Heme bound amylin: Spectroscopic characterization, reactivity, and relevance to type 2 diabetes

Article abstract of DOI:10.1021/ic4001413

Deposition of human amylin or islet amyloid polypeptide (hIAPP) within the β-cells of the pancreatic islet of Langerhans is implicated in the etiology of type 2 diabetes mellitus (T2Dm). Accumulating evidences suggest that increased body iron stores, iron overload, and, in particular, higher heme-iron intake is significantly associated with higher risk of Type 2 diabetes mellitus (T2Dm) (PloS One2012, 7, e41641). Some key pathological features of T2Dm, like iron dyshomeostasis, iron accumulation, mitochondrial dysfunction, and oxidative stress are very similar to the cytopathologies of Alzheimer's disease, which have been invoked to be due to heme complexation with amyloid β peptides. The similar etiology and pathogenic features in both Alzheimer's disease (AD) and T2Dm indicate a common underlying mechanism, with heme playing an important role. In this study we show that hIAPP can bind heme. His18 residue of hIAPP binds heme under physiological conditions and results in an axial high-spin active site with a trans-axial water derived ligand. Arg11 is a key residue that is also essential for heme binding. Heme(Fe²⁺)-hIAPP complexes are prone to produce partially reduced oxygen species (PROS). The His18 residue identified in this study is absent in rats which do not show T2Dm, implicating the significance of this residue as well as heme in the pathology of T2Dm.

Full text of DOI:10.1021/ic4001413

Article
pubs.acs.org/IC
Heme Bound Amylin: Spectroscopic Characterization, Reactivity, and
Relevance to Type 2 Diabetes
Soumya Mukherjee and Somdatta Ghosh Dey*
Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata, India 700032
S
* Supporting Information
ABSTRACT: Deposition of human amylin or islet amyloid polypeptide
(hIAPP) within the β-cells of the pancreatic islet of Langerhans is implicated in
the etiology of type 2 diabetes mellitus (T2Dm). Accumulating evidences
suggest that increased body iron stores, iron overload, and, in particular, higher
heme-iron intake is significantly associated with higher risk of Type 2 diabetes
mellitus (T2Dm) (PloS One 2012, 7, e41641). Some key pathological features
of T2Dm, like iron dyshomeostasis, iron accumulation, mitochondrial
dysfunction, and oxidative stress are very similar to the cytopathologies of
Alzheimer’s disease, which have been invoked to be due to heme complexation
with amyloid β peptides. The similar etiology and pathogenic features in both
Alzheimer’s disease (AD) and T2Dm indicate a common underlying
mechanism, with heme playing an important role. In this study we show that
hIAPP can bind heme. His18 residue of hIAPP binds heme under physiological
conditions and results in an axial high-spin active site with a trans-axial water derived ligand. Arg11 is a key residue that is also
essential for heme binding. Heme(Fe2+)-hIAPP complexes are prone to produce partially reduced oxygen species (PROS). The
His18 residue identified in this study is absent in rats which do not show T2Dm, implicating the significance of this residue as
well as heme in the pathology of T2Dm.
Emerging scientific evidences have undisputedly established
the potential link of body iron stores and iron influenced
glucose metabolism in the etiopathogenesis of T2Dm.20−22
Iron is a strong pro-oxidant and participates in the generation
of reactive oxygen species such as hydroxyl radicals when
present in excess and unshielded, resulting in the consequent
elevation in oxidative stress.23 Iron-induced oxidative stress on
the β-cells along with iron deposition in the pancreatic β-cells
results in their apoptosis which consequently leads to insulin
deficiency.24−26 Insulin resistance is also induced by iron
deposition inhibiting glucose uptake in skeletal muscles and
adipose tissues, with reduction in liver capacity to extract
insulin, resulting in an abnormal hepatic glucose produc-
tion.27,28 Compared to other cell types, pancreatic β-cells are
extremely susceptible to damage by partially reduced oxygen
species (PROS),17 probably because the islet cells almost
exclusively rely on mitochondrial metabolism of glucose for
glucose-induced insulin secretion and contain relatively very
low levels of several PROS detoxifying systems.25,29
1. INTRODUCTION
Type 2 diabetes mellitus (T2Dm) is the most common chronic
metabolic disorder characterized by dysfunction in both insulin
action and insulin secretion from the β-cells of pancreatic islets
of Langerhans, resulting in hyperglycemia.1 Recent literature
shows that protein aggregation, amyloidosis, and fibril
formation are the central processes in many diseases like
Alzheimer’s disease (AD), Parkinson Disease, and T2Dm.2−4
The invariant histopathological feature of patients suffering
from T2Dm is the presence of fibrillar amyloid deposits in and
around the β-cells of the pancreas.1,4,5 The principal constituent
of these amyloid deposits is a polypeptide hormone called
amylin or Islet Amyloid Polypeptide Protein (IAPP) compris-
ing of 37 amino-acid residues.6,7 IAPP is coproduced and
cosecreted with insulin by the pancreatic β-cells and have
complementary hormonal activities.8,9 Human IAPP (hIAPP) is
a highly amyloidogenic peptide, with residues 20−29 forming
the part of its amyloidogenic core.10 Several reports indicate
that aggregation of hIAPP(1−37) is responsible for the death of
the pancreatic β-cells;4,11,12 however, it is increasingly becoming
apparent that it is the smaller oligomers that have the ability to
induce membrane instability and β-cell apoptosis.13−16 These
soluble oligomers can induce oxidative stress by the
spontaneous generation of PROS, for example, H2O2, and
other free radicals.17,18 The H2O2 generated is freely permeable
across cell membranes and in the presence of reduced
transition metal ions (Fe2+, Cu+) can generate cytotoxic
hydroxyl radicals.19
AD, the most common form of dementia in the elderly, is a
neurodegenerative disorder.30 AD is marked by the formation
of senile plaques composed of aggregated amyloid beta (Aβ)
peptides and the generation of PROS in the brain.2,31 There is
excessive accumulation of iron (especially heme b) in the brain
of AD patients in the same region that are characterized by Aβ
Received: January 17, 2013
Published: April 23, 2013
© 2013 American Chemical Society
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deposition.32 Such abnormal increase in the heme concen-
tration in the brain of AD patients has been associated with the
complexation of heme with Aβ peptides. The active site
environment and enzymatic activity of the heme-Aβ complexes
have been widely investigated to show that they behave as
peroxidases, and the mechanism of generation of neurotoxic
PROS has been elucidated.32−34 This complexation leads to
heme depletion and ultimately results in major symptoms like
elevation in iron intake, increase of stress-related enzymes or
proteins such as heme oxygenase-1 and transferrin, high
bilirubin concentration,35,36 abnormal iron homeostasis, decay
of iron regulatory proteins, dysfunction of mitochondrial
complex IV,31,37 oxidative stress, and so forth. Incidentally
the symptoms of AD are also common pathological features of
T2Dm.38,39 Given the overwhelming similarities between AD
and T2Dm with respect to the involvement of iron and
generation of oxidative stress, and the fact that evidence
indicate that heme binding to Aβ peptides is a significant
contributor for the pathology of AD, heme binding to hIAPP
may lead to the cytopathologies of T2Dm resulting in the β-cell
dysfunction.
Recent prospective cohort studies and systematic meta-
analysis have positively associated high heme iron intake, the
major dietary resource of body iron stores, with high risk of
developing T2Dm.40−42 Studies have also linked high heme
iron intake with increased possibility of developing gestational
diabetes.43 Women diagnosed with gestational diabetes have a
22−60% chance of developing T2Dm in the future.44,45 Thus,
there could be a crucial involvement of heme in the T2Dm
pathology, which would open up a new dimension in T2Dm
research and may hold a key toward development of a cure. In
this study we show that heme can bind hIAPP and investigate
the active site environment of these complexes. The three well-
known amino-acid residues present in hIAPP(1−37) are
histidine (His18), cysteine (Cys2 and Cys7), and tyrosine
(Try37) that are commonly involved in binding heme
biologically. Arginine residue, though it does not bond directly
with the heme-Fe, is often involved in secondary interactions
near the active site, stabilizing it and enhancing the
reactivity.33,46 The hIAPP(1−37) has a single arginine residue
(Arg11). Different spectroscopic techniques, absorption,
resonance Raman (rR), and electron paramagnetic resonance
(EPR), have been used to identify the amino acid residue of the
hIAPP peptide that binds to heme, using native hIAPP(1−37)
peptide, various isolated peptide fragments, and site directed
mutants. CO binding to the reduced heme-hIAPP complexes
provides insight into the nature of the active site environment,
along with the elucidation of the key second sphere residue. We
evaluated the effect of pH on these heme bound hIAPP peptide
complexes to elucidate the nature of the exchangeable sixth
ligand. Finally, we demonstrate and quantify that heme bound
hIAPP can generate cytotoxic PROS.
(7−9.5). All the peptide stock solutions for absorption spectra were
made in 100 mM pH 7 phosphate buffer, hIAPP(1−37) peptide stock
was made in 5 mM pH 5.5 Mes buffer. A stock of heme solution was
made by dissolving hemin chloride in 1 M NaOH solution, and the
concentration of the heme solution was determined spectrophoto-
metrically (ε385 = 58.44 mM−1 cm−1).47
Peptide stock solutions were 0.5 mM and heme stock solution was 5
mM. Heme-hIAPP complexes were prepared by incubating 1 equiv of
hIAPP and 0.8 equiv of heme solutions for ∼6 h. Absorption spectra
were recorded by adding 20 μL of the heme-IAPP complex solution in
1 mL of 100 mM pH 7 phosphate buffer. For the pH dependent
absorption data, the peptide stock solution was added to 1 mL of 100
mM of the required buffer needed for the particular pH range. All the
spectral data were obtained by an UV−vis diode array spectropho-
tometer (Agilent 8453).
The pH of the heme-hIAPP complexes for EPR spectra were
calibrated to pH 7 using 1 M H2SO4. EPR spectra were obtained by a
Jeol (JES FA200) spectrophotometer. 0.5 mM concentration samples
were run at 77 K in a liquid nitrogen finger dewar flask.
Resonance Raman spectroscopy data were obtained at room
temperature using a Trivista 555 spectrograph (Princeton Instru-
ments) and 413.1 nm excitation from a Kr+ laser (Coherent, Sabre
Innova SBRC-DBW-K). Raman shifts were calibrated with naph-
thalene and indene. The wavenumber accuracy was 1 cm−1 for well-
defined peaks. The laser power was varied between 10 and 15 mW.
To obtain ferrous and ferrous-CO absorption spectra, all heme-
hIAPP samples were degassed by purging with argon in anaerobic vials
for ∼10 min. Absorption spectra were collected with heme-hIAPP
samples (20 μL) in degassed 100 mM pH 7 phosphate buffer.
Reduction of Fe3+ protein samples were carried out in anaerobic
cuvettes by adding an anaerobically prepared stock solution of 20 mM
sodium dithionite. CO adducts of heme(Fe2+)-hIAPP complexes were
prepared by purging CO(g) through the headspace of septum-sealed
cuvette with gentle agitation of the samples, until no further change
was noticed in absorption spectra. CO(g) was generated by adding
concentrated H2SO4 to ammonium formate and then passing the
generated gas through 4 M KOH.
Cyclic Voltammetry (CV) was performed on a CH Instruments
potentiostat (model CHI710D). An ingenious solution cell was
prepared where a Teflon hollow disk was tightly fit on a graphite
working electrode (∼100 μL volume). A Pt wire was used as the
counter electrode, while an Ag wire was used as the reference
electrode. Heme-hIAPP buffered solutions with 1 M KCl were
prepared. The Fe3+/Fe2+ potential for a 1 mM K4[Fe(CN)6] solution
in 1 M KCl was determined to be 200 mV using this setup, whose
actual value is +450 mV versus NHE. Thus, the E-values measured
against this Ag reference electrode were corrected by +250 mV to scale
them versus NHE. The experiments were performed with degassed
buffers in a glovebox to eliminate the O2 reduction by the heme-hIAPP
complex as well as from the graphite working electrode. Scan rate of 1
V/s was used to obtain the Fe3+/Fe2+ potential. Heme CVs have been
obtained at a high pH (pH ∼ 8−13) which is higher than the pKa of
the heme site (vide infra) and is thus the pure and physiologically
relevant form.
3,3′,5,5′-Tetramethylbenzidine (TMB) was used as the substrate for
peroxidase activity measurement. Ten milligrams of TMB was
dissolved in 0.5 mL of glacial AcOH and 10 mL of AcOH/NaOAc
buffer (1 M, pH 4.5). The solution was diluted to 25 mL with
nanopure water and 100 μL, 30% H2O2 was added, followed by 10 μL
of 0.05 mM of the protein sample. Kinetic traces were obtained by
monitoring the increase of the 652 nm absorption band.
Catalase activity was measured by following the rate of decay of
H2O2 at 240 nm. 1.5 mL of deionized water and 100 μL of 0.05 M
H2O2 were mixed in a cuvette. Twenty microliters of 0.5 mM heme-
hIAPP was added to it. All spectra were recorded in a kinetic mode,
with continuous stirring at 25 °C.
2. EXPERIMENTAL METHODS
All reagents were of the highest grade commercially available and were
used without further purification. Amylin (hIAPP) peptides of various
amino-acid residues (1−37, 1−19, 1−22, and 20−37) have been used
for this study. All site directed mutants are of hIAPP(1−19) peptide.
The mutants used were His18Asn, His18Gly, Arg11Asn, and double
mutant: Arg11Asn, His18Asn. All peptides were purchased from GL
Biochem (Shanghai) Ltd. with >95% purity. Hemin, xylenol orange
and buffers were purchased from Sigma, while sodium dithionite and
Mohr’s salt were purchased from Merck. Different buffers were used at
different pHs: MES for pH (5−6.4), HEPES and phosphate for pH
For PROS calculation, the following xylenol orange assay was
applied. A total of 4.9 mg of Mohr’s salt and 3.9 mg of xylenol orange
were dissolved in 5 mL of 250 mM H2SO4 and stirred for 10 min. A
200 μL portion of this solution was taken in 1.8 mL of nanopure water,
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Scheme 1. Amino Acid Sequence of (A) Human IAPP(1-37) Showing the Non-Amyloidogenic, Hydrophilic Region in Blue and
the Hydrophobic Region in Yellow, Highlighting the Amyloidogenic Segment in Pink, (B) Human IAPP(1-19) Highlighting the
Residues That Have Been Mutated in This Study; (C) Amino Acid Sequence of Human IAPP(1-22) without Disulfide Linkage;
(D) Rat- IAPP(1-37) Highlighting the Sequence Differences with Human IAPP(1-37) in Green
and a calibration curve for the quantitative estimation of H2O2 was
obtained for 0.05, 0.1, 0.5, 1, 2.5, 5, and 10 μM concentrations of H2O2
by recording their absorbance at 560 nm. The calibration curve was
expressed as absorbance at a fixed wavelength of 560 nm versus
concentration of H2O2 in micromolar units for a 2 mL volume.48 For
the detection of PROS of the heme-hIAPP complex, a blank was
obtained in the UV−vis spectrophotometer with 1.8 mL nanopure
water in a cuvette. A total of 50 μL of 0.5 mM heme-hIAPP complex
and 200 μL xylenol orange solution was added to the above cuvette
and absorbance was measured. This served as the control. The heme-
hIAPP complex was reduced using a minimal amount of dithionite
under anaerobic conditions, followed by their reoxidation by O2
(monitored by absorption). A total of 200 μL of 0.025 mM reoxidized
solution was added to the cuvette containing the control, and
absorbance of this solution was recorded. The value of absorbance of
Figure 1. Absorption spectra of heme incubated hIAPP at pH 7:
the above solution (after deducting the control) at 560 nm when
plotted on the calibration curve yielded the corresponding H2O2
concentration.
hIAPP(1−37), red; hIAPP(1−19), green; hIAPP(20−37), blue;
hIAPP(1−22) (peptide without the disulfide linkage between Cys2
and Cys7), brown and free heme, black.
3. RESULTS AND ANALYSIS
3.1. Choice of Peptides. The human islet amyloid
polypeptide (hIAPP) has 37 amino-acid residues. Two
fragments, the hydrophilic hIAPP(1−19) and the amyloido-
genic hIAPP(20−37) have been separately considered to
identify the heme binding domain (Scheme 1A). There are
three residues in hIAPP(1−19) that are known to bind heme
biologically. There is one histidine, His18 (His binds heme in
hemoglobin, myoglobin, cytochrome c oxidase, etc.) and two
cysteine residues, Cys2 and Cys7 (Cys binds heme in
cytochrome P450, nitric oxide synthase, etc.) (Scheme 1B).
However, Cys2 and Cys7 are linked by a disulfide bridge, and
hence the thiolate group of the cysteine residue will likely not
be available for heme coordination (vide infra). There is a
tyrosine residue, Tyr37, present in the amyloidogenic fragment
(Scheme 1A), also known to bind heme biologically in
catalases. To identify the heme binding residue, the following
site directed mutants of hIAPP(1−19) were considered: single
mutants His18Gly and His18Asn (Scheme 1B) to test the
possibility of histidine coordination, single mutant Arg11Asn,
double mutant Arg11Asn; His18Asn to understand the effects
of both Arg11 and His18 (Scheme 1B), and the peptide
fragment hIAPP(1−22) without the disulfide bridge (Scheme
1C).
an intensity decrease of the Soret band at ∼365 nm and a red
shift of the 385 nm band to 398 nm in hIAPP(1−37). Further,
the Q-band blue shifts from 618 to 605 nm with the
development of a new band at 693 nm relative to that of free
heme, indicating the formation of heme bound hIAPP(1−37)
complex (Figure 1, red). The peptide fragment hIAPP(1−19)
also shows similar spectral changes on incubating with heme,
though the Soret band shifts from 385 to 392 nm, (Figure 1,
green), thereby implicating that heme also binds to hIAPP(1−
19).49 However, heme does not bind to the peptide
hIAPP(20−37) as the heme incubated spectrum looks identical
to that of free heme (Figure 1, blue) even after prolonged
incubation. This implies that the heme binding residue lies in
the 1−19 fragment of the peptide sequence. This eliminates
Tyr37 as the possible heme binding residue.
The peptide hIAPP(1−22), without the disulfide bridge
between Cys2 and Cys7 also shows similar spectral changes
relative to native hIAPP(1−19) (Figure 1, brown). Since the
native peptide-heme complex and the heme complex of the
variant without the disulfide bridge (this variant has the thiolate
groups of Cys available for heme coordination) has similar
spectral features, it possibly indicates that heme does not
coordinate to the cysteine residues.
3.2. Absorption Spectroscopy. The absorption spectra of
1 equiv of heme incubated with 1 equiv of hIAPP(1−37) and
the isolated peptide fragments hIAPP(1−19) and hIAPP(20−
37) overlaid with free heme are presented in Figure 1. There is
Thus, there exists only His18 available to coordinate to
heme. Two single mutants His18Asn and His18Gly were each
incubated with 1 equiv of heme. Incidentally, the absorption
spectra of both mutants show the same characteristic features of
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the heme bound forms (Figure 2, orange and blue). Arginine
residues in close vicinity of the active site are known to play key
splitting. No high field, low spin signals were observed at this
temperature. Thus hIAPP peptides bind heme to form an axial
high-spin Fe3+ active site with rhombic distortion, similar to
those found in myoglobin, hemoglobin, and several perox-
idases.52,53 Note that the EPR spectrum of the double mutant
Arg11Asn,His18Asn is identical to that of free heme (Figure 3,
bright green), further indicating that both His18 and Arg11 are
essential for heme binding, consistent with the absorption data
(Figure 2).
3.4. Resonance Raman Spectroscopy. The resonance
Raman (rR) spectra in the high frequency region of the heme
incubated hIAPP(1−37) and hIAPP(1−19) are overlaid with
that of free heme in Figure 4. The oxidation state marker band,
Figure 2. Absorption spectra of heme incubated hIAPP(1−19), green,
and its mutants at pH 7: double mutant, Arg11Asn;His18Asn, bright
green; histidine mutants, His18Gly, orange; His18Asn, blue; and free
heme, black.
roles in heme based metalloenzymes or metallopeptide
complexes.50,51 Recent report in aldoxime dehydratase enzyme
(OxdA) also observed that Arg178 residue is an important
residue to anchor heme and heme content in the protein is low
in the absence of Arg178.46 There exists an arginine residue
(Arg11) in the hIAPP(1−19) fragment. To probe any possible
interaction of the arginine residue, a double mutant Arg11Asn;
His18Asn was selected and incubated with heme. Interestingly,
this mutant on incubating with heme shows spectral features
identical to that of free heme, indicating that it does not bind
heme (Figure 2, bright green). Thus from the absorption data
we infer that heme possibly binds to His18 only in the presence
of Arg11. Note that Arg11Asn mutant when incubated with
heme also binds heme (Supporting Information, Figure S1).
The fact that heme binds to hIAPP even in the absence of
His18 (His18Asn mutant) possibly indicates additional
interactions of the peptide with the heme cofactor (vide infra).
3.3. EPR Spectroscopy. Figure 3 shows the 77 K, X band
EPR spectra of the heme-hIAPP(1−37) and heme-hIAPP(1−
Figure 4. High frequency rR spectra of free heme, black, and heme-
hIAPP complexes: hIAPP(1−37), red, and hIAPP(1−19), green, at
pH 7 at room temperature.
ν4, appears at 1374 cm−1, as expected in ferric heme.54 The
vibrational frequencies sensitive to the core size of the heme
macromolecule are ν10, ν3, and ν2 and serve as the coordination
number and spin state marker bands.55 The ν3 and ν2 bands of
both heme-hIAPP(1−37) and heme-hIAPP(1−19) complexes
are observed at 1569 and 1491 cm−1, respectively, (Figure 4,
red and green respectively), while ν10, the depolarized spin state
marker band arises at 1628 cm−1 indicating a hexa-coordinated
high spin ferric species with an exchangeable weak field distal
ligand (such as H2O) as observed in chloroperoxidase,
cytochrome c peroxidase, horse radish peroxidase (HRP), and
so forth.56,57 There are present small intensities at 1587 (ν2)
and 1507 (ν3) cm−1 in the rR data of hIAPP(1−37), indicative
of formation of a relatively minor fraction of a six-coordinated
low spin ferric species, which is absent in free heme (Figure 4,
black).
3.5. Characterization of CO Binding to Ferrous Heme-
hIAPP Complexes. Exogenous ligands like carbon monoxide
(CO) can bind to the heme Fe from the distal site of the heme-
hIAPP complexes. CO is an extremely useful probe for heme
active sites.58 Studies on CO adducts of heme proteins have
been used to gain structural information, probe identity of the
proximal ligands, and identify interactions between distal
residues and CO, thereby providing insight into the nature of
the heme environment.59 The CO binding to heme-hIAPP
complexes, their mutants, and free heme was tested by
following their absorption spectra under anaerobic condition.
The absorption spectra of CO bound heme(Fe2+) complexes
of hIAPP(1−37), nonamyloidogenic segment hIAPP(1−19),
and amyloidogenic fragment hIAPP(20−37) are overlaid with
free heme(Fe2+)-CO in Figure 5. Binding of CO to reduced
heme complexes of hIAPP(1−37) (Figure 5A and 5B, red) and
Figure 3. EPR spectra of free heme, black and heme-hIAPP complexes
at pH 7: hIAPP(1−37), red; hIAPP(1−19), green; and double mutant
Arg11Asn,His18Asn, bright green in the high field regions at 77 K.
19) complexes overlaid with free heme. The EPR spectra of the
heme bound peptides are shifted to lower field relative to heme,
indicating the formation of heme-bound species. The spectrum
of the heme-peptide complexes shows an axial high spin (S = 5/
2) Fe signal with g ∼ 6.0 with the presence of small rhombic
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Figure 5. Absorption spectra of CO bound form of heme(Fe2+) complexes of hIAPP at pH 7: (A) hIAPP(1−37),red; hIAPP(1−19), green;
hIAPP(20−37), violet; and free heme, black. (B) Enlarged Q-band regions with assigned α and β bands.
Figure 6. Absorption spectra of CO bound form of heme(Fe2+)-hIAPP complexes at pH 7: hIAPP(1−19), green; histidine mutant His18Asn, blue;
double mutant Arg11Asn;His18Asn, pink; arginine mutant Arg11Asn, cyan; and free heme, black. (B) Enlarged Q-band regions with assigned α and
β bands.
Figure 7. Absorption spectra of heme-hIAPP complexes at different pHs: (A) hIAPP(1−37), (B) hIAPP(1−19), (C) His18Asn mutant, and (D)
Arg11Asn mutant. Inset: enlarged Q-band region.
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Figure 8. pKa plots of heme-hIAPP complexes of (A) (1−37), red; (1−19), green; and (B) His18Asn mutant of (1−19), green, and Arg11Asn
mutant, blue, with error bars. Six data points from Figure 7 have been used for each pKa determination.
Figure 9. (A) Anaerobic CV at 1 V/s of heme-hIAPP(1−37) (red solid line) and hemehIAPP(1−19) (red dotted line) at pH 8. (B) E° (vs AgCl)
plot with the increase of pH of heme-hIAPP(1−37), black, with error bars.
hIAPP(1−19) (Figure 5A and 5B, green) results in the Soret
maxima at 423 nm and α and β bands at 571 and 542 nm,
respectively, consistent with a hexacoordinate heme active site
with histidine residue as an axial ligand.60−62 Similar
spectroscopic features of the CO complex of heme(Fe2+)-
hIAPP(1−22) (peptide without the disulfide linkage) are also
observed (Supporting Information, Figure S1). This indicates
that heme binding to hIAPP does not alter even in the absence
of the disulfide bridge, consistent with the absorption data
(Figure 1, brown). CO binding to reduced heme incubated
complex of hIAPP(20−37) (Figure 5A and 5B, violet) results in
a Soret maximum at 408 nm and α and β bands at 566 and 535
nm, respectively, identical to the CO bound spectra of free
heme(Fe2+) (Figure 5A and 5B, black). This further supports
that heme does not bind to Tyr37 present in the hIAPP(20−
37) peptide fragment, consistent with our above results (Figure
1, blue).
mutant Arg11Asn;His18Asn results in the Soret maximum at
408 nm and α and β bands at 566 and 535 nm (Figure 6A and
6B, pink), respectively, identical to that of free heme(Fe2+)-CO
(Figure 6A and 6B, black). Thus it further supports the fact that
though heme binds to hIAPP peptides through the His18
residue, Arg11 residue is key for heme binding to hIAPP as
indicated by the absorption data of the double mutant
Arg11Asn;His18Asn (Figure 2, green) and its CO complex
(Figure 6A and 6B, pink).
3.6. pH Perturbation and Trans Axial Ligand. Since the
heme-hIAPP complexes bind exogenous ligands like CO, it
likely implies the presence of a sixth exchangeable ligand trans
to the axial His18 residue. The nature of the sixth ligand was
investigated by pH perturbations. The heme-hIAPP(1−37) and
hIAPP(1−19) complexes exhibit an increase in the Soret band
(∼ 365 nm) and a decrease in the Q-band (∼ 635 nm)
intensities and a blue shift of the Q-band on going from low to
high pH (Figure 7A and 7B) with a pKa of ∼7.3 0.2 (Figure
8, red and green). Similar spectral changes with changes in pH
are observed for the His18Asn mutant (Figure 7C), but the pKa
obtained was ∼7.15 0.2 (Figure 8, orange), lower than that in
native hIAPP peptides. The pKa of Arg11Asn mutant was ∼7.6
0.2 (Figure 7D) which is slightly higher than the wild type
hIAPP. The observed pKa of ∼7.3 likely indicates the presence
of a water derived ligand.33,56 This is also confirmed by the rR
data (Figure 4).
CO binding to heme(Fe2+) complex of His18Asn mutant
results in a Soret maximum at 410 nm and α and β bands at
568 and 538 nm, respectively (Figure 6A and 6B, blue). This is
drastically different from the spectrum of the CO complex of
native heme(Fe2+)-hIAPP(1−37) and heme(Fe2+)-hIAPP(1−
19) (Figure 6A and 6B, green). This again indicates that
probably His18 is the heme coordinating residue. The
absorption spectrum of CO bound heme(Fe2+)-Arg11Asn
mutant results in a Soret maximum at 418 nm and α and β
bands at 566 and 538 nm, respectively (Figure 6A and 6B,
cyan), which is also different than that of the wild type CO.
This is in accordance to our inference that Arg11 residue
strongly interacts with the active site. CO binding to the double
3.7. Cyclic Voltammetry. Cyclic Voltammerty (CV) of
native heme-hIAPP complexes shows reversible process under
anaerobic condition (Figure 9). Free heme has a reduction
potential of 0.20 V vs NHE.63 The heme-hIAPP(1−37) shows
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a Fe3+/Fe2+ redox process at −0.36 V (Figure 9A, red solid
line), while heme-hIAPP(1−19) show a Fe3+/Fe2+ redox
process at −0.34 V (Figure 9A, red dotted line) at pH 8.
The redox potential changes by ∼0.056 V per unit change in
pH in the pH range of 8−13 for heme-hIAPP(1−37) (Figure
9B). This indicates that the reduction of Fe3+ to Fe2+ involves a
proton coupled electron transfer (PCET) process involving one
proton.64−66 This is consistent with a Fe3+(H2O) →
Fe2+(HO−) redox process at the active site.
3.8. Reactivity. 3.8.1. Peroxidase Activity. Peroxidases are
heme based metalloenzymes with a coordinated histidine
residue containing a conserved arginine residue in the distal
pocket that provides protons for the O−O bond cleavage of
H2O2 in the catalytic cycle.50,67 The peroxidase activity of the
native heme-hIAPP complexes and free heme were tested by
following the catalytic oxidation of substrate, 3,3′,5,5′-
tetramethylbenzidine (TMB), by H2O2. The heme-hIAPP(1−
37) and heme-hIAPP(1−19) do not show any enhanced
peroxidase activity relative to that of free heme (Figure 10).
The Arg11 mutant also did not exhibit any peroxidase activity
relative to free heme (Supporting Information, Figure S3).
disproportionation of O2•−) has been detected by using the
xylenol orange assay (Materials and Methods Section). The
results indicate that heme(Fe2+)-hIAPP(1−37) produces 40
5% H2O2 and heme(Fe2+)-hIAPP(1−19) generates 38
5%
H2O2.
4. DISCUSSION
The results presented in this study indicate that the hIAPP
peptides associated with T2Dm bind heme, and the heme
binding domain resides in the nonamyloidogenic 1−19
segment of hIAPP. From the EPR and rR data it is evident
that heme-hIAPP complexes form an axial high spin Fe3+ active
site with an S = 5/2 ground state. There exist three heme
coordinating residues in the hydrophilic peptide fragment
hIAPP(1−19), His18, Cys2, and Cys7, that are known to bind
heme biologically. There is also a Tyr37 residue present in the
amyloidogenic peptide fragment IAPP(20−37) that can
possibly bind heme. Since the peptide fragment IAPP(20−
37) does not bind heme, even after prolonged incubation
(Figure 1, blue), Tyr37 is not the heme coordinating residue.
This is further supported by the lack of catalase activity of these
heme-IAPP complexes (Supporting Information, Figure S4),
implying that no tyrosine coordinated catalase type active site
results on heme binding to hIAPP peptides. Cys2 and Cys7 are
linked by a disulfide bond due to which the thiolate side chain
of cysteine is not available for heme binding to the peptides.
When heme was incubated with hIAPP(1−22), peptide without
the disulfide bond between Cys2 and Cys7, the resultant
absorption spectrum (Figure 1, brown) was identical to that of
the heme bound hIAPP(1−37) and hIAPP(1−19) complexes,
implying that cysteine probably does not coordinate to heme
bound hIAPP. This conclusion is further substantiated when
the CO complex of heme(Fe2+)-hIAPP(1−22) (peptide
without disulfide linkage) showed similar spectral features
(Supporting Information, Figure S2) to that of CO complex of
heme(Fe2+)-hIAPP(1−19) (Figure 5, green). Thus, His18 is
the only other residue available to coordinate heme. The CO
complex of heme(Fe2+)-hIAPP has the Soret band at 423 nm
and α and β bands at 571 and 542 nm respectively, indicating a
histidine ligated six coordinate heme active site.61,62,69
Interestingly, the absence of His18 (His18Asn and His18Gly
mutants, Figure 2) also indicates heme binding to the peptide.
However, the double mutant Arg11Asn;His18Asn does not
bind heme (Figure 2, bright green), reflecting that though
His18 is the heme coordinating residue, Arg11 plays a crucial
role in heme binding and Arg11 binds heme in the His18
mutants. This is further reflected by the absorption data of the
CO complex of the His18Asn mutant, which shows a shift in
the Soret band from 423 nm in the native hIAPP peptide to
410 nm in the mutant (Figure 6A and 6B, blue), indicating that
His18 is likely involved in heme binding. The CO complex of
the Arg11Asn mutant has the Soret maximum at 418 nm
(Figure 6A and 6B, cyan), relative to 423 nm in the native
hIAPP, further implicating that Arg11 is also involved in heme
binding. Further, the absorption data of the CO complex of
His18Asn;Arg11Asn double mutant is identical to that of free
heme (Figure 6A and 6B, pink), indicating that both His18 and
Arg11 are essential for heme binding to hIAPP peptides.
Arginine residues are known to act as key second sphere
residues in heme based metalloproteins and peptides, providing
protons for catalytic reactions.33,50 The Arg11 residue can
interact with heme in any of the following three ways: (i) bind
heme as an axial ligand along with His18, (ii) hydrogen bond
Figure 10. Kinetic traces for relative peroxidase activity, monitoring
the increase of the 652 nm absorbance intensity, for the native heme-
hIAPP complexes: hIAPP(1−37), red; hIAPP(1−19), green; and free
heme, black.
3.8.2. Catalase Activity. The catalase (tyrosine coordinated
heme enzyme) activity of the heme-hIAPP complexes show
that these heme complexes do not show any catalase activity
with respect to free heme (Supporting Information, Figure S4).
This indicates that Tyr37 does not coordinate to heme to form
any catalase type active sites.68 This further supports the
conclusion drawn from spectroscopy that the heme binding
domain lies in the peptide segment IAPP(1−19).
3.8.3. PROS Generation. The reduction of O2 by heme-
(Fe2+)-hIAPP complexes was investigated under homogeneous
condition. When 1 equiv of dithionite was added to the heme-
hIAPP peptide solutions, the resting Fe3+ site of heme was
reduced to Fe2+ (Supporting Information, Figure S5). When O2
was introduced to the either fully reduced heme(Fe2+)-
hIAPP(1−37) or heme(Fe2+)-hIAPP(1−19) complexes, it
reoxidized the Fe2+ to the Fe3+ form as indicated by absorption
spectroscopy (Supporting Information, Figure S5A and S5B).
In this process the O2 gets reduced. This reduction of O2 by the
heme(Fe2+)-hIAPP peptides can either produce superoxide
(O2•−), H2O2, or it could be a 4 e−/4 H+ process leading to the
formation of H2O. Any H2O2 formed during the O2 reduction
process (either due to the 2 e− reduction of O2 or by the
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with either the proximal His18 residue or the distal water
derived ligand, and (iii) hydrogen bond or ion pair (arginine is
positively charged and the heme propionate groups are
negatively charged at pH 7) with the propionate side chains
of heme. The first possibility is unlikely as there are no reported
cases of Arg binding to heme in biological systems, although
Arg is known to bind to the nonheme Fe active site of Biotin
Synthase.70,71 Furthermore, a six coordinate Fe3+ active site
with six nitrogen donor ligands is almost certain to be low spin
and not high spin as observed for this active site (Figure 3 and
4). The second possibility where Arg11 hydrogen bonds with
the NH of His18 (which is a hydrogen bond donor) is unlikely
as the lone pair of Arg11 will be protonated at pH 7, that is, it
can not accept a hydrogen bond. Hydrogen bonding to the
distal water derived ligand is quite likely. But this does not
explain the binding of heme to hIAPP even in the absence of
His18 (i.e., His18Asn and His18Gly mutants) and the relatively
small shift in pKa of the trans axial H2O ligand upon mutation
of the Arg11 residue. The third possibility, where Arg11
hydrogen bonds with the propionate side chain of heme along
with His18 coordination to heme, allows heme binding to both
His18Asn and His18Gly mutants (via Arg11 hydrogen bonding
or ion pairing with the propionate side chains of heme) and
Arg11Asn mutant (heme binding via His18). This has been
observed in the active site soluble Guanylate Cyclase (sGC),
where it has been shown that protoporphyrin IX (heme
without the Fe) can bind to the active site and be held tightly in
position by hydrogen bonding interactions between the
propionate side chain of heme and Arg139.72 Similarly, on
binding NO to sGC, the proximal histidine ligand (His105)
dissociates from the active side, while the heme is held by
hydrogen bonding interactions with Arg139. If the Arg11 group
is held near the propionate side chains of the heme, the
distance of the Arg11 from the active site would prevent it from
exerting the pull effect required for exhibiting peroxidase
activity (Figure 10). Similarly the lack of direct hydrogen
bonding interaction with the distal water ligand would lead to a
negligible shift of pKa for the Arg11 mutant (Figure 7D and
8B) relative to the wild type.
mitochondrial structure and function leads to profound effect
on mitochondrial iron concentration and heme homeostasis,
decreasing the activity of iron-containing enzymes and
complexes of the electron transport chain (ETC), which gives
rise to more fatty acid oxidation and increased risk of
developing T2Dm.77 Recent studies have shown that IAPP
deposits not only are exclusive in pancreatic islets but also are
found in kidneys,78 heart,79 with higher plasma concentration in
T2Dm patients than in controls. Hyperamylinemia have been
shown to induce risks like diabetic nephropathy and hampered
renal functions, altered cardiac myocyte structure and function
that contribute to progressive heart failure.78,79 Thus disrupted
heme homeostasis, compromised mitochondrial metabolism,
deposits of IAPP in organs like kidney, heart besides pancreatic
islets, along with our finding of heme binding to hIAPP under
physiological conditions and the associated PROS generation
provide an insight that heme may play a greater role in T2Dm
than previously comprehended.
Both AD and T2Dm have been demonstrated to share
common modes of toxic pathway through mitochondrial
dysfunction.39 Specifically, in both the diseases there occurs
significant reduction in complex IV activity, mitochondrial
respiration and increased PROS formation.39,80 Heme binding
to Aβ peptide associated with AD followed by subsequent
aberrations in the heme biogenesis pathway have been linked to
mitochondrial decay in AD.34,81 Similar epidemiological
features between AD and T2Dm suggest that heme binding
to hIAPP might be responsible for the loss of β-cells in T2Dm.
His13 residue in Aβ peptides binds heme under physiological
conditions and results in an axial high-spin active site with a
trans-axial water derived ligand, while acidic Agr5 residue
present at the distal side is responsible for the peroxidase
activity of these complexes.33 The active site environment of
heme-hIAPP is similar to that of heme-Aβ, as His18 binds
heme with a trans-axial water derived ligand. However, Arg11
possibly directly hydrogen bonds with the propionate side
chains of heme, thereby it is also essential for heme binding.
However, the heme-hIAPP complexes do not function as
peroxidase like heme-Aβ complexes, suggesting that the Arg11
is not available in the distal pocket to provide any proton
required to drive O−O bond heterolysis.
CV data at around physiological pH indicate that the heme
sites (E° = −0.34 V, i.e., −0.11 vs NHE) can be reduced by
physiological reducing agents like glutathione (E° = −0.25 V vs
NHE)73 and NADH (E° = −0.45 V vs NHE).74 The reduced
heme-IAPP complexes when exposed to O2 generate H2O2
which has been quantified. The 1 e− reduction of O2 produces
The hallmark of both AD and T2Dm is heightened oxidative
stress and PROS formation.38 While heme(Fe2+)-Aβ reduces
O2 by 2 e− to H2O2 quantitatively,48 heme(Fe2+)-hIAPP
produces ∼40% H2O2 through 1 e− reduction of O2. This is
because the Tyr10 residue of Aβ peptides is redox active and
donates an electron to O2 along with the reduced heme center
leading to a 2 e− reduction of O2 to H2O2. On the contrary, the
Tyr37 residue in hIAPP is not involved in O2 reduction by
reduced heme-hIAPP complex which can, therefore, reduce O2
•
superoxide, O2 ¯, which after disproportionation should yield
∼50% H2O2. The 2 e− reduction of O2 on the other hand
produces 100% H2O2. Experimentally ∼40% PROS has been
estimated for the heme-hIAPP complexes indicating that only 1
e− is being donated by the reduced metal center to O2 in
aqueous buffered solutions having physiological pH.
only by 1 e− to O2 . This is likely due to the close proximity of
−
The pancreatic β-cells are extraordinarily sensitive to damage
from PROS generation compared to other cells found in
skeletal muscles, liver, kidney, brain, and so forth.17,75 Thus
heme binding to hIAPP(1−37) and the PROS generation from
oxidation of the reduced heme-hIAPP(1−37) complex by
molecular O2 provide a vital explanation for the extreme
susceptibility of pancreatic β-cells toward oxidative stress and
the reduction of β-cell mass and its dysfunction in T2Dm.
Compromised mitochondrial activity and its reduced content,
heightened oxidative stress which consequently leads to altered
insulin sensitivity have been proposed to be an underlying
feature of obesity-driven T2Dm.76 The disruption of
the Tyr10 residue to the heme coordinating His13 residue in
heme-Aβ peptides. In the case of heme-hIAPP the heme
coordinating residue His18 is possibly not in the vicinity of the
Tyr37 residue.
It is important to note that rats are not affected by T2Dm.
Rat-IAPP is nonamyloidogenic and differs from hIAPP at 6
amino acid residues (Scheme 1D). Five sequence differences
mainly reside in the 20−29 peptide domain and are known to
reduce its amyloidogenicity, while the major change is the
replacement of His18 by an Arg in the nonamyloidogenic
part.10,82 The fact that the His18 residue involved in heme
binding to hIAPP is absent in rat-IAPP is very interesting and
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(9) Leighton, B.; Cooper, G. J. S. Nature 1988, 335, 632.
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the pathology of T2Dm.
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5. CONCLUSION
In conclusion, we have established that heme binds to human
IAPP. The heme binding domain lies in the 1−19 non-
amylodogenic segment of hIAPP. His18 of hIAPP peptides
binds heme at the proximal site. The open distal side of the
heme-hIAPP complex has an exchangeable water derived ligand
with a pKa 7.3. The Arg11 is a key residue that directly
hydrogen bonds with the propionate side chain of heme and
plays an important role in heme binding to hIAPP. These
findings illustrate the nature of the interaction of heme with
hIAPP peptides which is fundamental in unraveling the role
played by heme in T2Dm. Heme(Fe2+)-hIAPP complexes
reduce O2 by 1 e− to produce ∼40% H2O2. The electro-
chemical data around physiological pH suggests that heme-
hIAPP complexes can be reduced by physiological reductants
and are prone to generate cytotoxic PROS and oxidative stress.
The His18 residue of hIAPP, involved in heme binding, is
absent in rat IAPP. Incidentally, rats do not get affected by
T2Dm, further implying the importance of heme in the onset
and development of T2Dm.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Absorption spectrum of Arg11Asn mutant, CO bound heme-
hIAPP(1−22) complex, peroxidase activity of Arg11Asn
mutant, catalase activity plot of heme-hIAPP complexes, and
the 1 equivalent reduction by dithionite and consequent
reoxidation by O2 of heme-hIAPP complexes. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: icsgd@iacs.res.in.
Notes
■
(29) Lenzen, S.; Drinkgern, J.; Tiedge, M. Free Radical Biol. Med.
1996, 20, 463.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We thank IACS for funding this research. S.M. is thankful to
the Council of Scientific and Industrial Research, India, for a
Junior Research Fellowship. We thank Dr. Abhishek Dey for
helpful discussions along with use of the resonance Raman
instrument (DST, India, Grant-SR/IC-35/2009) and Sudipta
Chatterjee for help with CV experiments.
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