of 1.6 µM for Cu and 18 µM for Zn is
expected in L. patella with Prochloron and that copper is possibly divalent during exposure to sunlight
and monovalent during darkness.
Furthermore, knowledge about potential chelators other than the patellamides, in particular their
I
II
II
concentrations and binding affinities for Cu /Cu and Zn in the cells, is crucial for this study since most
likely a major part of the intracellular copper and zinc cations are complexed, leading to a much smaller
concentration of uncoordinated metal ions (probably as aqua complexes). Again, no data for
Prochloron or similar cyanobacteria (prochlorophyta) are available. Absolute metabolite
28
concentrations for E. coli have been published, and indicated that the concentrations of amino acids
like glutamate, glutamine, aspartate, valine and alanine, to mention but a few, are in the millimolar
28
range. Apart from amino acids, other metabolites like ATP, GTP and citrate are also present in similar
concentrations.28 In a study on the amino acid composition of the dried cyanobacteria Nostoc
spongiaeforme and Rivularia bullata, all peptides in the cell were hydrolyzed for sample preparation,
and the amount of amino acids reported does therefore not correspond to the concentration of amino
acids in solution. If one nevertheless uses these values and expects the native cyanobacteria to have
29
an approximate water content of 70 %, cytosol concentrations of the amino acids glycine, threonine
and leucine of 37.9 mM, 25.5 mM and 28.8 mM can be anticipated, and this resembles the
composition of all proteins and metabolites in cyanobacteria. The binding constants of 1:1 amino acid
II
Cu complexes are in the range of logK=7-9, and for 2:1 complexes, one expects logβ values between
30
II
1
2 and 16. These stability constants are significantly higher than those for patellamide Cu complexes,
2
typically in the range of logK
1
K
2
=4-6, depending on the patellamide backbone. Despite the lack of
exact knowledge of the amino acid concentrations in cyanobacteria and Prochloron in particular, one
1
3
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
II
can assume from the stability constants that the formation of Cu amino acid complexes should be
preferred over the formation of patellamide complexes. However, as mentioned earlier, the stability
constants greatly depend on the pH, the oxidation state of the copper cation and the relevant local
concentrations. In addition, so far no experiments have been carried out to elucidate whether or not
II
the Cu patellamide complexes might be integrated in a protein as a prosthetic group. That would not
be unexpected, since in nature the majority of metal complexes are embedded within the active site
of proteins.31-34 If that was the case, the stability constants measured previously for patellamides would
be challenged.2
Conclusion
In conclusion, the synthesis of the fluorescent-dye-derivatized patellamide H
4
pat-Atto550 is reported
II
together with optical spectroscopy and photophysical studies related to its Cu ion sensing. The
II
fluorescence of the ligand is Cu concentration dependent with a Stern-Volmer constant of
6
-1
K
s
= 1.35 x 10 M , which is of the same order of magnitude as the stability constants of similar model
2
patellamides. It is shown that the model peptide H
4
pat-Atto550 is taken up by Prochloron cells,
probably by a passive mechanism. The imaging via flow cytometry and confocal microscopy implies
II
that a Cu -patellamide complex forms in the Prochloron cells, as observed in vitro. The formation of
II
Cu complexes inside Prochloron cells indicates that the catalysis of various hydrolysis reactions
observed in vitro, carbonic anhydrase, phosphatase, lactamase and glycosidase,1, 11, 12 and possibly
others not yet explored, might be the metabolic purpose of the patellamides. The corresponding ZnII
complexes, which have also been shown to form and to be catalytically active are less likely to be of
3
II
II
biological relevance, and the Cu complexes might therefore be a rare case of Cu hydrolase enzymes.
So far, it is not clear, whether mono- or dinuclear complexes are formed in vivo, and their structures
II
are unknown. Structural information of the in vitro Cu -patellamide complexes was obtained by EPR
spectroscopy.2, 8, 9 Therefore, other reporter groups, spectroscopic techniques and mechanistic
experiments are needed to answer still open questions of these interesting systems.
Experimental Section
Materials and Methods
Some of the reactions reported were carried out under an inert atmosphere of argon or nitrogen using
standard Schlenk-techniques. Glassware was heated and dried under vacuum prior to use. All
chemicals were purchased from Sigma-Aldrich GmbH, ABCR GmbH & Co. KG and Merck at the highest
1
4
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
available purity. Dry solvents were purchased and used as delivered. For optimized comprehensibility,
names of compounds synthesized were simplified instead of usage of the exact IUPAC name.
High resolution mass spectra (HR-ESI MS) were recorded on a Finnigan MAT8230 and Joel JMS-700
spectrometer by Dr. Jürgen Gross and co-workers at the mass spectrometry facility in the Organic
Chemistry department of Heidelberg University on a ICR Apex-Qe and a JEOL JMS-700. Atmospheric
pressure chemical ionization mass spectra (APCI MS) were recorded on a Waters UPLC-SQD2 single
quadrupole.
1
Nuclear magnetic resonance spectra were recorded on a Bruker Avance III (15.1 T; 600 MHz for H)
spectrometer equipped with a cryoprobe. Chemical shifts δ are given in ppm and coupling constants J
1
13
in Hz. All spectra were calibrated using the residual H- or C-signals of the deuterated solvents.
Spectra were recorded at 298 K.
The following abbreviations were used to describe the multiplicities of the signals: s (singlet), d
(doublet), t (triplet), qn (quintet), m (multiplet). Signals were assigned using DEPT, HSQC and HMBC
spectra.
The purification of the dye conjugate H pat-Atto550 was performed with the HPLC (High Performance
4
Liquid Chromatography, Agilent, Waldbronn, serial number 1100), equipped with a binary pump
G1312A, a degasser G1322A, a diode array detector G1315 A and a fluorescence detector G1321A. The
chromatographic separation was performed using the reversed phase column from Knauer, Berlin
(250 mm, 4 mm inner diameter, ODS-Hypersil with pore diameter of 5 μm).
Statistical Analysis
A One-way ANOVA was used for statistical analysis. The required level of significance was defined to
be 5 % ( p ≥ 0.05 – not significant (n.s.), p ≤ 0.05 – significant (*), p ≤ 0.01 – highly significant (**),
p ≤ 0.001 extremely significant (***)).
Syntheses
The synthesis of H pat-Atto550 was accomplished following a method analogous to the previously
4
reported pseudo-peptide syntheses and is described in detail in the Supporting Information.
UV-vis and Fluorescence Spectra
As a copper(II) salt CuSO
4
x6 H O was used for all photophysical experiments and fluorescence
2
titrations.
UV-vis absorption spectra were recorded with the Cary 60 UV-vis spectrometer (Agilent) at room
temperature and fluorescence spectra (quenching experiments) of the H pat-Atto-550 - monitored
with the 'Fluorolog 3' fluorescence spectrometer (Horiba). The measurements were carried out in
4
1
5
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
quartz glass cuvettes. Fluorescence spectra (quenching experiments) of Atto550-NHS were recorded
with 'Cary Eclipse 500' fluorescence spectrometer (Varian, Darmstadt) with temperature control. The
measurements were carried out in dark quartz glass cuvettes (Suprasil©, Hellma, Müllheim).
Fluorescence lifetime measurements: Titration of H pat-Atto550 with Cu(II)
4
Fluorescence lifetimes were determined on a 'Fluotime 100' spectrometer with time-correlated single
photon count card 'TCSPC TimeHarp 200' from PicoQuant. The excitation wavelength was generated
by a pulsed diode 'PLS 500' (500 nm, 40 MHz) from PicoQuant with a pulse width of 300 ps tuneable
fibre coupled diode. The instrument response function was measured with colloidal SiO
2
nano-particles
(LUDOX®HS-40 (420816), Sigma-Aldrich). During the measurement, the excitation light was filtered
with suited high-pass filters, data were analysed with the programme 'FluoFit' from PicoQuant.
Fluorescence quenching experiments
The metal salt used (analytical grade, CuSO
O (0.01 M); a solution of H pat-Atto550 was adjusted to 1.8 µM and prepared from high
concentration aqueous solutions (doubly distilled water) by dilution into 100 mM TRIS buffer, pH 8.2,
doubly distilled H O.
To 200 μL of H pat-Atto550 in the required concentration, the metal salt in the required concentration
4 2
x6 H O 0.16 M aq. solution) was diluted in doubly distilled
H
2
4
2
4
was added in steps of 1 μL before measuring the fluorescence spectrum. The intensities were
corrected by the corresponding dilution factor; the reported data are averages of triplicates.
Cyanobacteria handling, Field Site and Sample Collection
For the experiments Prochlorothrix hollandica (SAG 10.89) and Synechococcus leopolienses (SAG 1402-
1
) were purchased from the Culture Collection of Algae at Göttingen University and stored on the
bench at room temperature and without direct light irradiation for a maximum of two weeks.
For Prochloron fom L. patella, individual patches and their coral rubble substrate were collected with
2
a hammer and chisel. Intact specimens (5-25 cm ) of 5-20 mm thick L. patella were collected at low
tide on snorkel on the outer reef flat and crest off Heron Island (First Point: S23°25′49.8, E151°57′15.8,
Blue Pools: S23°26′03.2, E151°55′18.1,) in May 2016 covering coral patches on the outer reef flat and
down to ∼4ꢀm depth on the reef crest. All samples were immediately transported in a bucket with sea
water back to Heron Island Research Station and subsequently kept in an outdoor aquarium, which
was continuously flushed with fresh aerated sea water (24–26°C) from the reef. As reported
17
previously, it is necessary to keep a continuous strong mixing of water in the aquarium to avoid the
degradation of the ascidians. The aquaria were covered by a shading cloth, to prevent excessive solar
irradiance.
1
6
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
Samples were then transported to the University of Queensland, St Lucia Campus, in a plastic bag, filled
with fresh aerated sea water and oxygen, and stored in a cold box at 25°C by plane and 6 h later put in
an aquarium with synthetic sea water and constant water flow. The seawater is generated from Tropic
35
Marine salt mixed with water (that was purified via reverse osmosis) to a salinity of 35 ppt (23-25°C;
the water is not sterilized but pumped through system of sediment and biological filters). Under these
conditions we were able to keep the sampled ascidians healthy and with Prochloron actively
photosynthesising for nine days.
Prochloron was collected using a pipette and by gently squeezing the middle section containing the
cloacal cavity harbouring the deep green symbiont.
Prior to all measurements the viability of the cells was tested with a ToxyPAM.36-38 Generally,
specimens showed a high maximum PSII quantum yield of >0.6 for several hours after such handling
indicative of fast recovery and minimal stress on Prochloron (Heinz Walz GmbH, Effeltrich).
1
2
cm x 1 cm patches were additionally cut from L. patella with a sterilized razor blade and stored in
0% glycerol solution at –80°C, transported back to St Lucia on dry ice and stored at –80°C. Healing
was observed for the patches which had been cut.
Protocol for the treatment of Prochloron (preparation of FCM and confocal microscopy experiments)
-
6
For the preparation of samples for FCM and confocal microscopy 40 μL of a 2.4 x 10 M solution
100 mM TRIS buffer, pH 8.2) of H pat-Atto550 were added to 30 μL of the respective cyanobacteria
at OD750=0.8 in BG 11 medium (sample a). For sample b and c 40 μL of a 2.4 x 10 M H
with 5 eq of copper(II) (CuSO x6H O) were used for the treatment of the cells. After heat shock
(
4
-
6
4
pat-Atto550,
4
2
treatment for 45 s at 40°C the samples were put on ice for 1 min. Washing with 50 μL buffer followed.
The cells were then resuspended in 10 μL of buffer and 90 μL of a 4 % PFA solution was added the cells
were incubated for 15 min. Again the cells were washed with 50 μL buffer and either (samples a and
b) resuspended in 50 μL of TRIS or (c) treated with 10 eq of cyclam for 10 min. Sample c was after that
also washed with 50 μL of buffer and resuspended in 50 μL of TRIS. All experiments were carried out
in triplicates. For FCM the final suspension were analyzed within 1 h after preparation.
For confocal microscopy the samples were placed in 8-well glass slides (ibidi GmbH, Lab-Tek) and the
solvent was slowly evaporated under reduced pressure in a desicator (1 h) and the cells were analyzed
immediately.
Flow cytometry (FCM)
The flow cytometry experiments were performed on a C6 cytometer (Accuri). Fluorescence excitation
was at λexc=520 nm and λexc=640 nm, and the respective emission was detected using a 585/42 nm
(FL1) and a 694/40 nm (FL2) filter. The mean ratio (and the respective SEM) of the fluorescence
1
7
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
observed at FL1 compared to FL2 is plotted. All flow cytometry measurements include minimum 4,000
events in the gated area and were carried out in triplicates.
Confocal Microscopy
All Prochlorothrix hollandica and Synechococcus leopoliensis samples were measured using a confocal
laser scanning microscope (Leica SP5), applying 63x water immersion objective. The image parameters
were chosen 2048x2048 by 16 bit at 200 Hz. Each picture represented an area of 246x246 µm,
representing a two-fold oversampling by a spatial resolution of 132 nm per pixel. Atto550 was excited
via pulsed whitelight laser (80 MHz, SuperK Extreme, Koheras) at 561 nm, intensity 70 %. Emission
detection was done using photomultiplier tubes (PMT) within 574-680 nm. At least 10 measurements
at random positions within one Lab-Tek chamber were conducted per condition.
Prochloron samples were measured using a confocal laser scanning microscope (ZEISS LSM 710)
applying 63x oil immersion objective (C-Apochromat 63x/1.2 W Korr). The image parameters were
chosen 1024x1024 by 16 bit at 100 Hz. Each picture represented an area of 135x135 µm, representing
a two-fold oversampling by a spatial resolution of 132 nm per pixel. Atto550 was excited via pulsed
white light laser at 561 nm, intensity 70 % (561 nm continuous wave Diode-Pumped Solid State Laser
DPSS on a Zeiss LSM 710 instrument). Emission detection was done using photomultiplier tubes (PMT)
within 556-606 nm and 624-735 nm. At least 10 measurements at random positions within one Lab-
Tek chamber were conducted per condition.
For quantification, Fiji (http://fiji.sc/) software was used. Images were filtered by median, followed by
background subtraction. Cells were selected using Otsu thresholding and subsequent particle analysis,
followed by calculation of median intensity and standard error.
Absorption measurement of the Prochlorothrix hollandica lysate
5
mL of Prochlorothrix hollandica (P.H.) from culture were centrifuged down at 18.000 g for 5 min. The
supernatant was discarded, and cells were resuspended to OD600=0.8 in TRIS buffer, pH 8.2.
Disintegration was done using a sonicator (BW2070, Bandelin electronics Berlin GmbH) for 5x10 s at
7
5% of maximum power. In between repeats, lysate was chilled on ice for 1 min. Cell debris was spun
down for 15 min at maximum speed. The supernatant was measured for absorbance properties (Cary
0 UV-vis spectrometer (Agilent) at room temperature) with or without CuSO (final concentration as
6
4
-
7
described in the experimental section (FCM and confocal microscopy): 2.9 x 10 M). As control the
same volume of TRIS buffer was supplemented.
1
8
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
Supporting Information
4
The Supporting Information covers details on the synthesis of H pat-Atto550, fluorescence lifetime
4
curves for the H pat-Atto550 conjugate, as well as raw confocal microscopy pictures on the uptake of
4
H pat-Atto550 by Prochloron. In addition, the results from the investigation of Synechococcus
leopoliensis and Prochlorothrix hollandica by FCM and confocal microscopy are given. Coordinates of
the computed structures and details on the computations are also presented in the Supporting
Information.
Acknowledgements
For financial support we are grateful to the German Science Foundation (DFG, HGS MathComp), ARC
and the University of Heidelberg. We thank Sandra König for support with flow cytometry, as well as
Prof. Peter Vize and Daniel Wuitchik for help with the collection of L. patella samples on SCUBA at
Heron Reef. We are grateful to Prof. Dr. Thomas Söllner for access to his labs, and to Prof. Dr. Jürgen
Müller's and Jake O'Brien's efforts to allow AE to use their ToxyPAM. The excellent technical support
by the staff at Heron Island Research Station, University of Queensland, as well as the support from
Dr. Steven Mason, Prof. Bernhard Degnan and Dr. Jabin R. Watson is acknowledged. We are grateful
for the computational resources provided by the bwForCluster JUSTUS, funded by the Ministry of
Science, Research and Arts and the Universities of the State of Baden-Württemberg, Germany within
the framework program bwHPC-C5. GS acknowledges the Australian Research Council for support in
form of a Future Fellowship (FT120100694).
References
1.
Comba, P.; Eisenschmidt, A.; Kipper, N.; Schiessl, J. J. Inorg. Biochem. 2016, 159, 70-75.
2
4
.
Comba, P.; Dovalil, N.; Gahan, L. R.; Hanson, G. R.; Westphal, M. Dalton Trans. (Dalton Perspective) 2014,
3, 1935-1956.
3.
Comba, P.; Eisenschmidt, A.; Gahan, L.; Hanson, G.; Mehrkens, N.; Westphal, M. Dalton Trans. 2016, 45,
18931-18945.
4
.
.
Hawkins, C. J. Pure and Applied Chemistry 1988, 60, 1267-1270.
Wright, S. H.; Raab, A.; Feldmann, J.; Krupp, E.; Jaspers, M. In Handbook of Marine Natural Products;
5
Fattorusso, E., Gerwick, W. H., Taglialatela-Scatati, O., Ed.; Springer: Heidelberg, New York, 2012; pp 861-892.
6.
van den Brenk, A. L.; Byriel, K. A.; Fairlie, D. P.; Gahan, L. R.; Hanson, G. R.; Hawkins, C. J.; Jones, A.;
Kennard, C. H. L.; Moubaraki, B.; Murray, K. S. Inorg. Chem. 1994, 33, 3549-3557.
1
9
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
7.
8.
9.
Haberhauer, G.; Rominger, F. Eur. J. Org. Chem. 2003, 3209-3218.
Comba, P.; Dovalil, N.; Hanson, G. R.; Linti, G. Inorg. Chem. 2011, 50, 5165-5174.
Comba, P.; Dovalil, N.; Gahan, L. R.; Haberhauer, G.; Hanson, G. R.; Noble, C. J.; Seibold, B.; Vadivelu, P.
Chem. Eur. J. 2012, 18, 2578-2590.
10.
Comba, P.; Dovalil, N.; Hanson, G.; Harmer, J.; Noble, C. J.; Riley, M. J.; Seibold, B. Inorg. Chem. 2014, 53,
12323-12336.
11.
12.
13.
Comba, P.; Gahan, L. R.; Hanson, G. R.; Maeder, M.; Westphal, M. Dalton Trans. 2014, 43(8), 3144-3152.
Comba, P.; Gahan, L. R.; Hanson, G. R.; Westphal, M. Chem. Comm. 2012, 48, 9364-9366.
Kühl, M.; Behrendt, L.; Trampe, E.; Qvortrup, K.; Schreiber, U.; Borisov, S. M.; Klimant, I.; Larkum, A. W.
D. Frontiers in Microbiology 2012, 3, 1.
1
4.
Ely, F.; Hadler, Kieran S.; Gahan, Lawrence R.; Guddat, Luke W.; Ollis, David L.; Schenk, G. Biochemical
Journal 2010, 432 (3), 565-573.
1
1
1
5.
6.
7.
Mitić, N.; Hadler, K. S.; Gahan, L. R.; Hengge, A. C.; Schenk, G. J. Am. Chem. Soc. 2010, 132, 7049-7054.
Hiller, R. G.; Larkum, A. W. D. Biochimica et Biophysica Acta 1985, 806, 107-115.
Kühl, M.; Behrendt, L.; Trampe, E.; Qvortrup, K.; Schreiber, U.; Borisov, S. M.; Klimant, I.; Larkum, A. W.
Front. Microbiol. 2012, 3, 402.
18.
Valeur, B.; Berberan-Santos, M. N. Molecular Fluorescence - Principles and Applications. 2nd Edition, pp.
5
12 ed.; Wiley-VCH: 2012.
19.
Comba, P.; Dovalil, N.; Haberhauer, G.; Kowski, K.; Mehrkens, N.; Westphal, M. Z. Anorg. Allg. Chem.
2013, 639 (8-9), 1395-1400.
20.
EMBL
https://www.embl.de/pepcore/pepcore_services/cloning/transformation/transformation_heatshock/
04.10.2016).
(
21.
Ormeod, M. G. Flow Cytometry A Practical Approach. Third Edition Reprinted 2003 ed.; Oxford
University Press: Great Clarendon Street, Oxford OX1 6DP, 2003.
2
2
2
2
2.
3.
4.
5.
Puranik, D. B.; Singh, A.; Chang, E. L. J. Coord. Chem. 1996, 39:3-4, 321-331.
Foster, A. W.; Osman, D.; Robinson, N. J. J. Biol. Chem. 2014, 289 (41), 28095-28103.
Porcheron, G.; Garenaux, A.; Proulx, J.; Sabri, M.; Dozois, C. FCIMB 2013, 3 (90),
De la Cerda, B.; Castielli, O.; Durán, R. V.; Navarro, J. A.; Hervás, M.; De la Rosa, M. A. Briefings in
Functional Genomics & Proteomics 2007, 6 (4), 322-329.
26.
Morris, L. A.; Jaspars, M.; Kettenes-van den Bosch, J. J.; Versluis, K.; Heck, A. J. R.; Kelly, S. M.; Price, N.
C. Tetrahedron 2001, 57, 3185-3197.
27.
Note that the oxygen saturation and pH were not measured inside Prochloron cells, but in close
proximity to it.
28.
Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van Dien, S. J.; Rabinowitz, J. D. Nat. Chem. Biol.
2009, 5 (8), 593-599.
2
0
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
29.
30.
31.
Sarma, T. A. Handbook of cyanobacteria. CRC Press: 2012.
Hallman, P. S.; Perrin, D. D.; Watt, A. E. Biochem. J. 1971, 121 (3), 549-555.
Kaim, W.; Schwederski, B. Bioanorganische Chemie - Zur Funktion chemischer Elemente in
Lebensprozessen. Teubner: Wiesbaden, 2005; Vol. 4th Edition.
32.
Ermler, U.; Grabarse, W.; Shima, S.; Goubeaud, M.; Thauer, R. K. Current Opinion in Structural Biology
1998, 8 (6), 749-758.
3
3.
4.
Krämer, R. Coord. Chem. Rev. 1999, 182 (1), 243-261.
Solomon, E. I.; Heppner, D. E.; Johnston, E. M.; Ginsbach, J. W.; Cirera, J.; Qayyum, M.; Kieber-Emmons,
3
M. T.; Kjaergaard, C. H.; Hadt, R. G.; Tian, L. Chem. Rev. 2014, 114 (7), 3659-3853.
3
5.
http://www.aquabluedistribution.com.au/productdetails/brandname/Tropic%20Marin/category/Salt/prdname
TM+Pro+Reef+750ltr%0Abucket+25kg (08.11.2016).
Tropic-Marine-Salt
/
36.
Campbell, D.; Hurry, V.; Clarke, A. K.; Gustafsson, P.; Öquist, G. Microbiol. Mol. Biol. Rev. 1998, 62 (3),
667-683.
3
7.
8.
Murchie, E. H.; Lawson, T. J. Exp. Bot. 2013, 64 (13), 3983-3998.
3
ToxyPAM
user
manual
http://www.lombardemarozzini.com/sites/default/files/toxyp_2e.pdf
(01.11.2016).
2
1
This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201700895
TOC
II
II
Cu turns off the light. To prove the formation of a Cu patellamide complex, a fluorescent-tag-
II
labeled patellamide was introduced to Prochloron to study its Cu binding behavior.
2
2
This article is protected by copyright. All rights reserved.