Structures and Catalytic Activities of Complexes between Heme and

Sep 20, 2018 - Heme in its ferrous and ferric states [heme(Fe2+) and heme(Fe3+), respectively] binds selectively to the 3′-terminal G-quartet of all...
0 downloads 0 Views 1MB Size
Download PDF
Subscriber access provided by Washington University | Libraries

Article

Structures and Catalytic Activities of Complexes between Heme and All Parallel-stranded Monomeric G-quadruplex DNAs Yasuhiko Yamamoto, Haruka Araki, Ryosuke Shinomiya, Kosuke Hayasaka, Yusaku Nakayama, Kentaro Ochi, Tomokazu Shibata, Atsuya Momotake, Takako Ohyama, Masaki Hagihara, and Hikaru Hemmi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00792 • Publication Date (Web): 20 Sep 2018 Downloaded from http://pubs.acs.org on September 22, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 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

Biochemistry

Structures and Catalytic Activities of Complexes between Heme and All Parallel-stranded Monomeric G-quadruplex DNAs

Yasuhiko Yamamoto,1-3* Haruka Araki,1 Ryosuke Shinomiya,1 Kosuke Hayasaka,1 Yusaku Nakayama,1 Kentaro Ochi,1 Tomokazu Shibata,1 Atsuya Momotake,1 Takako Ohyama,4 Masaki Hagihara,5 and Hikaru Hemmi6

1

Department of Chemistry, University of Tsukuba, Tsukuba 305-8571, Japan, 2Tsukuba Research Center

for Energy Materials Science (TREMS), University of Tsukuba, Tsukuba 305-8571, Japan, 3Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba 305-8577, Japan, 4NMR Division, RIKEN SPring-8 Center, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, 5Graduate School of Science and Technology, Hirosaki University, Hirosaki 036-8560, Japan, and 6Food Research Institute, NARO, Tsukuba 305-8642, Japan

* Corresponding author. Phone/Fax: +81-29-853-6521, E-mail: [email protected]

†

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education,

Culture, Sports, Science and Technology, Japan.

1 ACS Paragon Plus Environment

Biochemistry 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

Page 2 of 26

Abstract: Heme in both its ferrous and ferric states (heme(Fe2+) and heme(Fe3+), respectively) binds selectively to the 3’-terminal G-quartet of all parallel-stranded monomeric G-quadruplex DNAs formed from

inosine(I)-containing

sequences,

i.e.,

d(TAGGGTGGGTTGGGTGIG)

DNA(18mer)

and

d(TAGGGTGGGTTGGGTGIGA) DNA(18mer/A), through a - stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form 1:1 complexes (heme-DNA(18mer) and heme-DNA(18mer/A) complexes, respectively).

These complexes exhibited enhanced peroxidase

activities, compared with heme(Fe3+) alone, and the activity of the heme(Fe3+)-DNA(18mer/A) complex was greater than that of the heme(Fe3+)-DNA(18mer) one, indicating that the 3’-terminal A of the DNA sequence acts as an acid-base catalyst that promotes the catalytic reaction. In the complexes, a water molecule (H2O) at the interface between the heme and G-quartet is coordinated to the heme Fe atom as an axial ligand, and possibly acts as an electron-donating ligand that promotes heterolytic peroxide bond cleavage of hydrogen peroxide bound to the heme Fe atom, trans to the H2O, for the generation of an active species. The intermolecular nuclear Overhauser effects observed among heme, DNA, and Fe-bound H2O indicated that the H2O rotates about the H2O-Fe coordination bond with respect to both the heme and DNA in the complex. Thus, the H2O in the complex is unique in terms of not only its electronic properties, but also dynamic ones.

These findings provide novel insights as to the design of heme-deoxyribozymes and

-ribozymes.

2 ACS Paragon Plus Environment

Page 3 of 26 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

Biochemistry

INTRODUCTION

Conjugated proteins are thought to have evolved from simple proteins by accommodating non-protein materials known as cofactors to enlarge their repertoire of chemical functionalities.

Iron-protoporphyrin

IX complex (heme) is perhaps the most ubiquitous and abundant cofactor found in nature and the most functionally diverse.1

Heme (Figure 1A) is incorporated, as a prosthetic group, into various apoproteins to

form a series of hemoproteins that play vital roles in biological systems.

Despite such ubiquitous

occurrence of heme in nature, no functional role in vivo has yet been attributed to heme incorporated into nucleic acids. The RNA world hypothesis2 has provided a strong stimulus for exploring new catalytic properties of nucleic acids. Incorporation of cofactors into nucleic acids is expected to contribute to such a study, and heme is most likely an ancient compound and a likely player in the postulated RNA world.3 Travascio et al.4-7 found that DNA aptamers bound to heme exhibit peroxidase activities, and Poon et al.8 further demonstrated that heme-bound RNA aptamers as well as heme-bound DNA ones catalyze two-electron oxidation.

Thus, the heme-bound nucleic acids recapitulate at least two of the known

catalytic functions of contemporary hemoproteins, lending credence to heme’s link to a primordial RNA world.8 Hence, these heme-bound nucleic acids could be regarded as prototypes for redox-catalyzing ribozymes in the primordial RNA world.

Figure 1. Molecular structures of heme (A) and the G-quartet (B).

Although heme-bound nucleic acids and peroxidases share common catalytic activities, the functional groups involved in the catalytic reactions of these two heme systems should be completely different from each other.

Therefore, apart from their relevance to the primordial RNA world, elucidation of the 3 ACS Paragon Plus Environment

Biochemistry 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

Page 4 of 26

structure-function relationships of the catalytic nucleic acids is expected to lead to the discovery of a novel reaction mechanism that will contribute not only to exploration of the biological versatility of heme as a prosthetic group of nucleic acids, but also to tuning of the heme reactivity in the scaffold of the nucleic acid structure.

The regulation of heme Fe reactivity has been shown to be achieved through the heme

environment furnished by nearby functional groups,9-11 in addition to electronic tuning of the intrinsic heme Fe reactivity12-18.

Hence characterization of the heme structural environment in the heme-bound nucleic

acids is indispensable for elucidation of their catalytic activities. Heme in both its ferrous and ferric states (heme(Fe2+) and heme(Fe3+), respectively) binds to guanine-rich DNAs with relatively high affinities.19-27

Travascio et al.7 suggested that, in a

heme(Fe3+)-DNA aptamer complex, heme(Fe3+) binds onto a G-quartet formed from four guanine bases cyclically associated through Hoogsteen hydrogen-bonding28 (Figure 1B), with a water molecule coordinated to heme(Fe3+)27.

We have been focusing on characterization of the interaction between heme

and an all parallel-stranded tetrameric G-quadruplex DNA formed from a single repeat sequence of the human telomere, i.e., (d(TTAGGG))4.19-25,

The 4-fold rotationally symmetric structure of

27

(d(TTAGGG))4 is stabilized through the stacking of three consecutive G-quartets, i.e., the G4, G5, and G6 G-quartets, and its structural stability and simplicity allowed us to reveal to some extent the interaction between heme and G-quadruplex DNA.19-25, 27 We found that heme binds to the 3’-terminal G-quartet of (d(TTAGGG))4, through a - stacking interaction between the porphyrin moiety of the heme and the G-quartet, to form a stable 1:1 heme-(d(TTAGGG))4 complex that exhibits various spectroscopic properties remarkably similar to those of myoglobin (Mb).19-22,

24

Furthermore, we found that the

heme-(d(TTAGGG))4 complex exhibits functional properties similar to those of Mb such as binding of imidazole25 and carbon monoxide (CO)22,23,27, as exogenous ligands, to the heme(Fe3+)-(d(TTAGGG))4 and heme(Fe2+)-(d(TTAGGG))4 complexes, respectively.

Heme(Fe2+) coordinated to CO adopts a low spin

configuration with S = 0, i.e., a diamagnetic form, and hence the heme environment in the complex, which provides the basis for elucidating their functional properties, could be characterized in great detail through NMR structural characterization of a CO adduct of the heme(Fe2+)-(d(TTAGGG))4 complex.23, 27 In this study, we have extended our efforts to elucidate the structure-function relationships of heme-bound nucleic acids. We characterized the structures of unimolecular G-quadruplex DNAs formed from

inosine(I)-containing

sequences,

i.e.,

d(TAGGGTGGGTTGGGTGIG) 4

ACS Paragon Plus Environment

(DNA(18mer))

and

Page 5 of 26 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

Biochemistry

d(TAGGGTGGGTTGGGTGIGA) (DNA(18mer/A)), and their interaction with heme. The guanine to inosine substitution has been used in G-quadruplex DNA structure studies,29 and the introduction of inosine in the sequences considered in the study was used to improve the separation of imino 1H NMR signals useful for structural characterization. The formation of all parallel-stranded monomeric G-quadruplex DNAs from DNA(18mer) and DNA(18mer/A) was confirmed by circular dichroism (CD) and NMR analyses.

In addition, as in the case

of a CO adduct of the heme(Fe2+)-(d(TTAGGG))4 complex,19-25, 27 heme(Fe2+) binds selectively to the 3’-terminal G-quartet of G-quadruplex DNAs of DNA(18mer) and DNA(18mer/A) to form 1:1 complexes (heme(Fe2+)-DNA(18mer) and heme(Fe2+)-DNA(18mer/A) complexes, respectively), with coordination of CO to the heme Fe atom on the side of the heme opposite the G-quartet.

We also investigated the

peroxidase activities of the two complexes to examine the effect of the addition of the extra A at the 3’-terminal of the sequence on the activity. We found that the activity of the heme(Fe3+)-DNA(18mer/A) complex was greater than that of the heme(Fe3+)-DNA(18mer) one, indicating that the 3’-terminal A acts as an acid-base catalyst that promotes the catalytic reaction.

These findings provide novel insights as to the

design of heme-deoxyribozymes and -ribozymes.

MATERIALS AND METHODS Sample Preparation.

d(TAGGGTGGGTTGGGTGIG) (DNA(18mer)) and d(TAGGGTGGGTTGGGTG IGA)

(DNA(18mer/A)) purified with a C-18 Sep-Pak cartridge were purchased from Tsukuba Oligo Service Co. DNA(18mer) and DNA(18mer/A) were purified by ethanol precipitation and then desalted with Microcon YM-3 (Millipore, Bedford, MA).

The concentrations of DNA(18mer) and DNA(18mer/A) were determined

spectrophotometrically using the absorbance at 260 nm (molar extinction coefficients 260 = 2.01 × 105 and 2.16 × 105 cm-1M-1 for DNA(18mer) and DNA(18mer/A), respectively)).

Heme(Fe3+) was purchased from Sigma-Aldrich Co.

Preparation of the heme(Fe3+)-DNA(18mer) and heme(Fe3+)-DNA(18mer/A) complexes was carried out by mixing 1.0 mM heme(Fe3+), and 5.0 mM DNA(18mer) and DNA(18mer/A), respectively, as described previously.23

Carbon

monoxide (CO) adducts of the heme(Fe3+)-DNA(18mer) and heme(Fe3+)-DNA(18mer/A) complexes were prepared by reduction of heme(Fe3+) of the complexes with Na2S2O4 (Wako Pure Chemical Industries, Ltd.) and (±)-dithiothreitol (Wako Pure Chemical Industries, Ltd.) in the presence of CO gas.

50 mM potassium phosphate buffer, pH 6.80,

containing 300 mM KCl was used as the solvent throughout the optical measurements.

5 ACS Paragon Plus Environment

Biochemistry 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

Page 6 of 26

UV-Vis Absorption, Circular Dichroism and NMR Measurements.

Absorption spectra were recorded on a

Beckman DU640 spectrometer over the spectral range of 300-700 nm and with a sample concentration of 10.0 M. In order to characterize the complexation between heme(Fe3+) and the DNA, 2.0 µM heme(Fe3+) in 100 mM KCl and 5 mM potassium phosphate buffer, pH 6.80, together with 0.08 w/v% Triton X-100 and 0.5 v/v% dimethyl sulfoxide to prevent heme aggregation, was titrated against the DNA at 25 ºC.

A circular dichroism (CD) spectrum was recorded

with a JASCO J-820 spectrodichrometer, and the CD magnitude was expressed in terms of molar ellipticity ([] in deg M-1cm-1).

1

H NMR spectra were recorded on Bruker AVANCE 600 and 800 spectrometers (Bruker Instruments, Inc.,

Bellerica, MA, USA) operating at 1H frequencies of 600 and 800 MHz, respectively.

One-dimensional 1H NMR

spectra were obtained with a 20 ppm spectral width, 32 k data points, a 2 s relaxation delay, and 64 transients at 25 ºC. Water suppression was achieved by the watergate method.30, 31

The signal-to-noise ratio of the spectra was improved

by apodization, which introduced 0.3 Hz line-broadening.

Two-dimensional 1H-1H nuclear Overhauser effect

(NOESY) spectra of the CO adducts of the heme(Fe2+)-DNA(18mer) and heme(Fe2+)-DNA(18mer/A) complexes were acquired by quadrature detection in the phase-sensitive mode with a States-TPPI32, with a 15k Hz spectral width, 8k × 512 data points, a 2 s relaxation delay, and a mixing time of 300 ms at 25 ºC. Amplex Red Oxidation Kinetics.

The chromogenic substrate, Amplex Red (10-acethyl-3,7- dihydroxyphenoxazine),

was purchased from Sigma Aldrich, and dissolved in dimethylformamide to create a 10 mM stock solution. studies involving Amplex Red as a substrate were performed on a Beckman DU640 spectrometer.

Kinetic

DNA(18mer) and

DNA(18mer/A) in 50 mM K+ phosphate buffer, pH 6.80, were heat-denatured at 90 °C for 5 min., followed by cooling to 25 °C.

Heme(Fe3+) and then Amplex Red were added to 0.5 μM and 50 μM, respectively, to 20 μM DNA(18mer)

or DNA(18mer/A) in 50 mM K+ phosphate buffer, pH 6.80, at 25 °C. peroxide (H2O2) was added to 200 μM to the solution mixture.

To initiate the oxidation reaction, hydrogen

The reaction was monitored by following the

appearance of the oxidized product, 7-hydroxyphenoxazin-3-one (Resorufin), which absorbs light at ~570 nm.

The

initial slope (R0) of the time evolution of 570-nm absorbance was used as an index for the peroxidase activities of the complexes.

RESULTS Characterization of G-quadruplex DNAs Formed from DNA(18mer) and DNA(18mer/A).

Kuryavyi

et al.32 demonstrated that d(TAGGGTGGGTTGGGTGGGG) forms all parallel-stranded monomeric G-quadruplex DNA. DNA(18mer) and DNA(18mer/A) can be considered as counterparts of the sequence investigated by Kuryavyi et al.33

The CD spectrum of DNA(18mer) at pH 6.80 and 25 ºC exhibited

negative and positive Cotton effects at ~240 and ~260 nm, respectively, which are characteristic of all 6 ACS Paragon Plus Environment

Page 7 of 26 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

Biochemistry

parallel G-quadruplex DNA34 (Figure 2).

The formation of all parallel G-quadruplex DNA from

DNA(18mer) has confirmed through characterization of its conformation by

1

H NMR.

In the

downfield-shifted portion of the NMR spectrum of DNA(18mer) (traces A and A’ in Figure 3), ten imino proton signals, with doubling of intensity for the two signals at 11.11 and 11.21 ppm, were observed below > ~10 ppm, a chemical shift range characteristic of guanine and inosine imino protons involved in hydrogen bond formation with their carbonyl oxygen atoms, i.e., NH-OC hydrogen bonds,32 and hence the

Figure 2. CD spectra of d(TAGGGTGGGTTGGGTGIG) (DNA(18mer)) (solid line curve) and d(TAGGGTGGGTTGGGT GIGA) (DNA(18mer/A)) (broken line curve) in 100 mM KCl, 5 mM potassium phosphate buffer at pH 6.80 and 25 ℃.

observation of the twelve imino proton signals is consistent with G-quadruplex formation stabilized by three stacked G-quartets.

In the absence of an electron-donating NH2 side chain in inosine, the I17 imino

proton signal was downfield-shifted by more than ~2 ppm relative to those of guanines, i.e., G3-G5, G7-G9, G12-G14, G16, and G18, and thus the I17 imino proton signal served as a starting point for NMR signal assignments by means of nuclear Overhauser effects (NOEs) (Figure 4).29,

35

The observed NOEs

between imino NH protons, together with other NOE connectivities (see Figure S1 in the Supporting Information), identified formation of G-quartets composed of G3,G7,G12,G16, G4,G8,G13,I17, and G5,G9,G14,G18 (Figure 4). Supporting Information).

Other 1H signals of DNA(18mer) were also assigned (Table S1 in the

DNA(18mer/A) has been similarly characterized using CD and 1H NMR

spectroscopies (Figure 2 and trace C in Figure 3, and also see Figures S2-S6 in the Supporting Information), and it was found that the structure of the all parallel G-quadruplex DNA from DNA(18mer) is not greatly affected by the addition of the extra A at the 3’-terminal of the sequence. Shifts of the assigned 1H signals of DNA(18mer/A) are compared with those of DNA(18mer) in Table 1 (also see Tables S1 and S2 in the 7 ACS Paragon Plus Environment

Biochemistry 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

Figure 3. 600 MHz 1H NMR spectra of d(TAGGGTGGGTTGGGTGIG) (DNA(18mer)) (A) and a CO adduct of the heme(Fe2+)-DNA(18mer) complex (B) in 90% 1H2O/10% 2H2O, 100 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 25 ºC. The chemical shift ranges, 8.5 - 15 and –4.5 - –2.5 ppm, of traces A and B are expanded in traces A’ and B’, respectively. The corresponding shift ranges of spectra of d(TAGGGTGGGTTGGGTGIGA) (DNA(18mer/A)) and a CO adduct of the heme(Fe2+)-DNA(18mer/A) complex in 90% 1H2O/10% 2H2O, 300 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 25 ºC are shown in traces C and D, respectively. Assignments of guanine and inosine imino, heme meso, and axial H2O (axH2O) proton signals are indicated with the spectra. Nn, where N is guanine (G) or inosine (I), and n is the residue number, represents imino proton signals, and the signal due to the heme-DNA complex is indicated by subscript C.

Figure 4. Portions of the NOESY spectrum of d(TAGGGTGGGTTGGGTGIG) (DNA(18mer)) in 90% 1H2O/10% 2H2O, 100 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 15 ºC. A mixing time of 300 ms was used to record the spectrum. The cross-peaks used for assignments of guanine and inosine imino proton signals are indicated. The imino proton signal assignments are shown in the 1D spectrum illustrated at the top. The structure of an all parallel-stranded G-quadruplex DNA formed from DNA(18mer) is illustrated in the inset. 8 ACS Paragon Plus Environment

Page 8 of 26

Page 9 of 26 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

Biochemistry

Supporting Information). The I17 and G18 imino proton signals of DNA(18mer/A) were upfield-shifted by 0.19 and 0.37 ppm relative to the corresponding ones of DNA(18mer), whereas the differences in shift of the other imino proton signals between DNA(18mer) and DNA(18mer/A) were relatively small, i.e., 0.01 – 0.09 ppm (Table 1). Finally, the thermal stabilities of the G-quadruplex DNAs of DNA(18mer) and DNA(18mer/A) were similar to each other (see Figure S7 in the Supporting Information),

Determination of the Stoichiometries and Association Constants of the Heme(Fe3+)-DNA(18mer) and Heme(Fe3+)-DNA(18mer/A) Complexes. Heme(Fe3+) at pH 6.80 was spectrophotometrically titrated with DNA(18mer) and DNA(18mer/A) in order to characterize the complexation reactions (see Figure S8 in the Supporting Information). Upon addition of the DNAs, the Soret band of heme(Fe3+) exhibited ~230% hyperchromism, associated with a red-shift of 6 nm, and isosbestic points at ~375 and ~420 nm. The observation of isosbestic points indicated the formation of 1:1 heme-DNA complexes.

The

absorption changes were analyzed as described by Wang et al.36 in order to obtain association constants (Ka). 1.6 ± 0.1 and 2.7 ± 0.6 M-1 were obtained for the Ka values of the heme(Fe3+)-DNA(18mer) and 9 ACS Paragon Plus Environment

Biochemistry 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

Page 10 of 26

heme(Fe3+)-DNA(18mer/A) complexes, respectively (see Figure S8 in the Supporting Information). The Soret bands of the heme(Fe3+)-DNA(18mer) and heme(Fe3+)-DNA(18mer/A) complexes exhibited maxima at 404 nm (see Figure S8 in the Supporting Information), which was similar to that of the heme(Fe3+)-(d(TTAGGG))4 complex20. 1

H NMR Spectra of CO Adducts of the Heme(Fe2+)-DNA(18mer) and Heme(Fe2+)-DNA(18mer/A)

Complexes. The downfield- and upfield-shifted portions of the 600 MHz 1H NMR spectrum of a CO adduct of the heme(Fe2+)-DNA(18mer) complex are shown in traces B and B’ in Figure 3.

Similarly to

the case of DNA(18mer), the imino proton signals of a CO adduct of the heme(Fe2+)-DNA(18mer) complex were assigned by monitoring NOEs (Figures 5-7), and the shifts of the signals are compared with those of the corresponding ones of DNA(18mer) in Table 1. All the imino proton signals of DNA(18mer) were upfield-shifted by the heme binding.

Through the interaction with a CO adduct of heme(Fe2+), the

imino proton signals of the guanosines of the 5’-terminal G-quartet (G-quartet(5’)), i.e., G3, G7, G12, and G16, were upfield-shifted by ~0.5 ppm, those of the guanines and inosine sandwiched between the terminal

Figure 5. Portions of the NOESY spectrum of a CO adduct of the heme(Fe2+)-DNA(18mer) complex in 90% 1H2O/10% 2H2O, 100 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 15 ºC. A mixing time of 300 ms was used to record the spectrum. The signals denoted by an asterisk in the top 1D spectrum are due to free DNA(18mer). The imino proton signal assignments are shown in the 1D spectrum illustrated at the top. The cross-peaks used for assignments of imino proton signals are indicated, and those denoted by a blue dotted-line ellipse are due to magnetization transfer through interconversion between the free and heme(Fe2+)-bound states of the DNA. 10 ACS Paragon Plus Environment

Page 11 of 26 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

Biochemistry

Figure 6. Portions of the NOESY spectrum of a CO adduct of the heme(Fe2+)-DNA(18mer) complex in 90% 1H2O/10% 2H2O, 100 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 15 ºC. A mixing time of 300 ms was used to record the spectrum. The cross-peaks used for assignments of heme proton signals are indicated. The heme meso proton signal assignments are shown in the 1D spectrum illustrated at the top, and subscripts a and b represent the two different orientations of heme with respect to the DNA in the complex (see Figure S9C in the supporting information).

Figure 7. NOE connectivities associated with axial H2O (axH2O) proton signals, i.e., axH2Oa and b 2+ 1 2 axH2O , of a CO adduct of the heme(Fe )-DNA(18mer) complex in 90% H2O/10% H2O, 100 mM KCl, 50 mM potassium phosphate buffer at pH 6.80 and 15 ºC (A). The axH2O signals exhibited connectivities with guanine imino proton ones of the 3’-terminal G-quartet of the G-quadruplex formed from DNA(18mer), i.e., G5, G9, G14, and G18, in the shift range of ~8.3 - ~8.7 ppm. Weak connectivities between axH2O signals and heme methyl and meso proton ones in shift ranges of ~2.7 ~3.3 and ~9.2 - ~9.5 ppm, respectively, were also observed. Schematic representation of the complex (B), and the interface between the heme and the DNA of the complex (C). In the complex, axH2O is sandwiched between the heme and the 3’-terminal G-quartet planes, and the G-quartet composed of only guanine bases and the porphyrin moiety is illustrated for clarity. The orientation of axH2O with respect to the 3’-terminal G-quartet (D) and the heme (E) in the complex. The theoretical calculation suggested that axH2O is coordinated to the heme Fe atom through donation of one lone pair of electrons of the oxygen atom to the empty dz2 orbital of low-spin Fe2+, and then two hydrogen bonds are formed between axH2O and carbonyl oxygen atoms of the guanine bases.27 Some of the NOE connectivities observed in the spectrum are indicated by curved double arrows. The apparent non-selective NOE connectivities indicated that axH2O at the interface rotates about the coordination bond with respect to both the G-quartet and heme. In E, one of the two heme orientations is shown (see Figure S9C in the supporting information). 11 ACS Paragon Plus Environment

Biochemistry 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

G-quartets (G-quartet(mid)), i.e., G4, G8, G13, and I17, by ~1.0 ppm, and those of the guanosines of the 3’-terminal G-quartet (G-quartet(3’)), i.e., G5, G9, G14, and G18, by ~2.5 ppm (Table 1 and see also Figure S9 in the Supporting Information).

In the absence of an unpaired electron in a CO adduct of heme(Fe2+),

the upfield shift changes observed for the imino proton signals are attributed primarily to the ring current effect of the heme porphyrin moiety37.

The observed heme ring current-induced shift changes (rc)

clearly indicated that a CO adduct of heme(Fe2+) binds selectively to the G-quartet(3’) of the G-quadruplex of DNA(18mer). The observation of two sets of imino proton signals, one from the DNA and the other from the complex, in the spectra of DNA(18mer) in the presence of various stoichiometric amounts of a CO adduct of heme(Fe2+) (see Figure S10 in the Supporting Information) indicated that the heme(Fe2+) binding to DNA(18mer) is slow on the NMR time scale, and the relative fraction of DNA(18mer) bound to heme(Fe2+) was consistent with the binding affinity of the heme(Fe2+) to DNA(18mer) estimated optically. Moreover, as in the case of a CO adduct of the heme(Fe2+)-(d(TTAGGG))4 complex,23 the guanine imino proton signals of the G-quartet(3’) of the complex, i.e., G5, G9, G14, and G18 ones, were observed as ~1:1 doublet peaks due to the so-called heme orientational disorder38 (Table 1 and also see trace C in Figure S9

12 ACS Paragon Plus Environment

Page 12 of 26

Page 13 of 26 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

Biochemistry

in the Supporting Information). Similarly, a signal due to a water molecule coordinated to heme Fe2+ (axH2O), trans to the Fe2+-bound CO, observed at ~-3.5 ppm also exhibited splitting (trace B’ in Figure 3 and Figure 7). The splitting of these signals ranged from 0.03 ppm to 0.07 ppm, indicating that the time scale of the interconversion between the two heme orientations is