Structural characterization of a carbon monoxide adduct of a heme–DNA complex

Abstract

The structure of a carbon monoxide (CO) adduct of a complex between heme and a parallel G-quadruplex DNA formed from a single repeat sequence of the human telomere, d(TTAGGG), has been characterized using ¹H and ¹³C NMR spectroscopy and density function theory calculations. The study revealed that the heme binds to the 3'-terminal G-quartet of the DNA though a π-π stacking interaction between the porphyrin moiety of the heme and the G-quartet. The π-π stacking interaction between the pseudo-C₂-symmetric heme and the C₄-symmetric G-quartet in the complex resulted in the formation of two isomers possessing heme orientations differing by 180° rotation about the pseudo-C₂ axis with respect to the DNA. These two slowly interconverting heme orientational isomers were formed in a ratio of approximately 1:1, reflecting that their thermodynamic stabilities are identical. Exogenous CO is coordinated to heme Fe on the side of the heme opposite the G-quartet in the complex, and the nature of the Fe-CO bond in the complex is similar to that of the Fe-CO bonds in hemoproteins. These findings provide novel insights for the design of novel DNA enzymes possessing metalloporphyrins as prosthetic groups.

Characterization of the Interaction between Heme and a Parallel G-Quadruplex DNA Formed from d(TTAGGGT)

“…2.6 ppm of the G4, G5, and G6 NH signals, respectively ( Table 1 (Table 1). 11 In the case of the diamagnetic CO adduct of the heme(Fe 2+ ) DNA complex, the ¦¤ H(II) values observed for the DNA proton signals were attributed primarily to the ring current effect of the porphyrin moiety of heme(Fe 2+ ), and calculation using the reported equation for the ring current effect of a porphyrin, 35 together with a simple model for the DNA structure constructed on the basis of an X-ray crystallographic study of stacked Gquartets, 36 indicated that the heme(Fe 2+ ) located at 0.34 nm from the G6 G-quartet satisfies the ¦¤ H(II) values for the G4, G5, and G6 NH signals (see Figure SI13 in the Supporting Information). Consequently, similarities in the ¦¤ H(II) values for the G4, G5, and G6 NH signals between the heme(Fe 14 a) Shifts of signals, labeled G4(free), G5(free), G6(free) in Figure 7, spectrum B¤, due to the DNA in 50 mM KCl and 50 mM potassium phosphate buffer, pH 7.00, at 25°C.…”

“…Upon the stacking of the pseudo-C 2 symmetric heme onto the C 4 symmetric G6 G-quartet, the orientation of the porphyrin moiety of the heme relative to the G6 G-quartet depends upon which side of the heme plane, i.e., either the obverse or reverse heme orientation, interacts with the G6 G-quartet, because the steric contacts between heme vinyl side chains and DNA G6 sugars could be slightly altered on the 180°rotation of the pseudo-C 2 symmetric heme around the 5-H15-H axis. 11 The observation of single G6 imino NH proton signals for each of the obverse and reverse heme orientations ( Figure 7B¤) indicated that the time scale of the interconversion among the four optimal orientations of the heme is fast enough to average out the possible shift differences among the four G6 imino NH proton signals of the complex. On the other hand, the small splitting of the G6 NH signal ( Figure 7B¤) indicated that the time scale of the heme flipping on the G6 G-quartet of the complex, i.e., the interconversion between the obverse and reverse heme orientations, is rather slow.…”

“…As has been reported for the heme(Fe 3+ )(d(TTAGGG)) 4 complex, most heme(Fe 2+ ) proton signals, together with some DNA ones, split into two peaks with an intensity ratio of ca. 1:1 (Table 2 and see Figures SI6SI10 in the Supporting Information), due to the two possible heme orientations differing by 180°rotation about the 5-H15-H axis, with respect to the DNA, in the complex, 11 i.e., the obverse and reverse heme forms of the hemeDNA complex (see Figure SI12 in the Supporting Information). Upon the stacking of the pseudo-C 2 symmetric heme onto the C 4 symmetric G6 G-quartet, the orientation of the porphyrin moiety of the heme relative to the G6 G-quartet depends upon which side of the heme plane, i.e., either the obverse or reverse heme orientation, interacts with the G6 G-quartet, because the steric contacts between heme vinyl side chains and DNA G6 sugars could be slightly altered on the 180°rotation of the pseudo-C 2 symmetric heme around the 5-H15-H axis.…”

“…Heme was purchased from Sigma-Aldrich Co. For NMR sample preparation of the heme(Fe 3+ )(d(TTAGGGT)) 4 complex, 360¯L of 2.8 mM DNA, as a quadruplex form, in 420 mM KCl and 70 mM potassium phosphate buffer, pH 7.0, was mixed with 50¯L of 10 mM heme dissolved in 50 mM KOH, and then the pH of the resulting solution was adjusted to 7.0 using 0.2 M HCl or 0.2 M KOH, if necessary. 11,12 The CO adduct of the heme(Fe 2+ )DNA complex was prepared by reduction of heme Fe 3+ of the heme(Fe 3+ )DNA complex with 20¯L of 500 mM Na 2 S 2 O 4 (Wako Pure Chemical Industries, Ltd.) in the presence of CO gas (Japan Air Gases), 12 and then 20¯L of 500 mM («)-dithiothreitol (Wako Pure Chemical Industries, Ltd.) was added to the sample to keep the heme of the complex in the reduced state. Thus, the final concentrations of both DNA and heme in the solution mixture were 2 mM.…”

“…In this study, a complex (heme(d(TTAGGGT)) 4 complex) between heme and G-quadruplex DNA formed from d(TTAGGGT), i.e., (d(TTAGGGT)) 4 , has been characterized, and the obtained results were compared with those for the heme(d(TTAGGG)) 4 complex 11,12 in order to elucidate the effects of the extra T at the 3¤-terminal of the DNA on the binding stoichiometry, the structure of the complex, and the heme electronic structure in the complex. The study demonstrated that heme, irrespective of the heme Fe oxidation state, and (d(TTAGGGT)) 4 form only a 1:1 complex, through stacking of the heme between G6 and T7 of the DNA.…”