The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme

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The Iron Chaperone Protein CyaY from Vibrio cholerae is a Heme-Binding Protein Takeshi Uchida, Noriyuki Kobayashi, Souichiro Muneta, and Koichiro Ishimori Biochemistry, Just Accepted Manuscript • Publication Date (Web): 24 Apr 2017 Downloaded from http://pubs.acs.org on April 24, 2017

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Biochemistry

The Iron Chaperone Protein CyaY from Vibrio cholerae is a Heme-Binding Protein

Takeshi Uchida*1,2, Noriyuki Kobayashi2, Souichiro Muneta2, and Koichiro Ishimori1,2

1

Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan

2

Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo 060-8628, Japan

Corresponding Author *Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo 060-0810, Japan. E-mail: [email protected].

Funding Information This study was supported in part by Grants-in-Aid for Scientific Research (16K05835 to T.U. and 15H00909 to K.I.) from the Ministry of Culture, Education, Sports, Science, and Technology (MEXT) of Japan.

1 ACS Paragon Plus Environment

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ABBREVIATIONS: Fe-S, iron-sulfur; ROS, reactive oxygen species; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass; VcCyaY, CyaY from Vibrio cholerae; WT, wild-type.


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Biochemistry

ABSTRACT CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe2+ into protoporphyrin IX. Here, we found that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 µM. Circular dichroism spectra of CyaY suggested that heme binding to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.

KEYWORDS: heme, iron, enzyme, heme biosynthesis


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INTRODUCTION Iron is a trace element essential for nearly all life on earth. In eukaryotes, frataxin plays an essential role in iron homeostasis.1–3 Defects in frataxin can result in the accumulation of high concentrations of iron, which are toxic to cells owing to production of reactive oxygen species (ROS). This is because frataxin mediates iron transfer to the iron-sulfur (Fe-S) cluster scaffold protein Isu1,4–7 which assembles both [2Fe-2S] and [4Fe-4S] clusters consisting of iron and sulfur atoms transferred by frataxin and the desulfurase Nfs, respectively. Frataxin also delivers iron to ferrochelatase, which catalyzes insertion of iron to protoporphyrin IX to produce heme.8–10 In this context, loss-of-function mutations in the gene encoding frataxin lead to the neurodegenerative disease, Friedreich’s ataxia, as a result of ROS damage.11 Delivery of iron is not the only function of frataxin, which is a multifunctional protein: iron homeostasis,12 iron-storage,10,13 protection against oxidative damage14 as well as iron chaperone.15 Although it is well known that frataxin deficiency causes phenotype including loss of Fe-S cluster proteins, there is no consensus regarding the main function of frataxin. Frataxin is evolutionarily conserved in mammals, yeast, nematode, and Gram-negative bacteria. In bacteria, the frataxin homologue, CyaY, has been found in Escherichia col 16 and Acidithiobacillus ferrooxidans.17 CyaY from E. coli shares 30% sequence identity with the C-terminal region of human frataxin (residues 91–210) (Supplemental Figure S1). CyaY, like frataxin, binds to the desulfurase, IscS. However, binding of CyaY to IscS slows the kinetics of Fe-S cluster formation on the scaffold protein IscU in an iron-dependent manner,18–20 which is in sharp contrast to eukaryote frataxin. Instead, binding of CyaY to Fe-S cluster assembly strengthens the affinity of the assembly, indicating that CyaY function as a regulator of the assembly.21 Furthermore, deletion of the cyaY gene does not affect cellular metabolism.22 These results raised a possibility that CyaY from bacteria is not an iron chaperone protein despite sequence and structural similarity, but has functions different from


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Biochemistry

that of eukaryotic frataxin. Although CyaY is an iron-binding protein, its affinity for iron is relatively low; for example, the dissociation constant (Kd,Fe) of CyaY from E. coli for iron is 3.8 µM.23 However, this characteristic would be suitable for a chaperone protein, where reversible iron binding is an important functional property. Furthermore, anaerobic addition of Fe2+ causes CyaY to form a trimer, which further forms larger oligomers upon oxidation of the bound iron.16,23 These results also suggest another function of CyaY: formation of iron-loaded multimers that scavenge iron, like ferritin. CyaY is further known to interact with ferrochelatase, as frataxin does.24 Although heme is a cofactor of hemoproteins, such as hemoglobin, catalase and P450, it is also known to act as an effector molecule.25 Here, we investigated the possibility that heme binding to proteins involved in heme biosynthesis induces a feedback mechanism on the heme biosynthetic process. In our previous study, we found that HutZ from Vibrio cholerae is a heme-degrading enzyme, which degrades heme and extract iron.26,27 Because CyaY is an iron chaperone protein, we investigated whether CyaY receives iron from HutZ directly, but the enzymatic activity of HutZ was not influenced in the presence of CyaY. However, in this study, we serendipitously found that CyaY binds 2 equivalents of heme. Mutational analyses suggested that Tyr67 and Cys78 are heme-binding residues. Upon binding of heme to CyaY, the Kd,Fe of CyaY increased, indicating a decrease in the affinity of CyaY for iron, a change that would facilitate iron transport from CyaY to the Fe-S cluster assembly machinery or ferrochelatase. Therefore, we postulate that heme itself serves to regulate biosynthesis of Fe-S cluster or heme through binding to CyaY.

EXPERIMENTAL PROCEDURES Materials.

The chemicals used in this study were purchased from Wako Pure Chemical


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Industries (Osaka, Japan), Nacalai Tesque (Kyoto, Japan), or Sigma-Aldrich (St. Louis, MO, USA), and used without further purification. Protein Expression and Purification.

The cyaY gene was synthesized by Eurofins

Genomics (Tokyo, Japan) according to the cDNA sequence reported in the NCBI data base [GI: 2612963 (http://www.uniprot.org/uniprot/Q9KVL8)], with codon optimization for expression in E. coli. The gene was cloned into a pGEX-6P-1 vector (GE Healthcare, Uppsala, Sweden) using the BamHI and NotI sites, yielding a construct of the cyaY gene fused to an N-terminal glutathione S-transferase (GST) tag. E. coli carrying CyaY expression plasmids were grown at 37 °C in LB broth supplemented with 50 µg/mL ampicillin. After cells reached an optical density at 600 nm (OD600) of 0.8, expression of CyaY was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside and cells were further incubated at 28 °C overnight. The cells were harvested by centrifugation and suspended in PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4). The cell pellet (~5.0 g) obtained from 1 liter of culture was thawed and incubated with 1 mg/mL lysozyme and DNase on ice for 60 minutes. Thereafter, the sample was sonicated using a Branson 250 sonifier (Danbury, CT, USA) and centrifuged at 40,000 × g for 30 minutes. GST-tagged CyaY was purified by affinity chromatography using COSMOGEL GST-Accept (Nacalai Tesque, Kyoto, Japan). The eluted protein was treated with Turbo3C protease (Accelagen, San Diego, CA, USA) at 4 °C for 16 hours to remove the GST-tag. After cleavage, the reaction mixture was again applied to the GST-Accept resin to remove uncleaved protein, cleaved GST-tag, and GST-tagged protease. The flow-through fraction was applied to a gel-filtration column (HiLoad 16/60 Superdex 200 pg; GE Healthcare) equilibrated with 50 mM Tris-HCl/150 mM NaCl (pH 8.0). Mutagenesis of CyaY was conducted using a PrimeSTAR Mutagenesis Basal kit from Takara Bio (Otsu, Japan). DNA oligonucleotides employed for mutation, purchased from


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Biochemistry

Eurofins Genomics, are listed in Supplemental Table S1. The mutated genes were sequenced (Eurofins Genomics) to ensure that only the desired mutations were introduced. Measurement of CyaY Heme-Binding Ability. Heme binding was tracked by difference spectroscopy in the Soret region of UV-vis absorption spectra. Aliquots of 0.5 mM hemin in 0.1 M NaOH were added to both the sample cuvette containing 5 µM CyaY and reference cuvette containing buffer only. Spectra were recorded 3 minutes after addition of each heme aliquot. The absorbance difference at 373 nm was plotted as a function of the heme concentration, and the apparent dissociation constant (Kd,heme) was calculated using equation 1,

[ P ] + [ H] + K d,heme − ([ P ] + [ H] + K d,heme ) ∆A = ∆Amax 2 [P]

2

− 4 [ P] [ H]

(1)

where ∆A is the change in absorption difference between sample and reference cell upon addition of hemin, ∆Amax is the maximum of the absorption difference upon addition of hemin, [P] and [H] represent total protein and hemin concentrations, respectively. Spectroscopy.

A Bruker

autoflex

matrix-assisted

laser

desorption

ionization

time-of-flight mass (MALDI-TOF MS) spectrometry (Bruker Daltonics, Billerica, MA, USA) operated in the reflector mode was used to obtain the mass of the protein sample. Spectra were externally calibrated using Protein calibration standard I of a Starter Kit for MALDI-TOF MS (Bruker Daltonics). Optical spectra of purified proteins were recorded with a V-660 UV-vis spectrophotometer (Jasco, Tokyo, Japan) at 25 °C. Resonance Raman spectra were obtained using a single monochromator (SPEX500M; Jobin Yvon, Edison, NJ, USA) equipped with a liquid nitrogen-cooled CCD detector (Spec-10:400B/LN; Roper Scientific, Princeton, NJ, USA). Samples were excited at wavelengths of 413.1 and 441.6 nm using a krypton ion laser (BeamLok 2060; Spectra Physics, Santa Clara, CA, USA) and helium-cadmium laser 7 ACS Paragon Plus Environment

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(IK5651R; Kimmon Koha, Tokyo, Japan), respectively. The laser power at the sample point was adjusted to ~5 mW for the ferric and ferrous forms, and to 0.1 mW for the CO-bound form to minimize photodissociation. Raman shifts were calibrated with indene, CCl4, acetone, and an aqueous solution of ferrocyanide. The accuracy of the peak positions of well-defined Raman bands was ±1 cm–1. Sample concentration for Raman experiments was approximately 10 µM in 50 mM Tris-HCl/150 mM NaCl (pH 8.0). Circular dichroism (CD) spectra in the far-UV region were measured with a J-1500 CD spectrometer (Jasco) over the spectral range of 190 to 250 nm at room temperature. Spectra were acquired at 0.5-nm intervals with a scan rate of 25 nm/min using a quartz cuvette with a path length of 10 mm; values presented are the average of three scans. The sample concentration was 2 µM in 100 mM sodium phosphate/50 mM NaCl (pH 6.0). A buffer spectrum measured under the same condition was subtracted to obtain the actual sample spectrum. The heme dissociation rate from VcCyaY was obtained spectrophotometry from a reaction solution containing 4 µM heme-VcCyaY and 60 µM apomyoglobin in 50 mM Tris-HCl/150 mM NaCl (pH 8.0) at 25 °C. Apomyoglobin was prepared by extracting heme using the acid/butanone method.28 Further addition of apomyoglobin did not affect the dissociation rate constants, indicating that the reaction proceeded under pseudo first-order conditions. The reaction was monitored by measuring the increase in absorbance at 409 nm after the addition of apomyoglobin. Measurements were taken with a Jasco V-660 UV-vis absorption spectrophotometer. The data were best fit to a double-exponential equation using Igor Pro (WaveMetrics, Portland, OR, USA). Size-Exclusion Chromatography.

Protein oligomerization was analyzed using an ENrich

SEC 650 gel-filtration column (Bio-Rad, Hercules, CA, USA) equilibrated in 50 mM Tris-HCl/150 mM NaCl (pH 8.0). The elution profile was monitored at 280 nm for


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Biochemistry

protein-containing fractions and 380 nm for heme-containing fractions. Standards with known molecular mass (thyroglobulin, 669,000 Da; ferritin, 440,000 Da; catalase, 232,000 Da; aldolase, 158,000 Da; bovine serum albumin, 66,000 Da; ovalbumin, 43,000 Da; chymotrypsinogen, 25,000 Da; ribonuclease A, 13,700 Da; and blue dextran, 2,000 kDa) were applied to the column (Gel Filtration Calibration Kits; GE Healthcare), and their elution volumes were determined at 280 nm. Metal-Binding Assay.

The affinity of iron was determined by measuring quenching of

tryptophan fluorescence (excitation at 295 nm and emission at 340 nm).15 Purified protein (2 µM) in 50 mM Tris-HCl/150 mM NaCl (pH 8.0) was placed in a quartz cuvette, after which aliquots of a freshly prepared solution of concentrated ferrous ammonium iron(II) (10 mM) were added stepwise to the protein solution under anaerobic condition. Fluorescence spectra were obtained 1 hour after each addition of Fe2+ using an FP-8500 spectrofluorimeter (Jasco). The dissociation constant (Kd,Fe) of Fe2+ for the protein was calculated by fitting the data to equation 2,

[ P] + [ M ] + K d,Fe − F0 − F = F0 − Fmin

([ P ] + [ M] + K ) d,Fe

2 [ P]

2

− 4 [ P] [M]

(2)

where F is the observed fluorescence at 340 nm, F0 is the initial fluorescence in the presence of Fe2+, Fmin is the minimum fluorescence intensity at 340 nm upon addition of a large concentration of Fe2+, and [P] and [M] represent total protein and Fe2+ concentrations, respectively. Although multiple metal-binding sites exist,15,23 each apparent dissociation constant is assumed to be the same. The data presented were averaged from three independent measurements.

RESULTS Cloning, Expression and Purification of VcCyaY.

The E. coli strain BL21(DE3) was used


Biochemistry

for overexpression of CyaY from V. cholerae (hereafter termed VcCyaY). The purified protein was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which showed that VcCyaY migrated as a single band with an approximate molecular mass of 18 kDa (Figure 1A), a value slightly larger than the calculated molecular mass for VcCyaY (12 kDa). A size-exclusion chromatography analysis indicated that the protein existed as a monomer (~15 kDa) (Figure 1B). The molecular mass of the purified VcCyaY was determined to be 12,141 Da using MALDI-TOF mass spectroscopy, which coincided well with the calculated mass of VcCyaY containing five extra amino acid (Gly-Pro-Leu-Gly-Ser) at the N-terminus (12,140.4 Da) (Supplemental Figure S2), indicating that the protein was purified without modification or undesirable cleavage. (B

(A) (M)

q

6.5

6.0

80 50 40

20

400

5.5

logMW

30

500

300 5.0 200 4.5

15

Absorbance / mAu

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 4.0

10 0

3.5 40

60

80

100

Elution Volume /mL

Figure 1. Purification of VcCyaY. (A) SDS-PAGE gel of purified VcCyaY (lane 1) and molecular mass markers (lane M). (B) Determination of the molecular mass of VcCyaY by gel-filtration column chromatography. Analytical gel filtration was performed using a Superdex 200 pg column equilibrated with 50 mM Tris-HCl/150 mM NaCl (pH 8.0) with a flow rate of 1 mL/min. The plot shows the log of molecular weight standards versus elution volume, including the following protein standards: thyroglobulin, 50.0 ml; ferritin, 56.4 ml; catalase, 66.7 ml; aldolase, 67.5 ml; albumin, 76.3 ml; ovalbumin, 81.8 ml; chymotrypsinogen A, 92.5 ml; and ribonuclease A, 97.6 ml. 10 ACS Paragon Plus Environment

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Biochemistry

The purified protein was colorless, suggesting that it was purified without any pigments (i.e., heme), an interpretation supported by the absorption spectrum, which lacked any features in the visible region (Figure 2A). Addition of an aliquot of hemin solution to VcCyaY resulted in the appearance of a broad Soret band at about 377 nm. This peak position and the shape of the Soret band were slightly, but detectably, different from those of free hemin (λmax, ~387 nm), indicating that heme specifically binds to VcCyaY (Figure 2A). Next, the stoichiometry of binding and the binding constant of VcCyaY for heme were obtained. A solution containing a fixed concentration of VcCyaY was titrated with increasing amounts of heme. Absorption spectra obtained after subtracting spectra for buffer titrated with the same amount of heme are shown in Figure 2B. The absorbance difference at 373 nm, plotted against heme concentration, increased with an increase in the heme concentration to 2.0 equivalents (inset), suggesting a binding stoichiometry of approximately 1:2. However, it is difficult to determine the exact value using absorption spectra owing to the presence of two different Soret peaks (373 and 419 nm). The apparent dissociation constant (Kd,heme) was calculated by fitting the absorbance change to equation 1 to be 21 ± 6 nM. However, the obtained Kd,heme value is not representative of the true Kd value because the binding curve is obtained under the condition that no free heme exists during the titration.


Biochemistry

(A)

(B 3 8 7

A b s o r b a n c e

0 .1 2 0 .1 0

3 7 7

0 .0 8 0 .0 6 0 .0 4 0 .0 2 0 .0 0 3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

W a v e le n g t h /n m

(C

(D) 1 .2

4 1 5

1 .0 0 .8

3 8 9

0 7 4 1 75 5 3 6 5 6 4

0 .6

6 1 9

0 .4

5 3 2 5 5 8

0 .2

x 5

0 .0

Absorbance (409 nm)

3 7 5 A b s o r b a n c e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.65 0.60 0.55 0.50 0.45 0.40

3 0 0

4 0 0

5 0 0

6 0 0

7 0 0

0

1000

2000

3000

Time /second

W a v e le n g t h /n m

Figure 2. Electronic absorbance spectra of heme-titrated VcCyaY. (A) Absorption spectra of purified apo-VcCyaY (solid line), heme-VcCyaY (dotted line), and a heme-complex of VcCyaY (dashed-dashed line). (B) Absorption difference spectra of heme-binding to VcCyaY. Inset: differences at 373 nm following incremental addition of heme (1–15 µM) to VcCyaY (5

µM) in 50 mM Tris-HCl/150 mM NaCl (pH 8.0), measured against a blank containing buffer alone. (C) Optical absorption spectra of heme-VcCyaY. Ferric (solid line), ferrous (dotted line), and ferrous-CO (dashed-dashed line) complexes in 50 mM Tris-HCl/150 mM NaCl (pH 8.0). (D) Representative time course of heme transfer from heme-VcCyaY to apomyoglobin (black). The reaction was monitored after initiation of the reaction by mixing heme-VcCyaY (4 µM) with apomyoglobin (60 µM) in 50 mM Tris-HCl/150 mM NaCl (pH 8.0). The red line represents the fitted curve.

UV-visible Absorption Spectra of Heme-Reconstituted CyaY.

To obtain information on

heme coordination of heme-bound VcCyaY, we added a 2.1-fold molar excess of hemin, and then removed unbound hemin using a gel-filtration column to form the heme-VcCyaY complex. The absorption spectra of heme-VcCyaY revealed a Soret band at 375 nm and 12 ACS Paragon Plus Environment

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Biochemistry

Q-bands at 507 and 619 nm (Figure 2C). This spectrum was similar to those of heme-bound Bach1 (371 nm)29 and peroxiredoxin 1 (370 nm)30, in which cysteine serves as a heme ligand. Upon anaerobic reduction of the ferric heme-VcCyaY with sodium dithionite, the Soret band was split into two bands at 389 and 417 nm, and broad visible bands appeared at 532 and 558 nm. These peak positions were almost identical to those observed for heme-HutX, whose axial heme ligand is tyrosine.31 Addition of CO to the reduced heme shifted the Soret maximum to 415 nm, and Q-bands maxima to 536 and 564 nm; these values resemble those of CO-bound myoglobin and hemoglobin,32 indicating that histidine or a neutral ligand is coordinated to heme-VcCyaY. The heme dissociation rate constant (koff,heme) depends on the nature of the heme axial ligand: the koff,heme for myoglobin, which possesses histidine as a heme ligand, is on the order of 10–7 s–1 33; the koff,heme of heme-HutX, with tyrosine as its proximal ligand, is on the order of 10–3 s–1 31; and the koff,heme of heme-albumin, with cysteine as a proximal ligand, is on the order of 10–2 s–1.34 To gain further insight into the heme ligand of VcCyaY, we measured the heme dissociation rate constant (koff,heme), obtained by addition of >10-fold excess of apomyoglobin to a solution containing heme-VcCyaY. The time course of heme transfer from VcCyaY to apomyoglobin was monitored at 409 nm (Figure 2D). The data were well fit to a double-exponential function, yielding a koff,heme1 of (0.77 ± 0.09) × 10–3 s–1 (~70%) and a koff,heme2 of (1.2 ± 0.4) × 10–2 s–1 (~30%). The presence of two phases reflects the existence of two different heme-binding sites in VcCyaY. The values are closer to those of HutX or albumin than those of myoglobin, supporting the conclusion that the heme ligands of VcCyaY are cysteine and/or tyrosine, but not histidine. Resonance Raman Spectra of Heme-VcCyaY. Resonance Raman spectroscopy is a useful tool for elucidating the heme coordination structure.35,36 Thus, we next measured resonance Raman spectra of heme-VcCyaY. The spin-state marker band, ν3, which is observed at


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1480-1510 cm-1 for most heme proteins,36 appeared at 1490 cm-1 for ferric heme-VcCyaY (Figure 3A, spectrum d). This frequency is typical of a 5-coordinate ferric high-spin heme. Furthermore, the large intensity of the ν3 band compared with the ν4 band (1372 cm-1) indicated that an anionic ligand, such as tyrosine or cysteine, was coordinated to heme, rather than histidine.37–39 These results coincide with the conclusions drawn from absorption spectra (Figure 2C) and koff,heme values (Figure 2D). For the ferrous heme, the ν3 band was composed of two bands, one at 1470 and one at 1499 cm-1 (Figure 3A, spectrum e). The 1470 cm-1 band is characteristic of the 5-coordinate high-spin ferrous heme. The frequency of the 1499 cm-1 band was slightly higher than that of the typical 6-coordinate low-spin heme (~1490 cm–1), but generally similar to that observed for tyrosine-coordinated ferrous heme,31,38,40 indicating that ferrous heme-CyaY contains a 4-coordinate heme, not a 6-coordinate heme. The position of the frequencies of νFe-CO and νC-O on the correlation plot between νFe-CO and νC-O provides useful insight into the donor strength of the trans ligand of iron-bound CO.41 In the spectrum of the CO-bound form, two isotope-sensitive bands appeared at 495 and 1965 cm-1, which shifted to 485 and 1873 cm-1, respectively, upon

13 18

C O replacement (Supplemental Figure

S3). Therefore, the 495 and 1965 cm-1 bands are assignable to Fe-CO (νFe-CO) and C-O (νC-O) stretching modes, respectively. The plot for VcCyaY fell on the line with proteins possessing a neutral ligand (Figure 3B), which is consistent with absorption spectra data (Figure 2C).


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Biochemistry

(A)

(B

Figure 3. Resonance Raman spectra of the heme-VcCyaY complex. (A) Resonance Raman spectra of ferric (a, d), ferrous (b, e), and CO-bound (c, f) forms of the heme-VcCyaY complex. The protein concentration was 10 µM in 50 mM Tris-HCl/150 mM NaCl (pH 8.0). The excitation wavelength for ferric and CO-bound forms is 413.1 nm, and that for the ferrous form is 441.6 nm. (B) Correlation plot of frequencies of νFe-CO versus νC-O. The two solid lines correspond to correlations for proximal imidazoles (solid circles), proximal imidazolate (solid triangles), and thiolate-ligated hemoproteins (solid diamonds). Data points for the oxidase superfamilies are depicted as solid squares. The data point for heme-VcCyaY is presented as an open circle in red. The data shown in the νFe-CO versus νC-O inverse correlation plot are taken from previous reports 41–43. 15 ACS Paragon Plus Environment

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Heme-Binding Sites of VcCyaY.

The observed spectroscopic data for heme-VcCyaY

suggested that cysteine, tyrosine, or histidine are possible heme ligands (Figures 2 and 3). VcCyaY has three histidine, two cysteine, and two tyrosine residues. Among these, His7, His68, His96, Tyr67, and Cys78 are predicted to be exposed to solvent (Figure 4A), according to the crystal structure of CyaY from E. coli (hereafter termed EcCyaY).44 Therefore, to identify the heme-binding site, we replaced each histidine with leucine, Tyr67 with phenylalanine, or Cys78 with serine, and examined heme-binding ability. The difference spectra of protein solutions (after subtracting spectra of buffer titrated with heme) for the three histidine mutants (H7L, H68L, and H96L) were almost identical to those of wild-type (WT) protein (Supplemental Figure S4A–C), indicating that the heme-binding sites in these mutants remained intact. In contrast, the absorbance difference at ~375 nm in the difference spectra of the Y67F and C78S mutants was significantly reduced (Figure 5). The titration curve of the C78S mutant suggested that a binding stoichiometry (Figure 5A, inset) is different from that observed for WT VcCyaY (Figure 2B, inset). In the Y67F mutant, the absorbance difference at 373 nm increased steadily with increasing concentrations of heme and showed no clear equivalent point (Figure 5B, inset). Mutation of both Tyr67 and Cys78 led to an almost complete loss of the absorbance difference at 377 nm, indicating loss of specific heme binding (Figure 5C, inset). However, the absorbance difference at 414 nm for theY67F/C78S mutant increased monotonously with increasing amounts of heme, which is in contrast to absorbance at 377 nm (Figure 5C). For the C78S and Y67F mutants, the dominant peak in the difference spectrum was 411 and 417 nm, respectively (Figure 5A, 5B), but not ~373 nm, as observed in WT VcCyaY. The species with the absorption maximum at 411-417 nm corresponds to the water-bound 6-coordinate heme, which would correspond to non-specific binding to VcCyaY. Thus, the mutants, in which Ty67 and/or Cys78 were replaced, can still bind heme, but possibly possess an extremely weak, non-specific heme-binding site. Collectively, these titration plots revealed that both Tyr67 and Cys78 16 ACS Paragon Plus Environment

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contribute to heme binding by VcCyaY.

(A)

(C

His68

2+

Fe -binding site

His7

Tyr67

Trp76 Cys7

Trp105

Trp59

His96

Heme-binding site (B

2+

Fe -binding site

Heme-binding site

Figure 4. Crystal structure of CyaY from E. coli. (A) Putative heme-binding site. (B) Fe2+-binding sites are mapped to the crystal structure of CyaY from E. coli (PDB entry 1EW444). Residues are numbered according to the amino acid sequence of VcCyaY.


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(A) C78S

(B) Y67F

(C) Y67F/C78S

Figure 5. Heme titration of mutant VcCyaY. Absorption difference spectra of heme binding to the (A) C78S, (B) Y67S, and (C) C78S/Y67S mutant forms of VcCyaY. Inset: differences at 373 nm following incremental addition of heme (1–15 µM) to VcCyaY (5 µM) in 50 mM Tris-HCl/150 mM NaCl (pH 8.0), measured against a blank containing buffer alone.


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Effect of Heme on the Affinity of VcCyaY for Iron. We found that CyaY, which is known as an iron chaperone protein, binds heme. Next, we examined the effect of heme binding on the metal affinity of VcCyaY using tryptophan fluorescence to monitor iron binding to VcCyaY, as reported for frataxin.15 VcCyaY contains three tryptophan residues (Trp59, Trp76, Trp103) (Figure 4C). Trp59 and Trp76 are located near the putative heme-binding site, whereas Trp103 is located toward the N-terminal α-helical region, which is a proposed metal-binding site.23,45,46 Therefore, the effect of iron addition on fluorescence intensity seems to reflect binding of iron near Trp103. The observed intensity changes at 340 nm were plotted against the molar ratio of iron ion to VcCyaY (Figure 6). Because it is difficult to obtain true dissociation constant owing to the presence of multiple iron-binding sites, apparent dissociation constant Kd,Fe for Fe2+ was determined by fitting the data to equation 2. The Kd,Fe of VcCyaY was 65.2 ± 2.0 µM, which increased to 87.9 ± 5.2 µM upon heme binding, showing that heme binding to VcCyaY decreased the affinity of VcCyaY for iron.


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Biochemistry

(A)

(B

(C

1.0 Relative Intensity at 345 nm

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

0.8 0.6 0.4 heme-CyaY 0.2 CyaY 0.0 0

200

400

600

800

1000

1200

3+

[Fe ] / [CyaY]

Figure 6. Fluorescence quenching analysis of Fe2+ binding to VcCyaY. Fluorescence spectra of (A) VcCyaY and (C) heme-VcCyaY complex, with excitation at 290 nm. A series of spectra showing quenching by added Fe2+. Plots of fluorescence at 340 nm against the concentration of Fe2+ for (B) VcCyaY and (D) heme-VcCyaY complex. Solid lines represent fits of the data to equation 2.


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Effect of Heme Binding on the Structure of VcCyaY.

To understand why the affinity of

VcCyaY for iron decreased upon heme binding, we investigated whether heme binding induces a structural change. To compare the secondary structure of VcCyaY, we measured far-UV CD spectra of the protein. The CD spectra of VcCyaY exhibited characteristic features, with a minimum at 208 nm and a negative shoulder at ~222 nm (Figure 7), indicative of the expected α-helical structure.47 Addition of heme to VcCyaY brought about a slight decrease in ellipticity at 222 nm, but left [θ]208 unaffected. A previous report proposed that the ratio of [θ]208 to [θ]222 reflects rearrangement of aromatic residues, in particular tryptophan.48–50 Therefore, heme binding to VcCyaY is likely accompanied by a minor rearrangement of aromatic residue(s).

Figure 7. Effect of heme on CD spectra of VcCyaY. CD spectra in the far-UV region of VcCyaY (2 µM) containing no heme or 1, 2, 4, and 8 equivalents of heme in 100 mM sodium phosphate/50 mM NaCl (pH 8.0).

It has been reported that Fe2+ binding to EcCyaY induces oligomerization of the protein.51 Thus, we examined whether heme binding affects the oligomerization state of VcCyaY using size-exclusion chromatography. Monitoring of the effluent at 280 nm showed that VcCyaY 21 ACS Paragon Plus Environment

Biochemistry

eluted at 15.5 mL (Figure 8A), which corresponds to a species with a molecular mass of ~20 kDa (line a). Mixing 2.0 equivalents of heme with VcCyaY caused the appearance of a new band at 13.5 mL corresponding to a species with a molecular mass of ~60 kDa, which corresponds to a trimer (or tetramer) (line b). The oligomer component contained heme, as evidenced by the absorbance at 380 nm, whereas the monomer component contained no heme. These results support the conclusion that heme mediates oligomerization of VcCyaY. The addition of 2.0 equivalents of heme resulted in conversion of as much as half of VcCyaY to an oligomer (Figure 8A), but further addition of heme did not affect oligomeric content. The same experiment conducted in the presence of Fe2+ showed that, in contrast to heme, addition of Fe2+ up to 20 protein equivalents did not alter the elution profile (Figure 8B). The band at 18.0 mL did not contain proteins judging from a SDS-PAGE gel, indicating this band is derived from absorption by iron.

(A)

(B 15.5

15.5

18.0

13.5

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5

10

15

2+

heme 2 eq

Fe 2 eq

0 eq

0 eq

20

25

5

10

15

20

25

Elution Volume /mL

Elution Volume /mL

Figure 8. Effect of heme and iron on the gel filtration elution profile. Titration of VcCyaY with increasing amounts of heme (A) and Fe2+ (B). The elution of protein was monitored by measuring absorption at 280 (blue) and 380 nm (red). The total protein concentration was 8 µM in Tris-HCl/150 mM NaCl (pH 8.0).


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Biochemistry

DISCUSSION Coordination Structure of Heme in VcCyaY.

CyaY is homologous to the human

mitochondrial protein frataxin, which functions as an iron chaperone.4,52 Frataxin donates iron to the Fe-S cluster assembly machinery11 and ferrochelatase.8–10 In this study, we demonstrated for the first time that VcCyaY binds heme as well as iron (Figure 2). The large intensity ratio of the marker band ν3 to ν4 in resonance Raman spectra for ferric heme-VcCyaY implies the coordination of an anionic atom, such as oxygen or sulfur, but not nitrogen, to the heme (Figure 3).37,55,56 A spectral deconvolution analysis of the absorption spectrum of heme-VcCyaY revealed that the Soret band consists of two components with maxima at 394 and 364 nm (Supplemental Figure S5). The absorption maximum at 394 nm is close to that observed for RV0203 (392 nm),57 HutX (390 nm)31 and MmpL3 (388 nm),58 in which tyrosine serves as an axial ligand. In contrast, the Soret maximum at 364 nm is close to that observed in Bach1 (371 nm)29 and peroxiredoxin 1 (370 nm)30 in which cysteine is proposed as a heme axial ligand. The absorption spectrum of ferrous VcCyaY, with Soret maxima at 388 and 412 nm, is not typical for ferrous heme. However, it is very similar to that observed for HutX31 and the H25Y mutant of heme oxygenase38, whose proximal histidine is replaced with tyrosine. The Raman spectrum of ferrous heme-VcCyaY was composed of a mixture of 4- and 5-coordinate species (Figure 3A). The appearance of the 4-coordinate species upon reduction also supports the coordination of an anionic ligand such as tyrosine or cysteine. The kd,heme of heme-VcCyaY also suggested that tyrosine and cysteine are heme ligands of VcCyaY (Figure 2D). The proposed tyrosine and/or cysteine coordination is further supported by our mutagenesis study, which showed that only mutants of Tyr67 and Cys78 exhibited heme-binding characteristics different from that of WT VcCyaY (Figure 5, Supplemental Figure S4). In the absorbance difference spectra for both Y67F and C78S mutants, the band intensity at ~377 nm was smaller than that at ~411 nm, indicating that water-bound 23 ACS Paragon Plus Environment

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6-coordinate heme is dominant (Figure 5B, C). Tyr67 is located close to Cys78. Thus, a water molecule would not be able to bind to heme owing to steric hindrance in WT VcCyaY. However, even if one of Tyr67 or Cys78 is replaced, it would bind to heme. Accordingly, Tyr67 and Cys78 are possible heme ligands of VcCyaY, to which two molecules of heme bind (Figure 4). Both Tyr67 and Cys78 are conserved in EcCyaY (Supplemental Figure S1). Figure 4 shows the corresponding residues in the crystal structure of EcCyaY (pdb entry 1EW4), which shows that Tyr67 and Cys78 are located close to each other. Although no crystal structure of iron-bound frataxin has been reported, the metal binding site of EcCyaY has been proposed based on NMR spectroscopy.23,45,46 The NMR signals of Arg20, Leu21, Asp22, and Asp23 disappeared completely, and those of Glu19, Trp24, Asp29, Asp31, and Ile32 were significantly broadened upon metal binding. Considering that the aspartate residues listed here (Asp22, Asp29, and Asp31) are highly conserved, and the carboxylate groups of the side chain are known to serve as iron ligands,59,60 these aspartate residues are considered to be the metal-binding site of frataxin family members. The corresponding residues in VcCyaY are indicated in red in Figure 4, which clearly shows that the heme-binding site is far from the metal-binding site. Accordingly, heme does not occupy the same sites that bind metal in VcCyaY. Role of Heme Binding to VcCyaY.

The apparent Kd,heme value of VcCyaY for heme is

estimated to be 21 nM (Figure 2A), which is in the same range as that for other heme-binding proteins, such as human serum albumin (Kd,heme = 34 nM),61 the ligand-binding domain of Rev-erbβ (Kd,heme = 25 nM),62 peroxiredoxin 1 (Kd,heme = 0.17 µM),30 and HutX (Kd,heme = 7.4 nM).31 This moderate affinity of VcCyaY for heme raises the possibility that heme acts as a regulatory molecule. According to fluorescence titration data, heme binding increased the Kd,Fe of VcCyaY


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Biochemistry

(Figure 6), indicating that heme binding facilitates the release of iron from VcCyaY. To determine how heme binding affects Kd,Fe, we examined secondary structural changes in VcCyaY upon heme binding using CD spectroscopy (Figure 7). The addition of heme slightly decreased the [θ]222 value, indicating that heme induces a conformational change in VcCyaY that affects the structure around aromatic residues, probably tryptophan.48–50 Heme binding also changed the oligomerization status of VcCyaY from monomer to trimer (or tetramer) (Figure 8). In yeast, frataxin is converted to a trimer under conditions in which the iron-to-protein ratio is 2, and assembles into much higher oligomers (24, 48 mer) at higher iron-to-protein ratios.63,64 This highly oligomerized form acts as an iron-storage protein, because it stores iron inside the oligomers as does ferritin, rendering iron unavailable for delivery to ferrochelatase or the Fe-S cluster machinery.65 In contrast, both the monomeric and trimeric forms of frataxin are active as iron-chaperone proteins. In the crystal structure of the trimeric form of the frataxin Y73A mutant, in which the mutation stabilizes the trimeric state, the acidic ridge, where putative iron-binding residues are clustered, is exposed to solvent.64,66 If the configuration of the heme-induced trimer of VcCyaY is the same as that of the iron-induced trimer, iron would be exposed to solvent, facilitating its transfer from VcCyaY to Fe-S cluster machinery or ferrochelatase. Accordingly, heme binding to VcCyaY induces a structural change, which leads to oligomerization of the protein. As a result, iron binding is destabilized (Figure 6), and iron transfer from VcCyaY to ferrochelatase or the Fe-S biosynthesis machinery might be facilitated. However, clarifying the precise mechanism of the heme-induced enhancement of heme transfer to ferrochelatase or the Fe-S biosynthesis machinery will require a crystal structural analysis of heme-bound VcCyaY. Biological Implications of Heme Binding to VcCyaY.

We found that heme binding to

VcCyaY, for which the Kd,heme is 21 nM (Figure 2), decreases the iron affinity of VcCyaY by


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approximately 1.5-fold (Figure 6). This finding suggests that synthesis of heme is modulated when the concentration of free heme is more than 20 nM. Biosynthesis of heme is composed of multiple steps.67,68 The first step is the formation of 5-aminolevulinic acid (ALA). It is reported that heme itself function as a regulator of ALA synthase (ALAS). Binding of heme to ALAS leads to decrease in the catalytic activity of ALAS69 because expression of excess of heme must be avoided owing to its cytotoxicity. CyaY is not an enzyme that is involved in heme synthesis, but it can modulate heme biosynthesis through ferrochelatase. Because both heme-binding residues (Tyr67 and Cys78) are conserved in CyaY from E. coli (Supplemental Figure S1), the property of heme binding would not be specific to VcCyaY.

In conclusion, although CyaY has been reported to bind a variety of divalent metal ions, this is the first report that VcCyaY is capable of binding heme. Heme binding to VcCyaY induces conversion of the monomeric form to the trimeric (or tetrameric) form, and modulates the strength of iron binding to VcCyaY. These changes would lead to acceleration of iron transfer from VcCyaY to ferrochelatase or IscS, thereby enhancing the formation of heme or Fe-S clusters.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications web site at http://pubs.acs.org. Sequence alignment of CyaY and human frataxin. (Figure S1). MALDI-MS and Resonance Raman spectra of spectrum of VcCyaY (Figures S2 and S3). Heme titration of mutant VcCyaY (Figure S4). Deconvolution of the Soret band of ferric heme-VcCyaY (Figure S5). Primers used for construction of expression vectors for mutants (Table S1).


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ACKNOWLEDGMENTS We thank Mr. Takumi Funamizu for assistance with MALDI-MS and CD measurements.

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Biochemistry

For Table of Contents Use Only

The Iron Chaperone Protein CyaY from Vibrio cholerae is a Heme-Binding Protein Takeshi Uchida, Noriyuki Kobayashi, Souichiro Muneta, and Koichiro Ishimori

COOH N N

COOH Fe

Tyr67

Vc CyaY

N N

heme Cys78 Fe

Ferrochelatase COOH

N

N H

COOH H N

N


heme

The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme

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