Vibrational relaxation of NO stretching modes in ferrous NO and ferric NO in model heme

Chemical Physics 422 (2013) 107–114

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Vibrational relaxation of NO stretching modes in ferrous NO and ferric NO in model heme Jaeheung Park 1, Taegon Lee 1, Manho Lim ⇑ Department of Chemistry, Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, Republic of Korea

a r t i c l e

i n f o

Article history: Available online 16 October 2012 Keywords: Femtosecond vibrational spectroscopy NO bound heme protein Thermal relaxation Vibrational relaxation of NO Geminate rebinding Photodeligation of NO bound heme protein

a b s t r a c t Femtosecond IR-pump–IR-probe spectroscopy was used to measure the vibrational lifetimes (T1) of NO stretching modes of ferrous NO near 1600 cm 1 and ferric NO near 1900 cm 1 at room temperature. The T1 of NO bound to the heme, ranging from 3.5 to 34 ps, is much shorter in ferrous NO. The vibrational relaxation (VR) of NO was independent of solvent used and excess imidazole concentration, suggesting that intramolecular VR into the internal vibrational modes of the probed molecule may be the dominant pathway for VR of the bound NO. With estimated T1 of the bound NO, we simulated transient spectra of NO bound to ferrous hemoglobin (HbII) after photodeligation of HbIINO and discussed the influence of the hot band on the determination of the dynamics of geminate rebinding of NO to HbII using the change in the magnitude of the fundamental band. Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction Although nitric oxide (NO) is a poisonous gas, it has been found to perform a number of physiological functions, including regulation of blood pressure, platelet inhibition, and neurotransmission [1–4]. Much of NO’s biological function comes from its interactions with heme proteins, such as soluble guanylate cyclase, cytochrome c (Cytc) oxidase, hemoglobin (Hb), and NO synthase enzymes [5–11]. Because the binding of NO to heme protein is an important process in the functioning of these proteins, knowledge of how the structure and dynamics of the protein influence the binding of NO is essential to understanding their functional mechanism. The dynamics of ligand binding to heme proteins such as Hb and myoglobin (Mb) has been used to probe how a protein’s motion and structure are related to its ligand binding function [12–21]. Under physiological conditions, NO deligated from nitrosylated ferrous Mb (MbIINO) or Hb (HbIINO) geminately rebinds on the picosecond time scale, and a conformational change proceeds upon deligation [14,18,22–30]. Thus, the dynamics of geminate rebinding (GR) of NO to these heme proteins after photodeligation of the nitrosylated protein has been investigated to reveal how NO binding is controlled by the structure and dynamics of the protein under physiological conditions [26,27,29,30]. The GR kinetics of a ligand is obtained by probing the change in the population of the deligated protein after photodeligation of a

⇑ Corresponding author. Tel.: +82 51 5102243; fax: +82 51 5167421. 1

E-mail address: [email protected] (M. Lim). These authors contributed equally to this work.

0301-0104/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chemphys.2012.09.030

ligated protein. Time-resolved visible absorption spectroscopy, which extracts this population change from transient electronic spectra collected upon photodeligation of a nitrosylated protein, has provided the kinetics of GR of NO to heme proteins [14,24,25,28,31,32]. However, it gives very little information on the location of deligated NO, which can be used to deduce the NO rebinding pathway [27,29]. Femtosecond time-resolved vibrational spectroscopy has been employed recently to obtain the location of deligated NO and its rebinding pathways in MbII and HbII as well as to measure the conformer-dependent kinetics of NO rebinding to the heme proteins [26,27,29,30]. In these experiments, MbIINO or HbIINO was photodeligated using an intense femtosecond visible pulse, and a time-delayed femtosecond IR pulse was used to probe the stretching mode of NO bound to the protein (near 1610 cm 1) and deligated from the protein (near 1860 cm 1). About 13% of the photodeligated NO was found to be vibrationally excited, and the vibrational relaxation (VR) time of the deligated NO within the protein was found to be about 200 ps [27]. Since the deligated NO geminately rebinds in a few picoseconds and GR kinetics of NO to heme protein was known to be independent of vibrational energy of NO [33], a significant portion of rebound MbIINO or HbIINO has vibrationally excited NO, resulting in a NO hot band (the v = 1 to v = 2 transition) in the rebound protein. When the hot band is present, kinetics of recovery of the NO fundamental band (the v = 0 to v = 1 transition) deviates from that of GR of NO. To obtain the change in the population of the deligated protein from transient vibrational spectra of bound NO with the hot band, one must extract the magnitude change of both the fundamental band and the hot band. The change in the magnitude of the hot band depends on the portion

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of the rebound ligand with vibrationally excited energy and the VR time of the bound ligand. The NO stretching mode in NO-bound ferrous heme proteins such as MbIINO and HbIINO overlaps the strong amide band of the protein, which evolves with time as the protein undergoes conformational relaxation upon deligation and sheds excess thermal energy deposited by the photolysis pulse [26,27,30]. Thus, typical time-resolved spectra of bound NO after photodeligation of nitrosylated ferrous heme proteins exhibit not only NO vibrational bands but also significant evolving background signals arising from conformational and thermal relaxation of the protein. The transient absorption from the hot band of bound NO tends to be buried in experimental noise on top of the background signal [26,30]. Consequently, while the fundamental band can be readily assigned by the use of 15NO isotope [26,27,30], it is extremely difficult to quantify the magnitude and decay time of the hot band in the measured time-resolved spectra of bound NO. Although the population of the deligated NO in the vibrationally excited state can be obtained by the measured transient spectra of the deligated NO [27,30], the VR of bound NO must be measured independently. Once the VR time and population of the excited NO are determined, transient vibrational spectra of bound NO can be fitted to obtain reliable parameters for the GR kinetics of NO. As mentioned, the stretching mode of NO bound to heme proteins is located on the shoulder of a strong amide band of the protein, and the absorbance of the NO band is more than 20 times smaller than that of the amide band of Mb or Hb [27,30]. Thus, it would be extremely difficult to measure the VR time of bound NO in MbIINO and HbIINO using a typical IR-pump–IR-probe method. On the other hand, the VR time of NO in a model heme, free from the protein’s amide band, can be readily determined by IR-pump–IR-probe spectroscopy. NO can bind to both ferrous and ferric heme and the NO mode in nitrosylated ferric heme is located near 1900 cm 1, far from the strong amide band. It is useful in deducing the VR time of NO in ferrous heme proteins from that in the ferrous model heme by comparing the VR time of NO in the ferric model heme with that in ferric heme proteins. The geometry of NO bound to the Fe atom in the heme depends on the bond character of the NO ligand in Fe–NO [34]. As shown in Fig. 1, ferrous hemin–NOs show a bent NO geometry, as in MbIINO. HbIINO also has a bent NO geometry [35]. However, NO is normal to the heme plane in a six-coordinated

(PPIX)FeII(1-MeIm)(NO)

(PPIX)FeIINO

MbIINO (PPIX)FeIII(1-MeIm)(NO) Fig. 1. Structures of NO-bound hemes. Green sticks: proximal ligands; gold sticks: heme; purple balls: Fe atoms; blue and red balls: NO. Gray ribbon represents the polypeptide backbone of MbIINO (Protein Data Bank entry 2FRJ). FeII: Fe2+; FeIII: Fe3+; PPIX: protoporphyrin IX; 1-MeIm: 1-methylimidazole. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

ferric heme–NO. Clearly, NO-bound Fe2+-complexed protoporphyrin IX (PPIX) with a 1-methylimidazole (1-MeIm) is a good model heme for MbIINO. The VR times of the ligand in ligated hemes were investigated for CO-bound heme to explore the heme–ligand bond dynamics and protein dynamics at the active site of heme proteins [36–38]. The VR time of CO in the major conformations of HbIICO and MbIICO was found to be shorter than that in CO-bound model heme. The vibrational energy of CO in these hemes was released primarily into intramolecular heme vibrations [36–38]. Although the VR times of NO in nitrosylated ferric Mb (MbIIINO) and Cytc (CtycIIINO) have been reported [39], to the best of our knowledge, those in nitrosylated ferrous heme proteins or model heme has not been investigated yet. Here we measured the VR time of the NO mode in nitrosylated ferrous and ferric model hemes and nitrosylated ferric heme proteins. We deduced the VR time of NO in MbIINO and HbIINO from that in nitrosylated ferrous hemin. With the deduced VR time and the known spectral parameters of the NO band [30], we simulated time-resolved vibrational spectra of bound NO after photodeligation of HbIINO and discussed how much the kinetics of GR of NO to the ferrous heme proteins is influenced by the presence of the hot band when the kinetics are obtained by fitting the magnitude change of the fundamental band in transient spectra of bound NO.

2. Materials and methods The femtosecond vibrational spectrometer used here was described elsewhere in detail [26,40]. Briefly, tunable mid-IR pulses were generated by difference frequency mixing of the signal and idler pulses of a home-built optical parametric amplifier (OPA) in a 1.5-mm-thick, type-I AgGaS2 crystal. The OPA was pumped by a commercial Ti:sapphire oscillator/amplifier system at a repetition rate of 1 kHz. A mid-IR pulse (110 fs, 1 lJ) with a typical spectral bandwidth of 180 cm 1 was filtered by a long-pass filter and separated into pump and probe pulses by a 1.5-mm-thick wedged BaF2 window. The spectrally broad IR pulse, centered at the N–O stretching mode of the molecule probed, used as pump and probe pulses as generated. The pump pulse, optically delayed by a computer-controlled translational stage, photoexcites the sample, and transient mid-IR absorbance of the sample is measured with the probe pulse. The isotropic absorption spectrum was collected by setting the polarization of the pump pulse at the magic angle (54.7°) relative to the probe pulse using two wire grid polarizers. To obtain vibrational spectra with high spectral resolution, the broadband transmitted probe pulse is routed through a 320-mm IR monochromator with a 150-l/mm grating, and the entire spectrum was detected simultaneously with a 64element N2(l)-cooled HgCdTe array detector mounted in the focal plane of the monochromator. In this configuration, the spectral resolution of the array detector is ca. 1.6 cm 1/pixel at 1920 cm 1 and 1.1 cm 1/pixel at 1620 cm 1. The pump-induced change in the absorbance of the sample, DA, is determined by chopping the pump pulse at half the repetition frequency of the laser and computing the difference between the pumped and unpumped absorbances. The pump spot was made 1.5 times larger than the probe spot to ensure spatially uniform photoexcitation across the spatial dimensions of the probe pulse. A moderate pump energy (<1 mJ) and decent beam size also minimize extended thermal effects. The instrument response function was typically 160 fs. Horse skeleton ferric myoglobin (MbIII), ferric Cytc (CytcIII), and NO gas (98.5%) were purchased from Sigma–Aldrich Co and used as received. To prepare NO-bound ferric protein samples (MbIIINO and CtycIIINO), the desired amount of the corresponding

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3. Results Fig. 2 shows time-resolved spectra of six-coordinated ferric and ferrous nitrosylated hemin, (PPIX)FeIII(1-MeIm)(NO) and (PPIX)FeII(1-MeIm)(NO), respectively, in CH2Cl2 following excitation with an intense IR pulse centered at the N–O stretching mode. Early in the pump–probe delay, two distinct features appear: a negative-going feature (bleach) at the frequency position of ground-state absorption, and a red-shifted transient absorption. The transient absorption results from the v = 1 to v = 2 transition of the N–O stretching mode due to vibrational excitation by the intense IR pulse which populates the v = 1 level. It is red-shifted by 30 ± 1 cm 1 for ferric heme and 28 ± 2 cm 1 for ferrous heme, reflecting the anharmonic frequency shift between the fundamental band and the hot band. Spectra of the bleach and transient

6

a

(PPIX)FeIII(1-MeIm)(NO) 4

ΔA(mOD)

2 0

1 ps 3.2 ps 10 ps 24 ps 42 ps 100 ps

-2 -4 -6 1940

1920

1900

1880

1860

E/hc (cm-1) 10

(PPIX)FeII(1-MeIm)(NO)

b

6

ΔA (mOD)

lyophilized ferric protein was dissolved in D2O buffered with 0.4 M potassium phosphate (pD 6.3). After extensive deoxygenation by purging with N2 gas, the protein solution was bubbled with a NO/N2 mixture gas for a short time to ligate about half of the protein with NO [41]. To eliminate NO2 produced by oxidation of NO, the mixture gas was bubbled through a 2 M NaOH solution before being introduced into the protein solution. To minimize reductive nitrosylation of the heme Fe, a slightly acidic buffer solution (pD 6.3) was used, and only half of the protein was nitrosylated [42]. The sample was then loaded in a gas-tight rotating sample cell with CaF2 windows and a path length of 100 lm. Nitrosylated hemin samples in various solvents were prepared as follows. Two different solvents (CH2Cl2 and CDCl3), 1-MeIm, FePPIX chloride, and Na2S2O4 were purchased from Aldrich and used as received. FePPIX chloride, a ferric hemin, was dissolved in the appropriate solvent (CH2Cl2 or CDCl3), and the hemin solution was extensively deoxygenated by purging with N2 gas. For the six-coordinated sample, after 1-MeIm in the same solvent was added to the hemin solution, the deoxygenated hemin solution with 1-MeIm was bubbled with NO gas for nitrosylation. For the ferrous sample, the hemin solution was reduced with an excess amount of freshly prepared Na2S2O4 solution under N2 gas before bubbling with NO gas. When NO was introduced to the deoxygenated hemin solution, it was also bubbled through a 2 M NaOH solution and subsequently through the corresponding solvent to remove any oxidized by-product of NO such as NO2. For the fivecoordinated ferrous sample, NO was bubbled into the reduced hemin solution. For proximal ligand coordination by 1-MeIm, a much greater excess quantity of 1-MeIm was required for ferrous hemin than for ferric hemin. The reagent concentrations used for the final sample were [FePPIX] = 8 mM and [1-MeIm] = 0.3–2 M for (PPIX)FeII(1-MeIm)(NO); [FePPIX] = 5 mM for (PPIX)FeII(NO); and [FePPIX] = 2 mM and [1-MeIm] = 0.01–0.2 M for (PPIX)FeIII(1MeIm)(NO). The ferrous and ferric samples were loaded into gastight rotating CaF2 cells with path lengths of 200 and 380 lm, respectively. During data collection, the sample cell was rotated sufficiently fast so that each photolyzing laser pulse illuminated a fresh volume of the sample. The temperature of the rotating sample cell was 294 ± 2 K, the room temperature of the lab maintained by an air controller. Throughout the experiment, the integrity and concentration of the samples were maintained by checking them with UV–vis and FT-IR spectroscopy. D2O was used in the protein sample to avoid strong water absorption in the spectral region of interest. Solvent used in the hemin sample is the one that dissolves an acceptable amount of nonpolar hemin without forming hemin aggregates. CH2Cl2 and CDCl3 solvents also provide a good spectral window in the IR spectral regions of interest near 1900 and 1600 cm 1.

2

-2

0.6 ps 1.0 ps 1.7 ps 3.2 ps 5.6 ps 10 ps

-6

-10 1650

1630

1610

1590

1570

E/hc (cm-1) Fig. 2. Representative time-resolved vibrational difference spectra of the NO stretching mode in (a) (PPIX)FeIII(1-MeIm)(NO) and (b) (PPIX)FeII(1-MeIm)(NO) in CH2Cl2. The bleach is due to the population loss in the v = 0 state of the NO mode, and the transient absorption is due to the population gain in the v = 1 state. Note that the time span is 10 times longer in (PPIX)FeIII(1-MeIm)(NO).

absorption were described well by a Gaussian function, suggesting that they are inhomogeneously broadened. These spectral features decay because of VR. Both the integrated area of the bleach and that of transient absorption decay exponentially with the same time constant, suggesting that pumping of higher vibrational levels (v = 2 or higher) is negligible in our experimental condition. The decay time of the transient spectra yields the VR time of the NO stretching mode, T1. As shown in Fig. 3, the times are 34 ± 1 ps for (PPIX)FeIII(1-MeIm)(NO) and 3.5 ± 0.4 ps for (PPIX)FeII(1MeIm)(NO) in CH2Cl2. Relaxation occurs an order of magnitude faster in ferrous hemin. When hemin samples in CDCl3 were probed, the spectral parameters of the NO band are slightly different from but its VR times are almost the same as those in CH2Cl2 (see Table 1). The six-coordinated hemin samples prepared with different concentration of 1-MeIm showed that VR times and the spectral parameters for the NO band of the samples were independent of the concentration of 1-MeIm explored. Transient spectra of five-coordinated ferrous hemin and NO-bound ferric heme proteins were also obtained and analyzed in the same way. Fig. 4 shows transient spectra of NO after photoexcitation of MbIIINO in D2O and decay kinetics of the integrated area of the bleach in the spectra. As summarized in Table 1, the NO band is

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8

a

(PPIX)FeIII (1-MeIm)(NO)

1

Mb(III)NO 4

ΔA (mOD)

NO (v = 1)

T1 = 34 ps

0.5

0 1 ps 3.2 ps 10 ps 17 ps 32 ps 100 ps

-4

(PPIX)FeII (1-MeIm)(NO) T1 = 3.5 ps

-8 0 0.1

1935 1

10

1915

1895

1875

E/hc (cm-1)

100

Time (ps)

b

Fig. 3. Population decay of the v = 1 state for the NO stretching mode in (PPIX)FeIII(1-MeIm)(NO) (black) and (PPIX)FeII(1-MeIm)(NO) (red) in CH2Cl2. Open circles are the normalized amplitude of transient bleach. The NO (v = 1) in the ordinate represents the fractional population of NO in the v = 1 state. The data are described well by an exponential function with a lifetime of T1 (solid line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

T1 = 29 ps

NO (v = 1)

located near 1920 cm 1 in ferric heme and near 1620 or 1670 cm 1 in ferrous heme. In ferrous hemin, the absence of the proximal MeIm shifts the band 50 cm 1 toward higher energy and slows the VR time by a factor of about three. As shown in Table 1, anharmonicity of NO band in the probed ferric hemes are very similar, almost independent of the presence of the protein matrix and solvent used but the VR time of NO in the ferric heme protein is slightly shorter than that in the model heme dissolved in CH2Cl2 or CDCl3. The bandwidth of NO in ferrous heme is much narrower when it is surrounded by the protein matrix, implying that the environment of ferrous NO in the protein is more homogeneous than the solvent.

1

0.5

0 0.1

1

10

100

Time (ps)

4. Discussion

Fig. 4. (a) Representative time-resolved vibrational spectra of NO after photoexcitation of MbIIINO in D2O with an intense IR pulse. (b) Kinetics of population decay of the v = 1 state for the NO stretching mode in MbIIINO in D2O. The normalized amplitude of transient bleach (open circles) is well described by an exponential function with a time constant of 29 ps (solid line).

The NO molecule has an unpaired electron in the p⁄ highest occupied molecular orbital with a bond order of 2.5. The stretching frequency of free NO, m(N–O), is 1876.09 cm 1 [43], whereas that of NO+ (NO oxidized by losing the unpaired p⁄ electron) is 2377 cm 1, and that of NO (NO reduced by gaining an electron in the p⁄ orbital) is 1470 cm 1 [44]. If NO attracts a greater electron density as it binds to metal, m(N–O) shifts to a frequency below 1876.09 cm 1,

and if NO donates electron density, it shifts to a higher frequency. When NO binds to the Fe3+ ion of ferric heme, it has been suggested that the p⁄ electron back-bonds into the Fe 3d orbital, and it was calculated to have the FeII–NO+ ground state [45], which is isoelectronic with FeIICO and has a linear Fe N O configuration. In ferrous heme, the unpaired p⁄ electron remains localized on NO,

Table 1 Vibrational lifetimes and spectral parameters of NO bound to various hemes. Molecules

T1 (ps)

mNO (cm 1)

m01

(PPIX)FeIII(1-MeIm)(NO)a

34 ± 1 36 ± 1 29 ± 1 29 ± 1 3.5 ± 0.4 3.5 ± 0.4 9±1 9±1 – –

1916 ± 2 1913 ± 2 1922 ± 1 1917 ± 1 1622 ± 2 1622 ± 2 1666 ± 2 1673 ± 2 1612 ± 1 1616 ± 1

30 ± 1 28 ± 1 29 ± 1 29 ± 1 28 ± 2 28 ± 2 28 ± 2 29 ± 2 – –

MbIIINO CytcIIINO (PPIX)FeII(1-MeIm)(NO)a (PPIX)FeIINO MbIINO [26] HbIINO [30]

m12 (cm 1)

Dm (v = 0?1) (cm 1)

Solvent

15 ± 1 14 ± 1 10 ± 1 14 ± 1 22 ± 2 29 ± 2 28 ± 2 28 ± 2 14 ± 1 10 ± 1

CH2Cl2 CDCl3 D2O (pH D2O (pH CH2Cl2 CDCl3 CH2Cl2 CDCl3 D2O (pH D2O (pH

6.5) 6.5)

7.3) 7.3)

a [1-MeIm] was varied from 0.02 to 0.2 M in (PPIX)FeIII(1-MeIm)(NO) and from 0.3 to 2 M in (PPIX)FeII(1-MeIm)(NO). T1 and the fitted spectral parameters were found to be independent of [1-MeIm] used.

111

-1

(PPIX)FeIII(1-MeIm)NO

-1

and the already-reduced Fe2+ ion decreases the NO bond order, lowering m(N–O) significantly and causing the NO ligand to bend with respect to the heme normal [34,46]. The measured vibrational frequency of NO near 1920 cm 1 in ferric heme is consistent with the reported N O stretching frequency for NO-bound histidinecoordinated ferric hemes, (His)FeIIINO, at 1903–1925 cm 1 [47]. In addition, 1622 and 1670 cm 1 are typical NO stretching frequencies for six- and five-coordinated NO-bound ferrous heme, respectively [26,34,41,46,48,49]. The observed vibrational frequency of NO confirms that the designated NO-bound hemin has a bonding geometry and electronic configuration similar to that of known nitrosylated heme molecules. The excess vibrational energy of a specific mode in a polyatomic molecule can be redistributed into its internal vibrational modes (intramolecular VR, IVR) and transferred into vibrational modes of the solvent. The time and pathway of VR depend on the coupling strength and energy matches between the initial and final states [50,51]. When the excited mode and acceptor mode are close in energy, there is a higher propensity for faster VR. Because higher molecular complexity in the solute generally introduces an additional IVR channel, the solute complexity tends to increase the contribution of IVR to vibrational energy relaxation [51]. As shown in Fig. 5, there are three absorption bands near 1720, 1670, and 1600 cm 1 other than NO band in the nitrosylated hemin molecules. These bands, stemming from hemin, are close enough to be an efficient acceptor mode for IVR of the NO mode in ferrous hemin. Although these vibrational bands in the hemin overlap more with the NO band in (PPIX)FeIINO than that in (PPIX)FeII(1MeIm)(NO), the NO mode in (PPIX)FeII(1-MeIm)(NO) has shorter VR time (3.5 ps) than that in (PPIX)FeIINO (9 ps). If energy transfer from the excited NO mode to these bands in hemin is a dominant channel for VR of the probe NO mode, faster VR in (PPIX)FeII(1MeIm)(NO) may arise from stronger coupling between the NO mode and the acceptor mode. (PPIX)FeII(1-MeIm)(NO) has the proximal ligand (1-MeIm) vibrational mode of which can be an acceptor mode. Interestingly, 1-MeIm has a strong absorption band at 1520 cm 1 that can serve as an efficient acceptor mode for solvent-assisted VR of the ferrous NO mode at 1622 cm 1. Although we cannot exclude the possibility that intermolecular VR of the NO mode to the excess 1-MeIm in solution and/or solvent is a dominant VR channel for the excited NO, VR time of NO independent of [1-MeIm] and solvent used suggests that IVR is the dominant relaxation pathway for the excited ferrous NO. The CO band in the model heme and heme proteins near 1950 cm 1 has no nearby absorption band [50,51] but IVR was suggested to be the predominant process for vibrational energy distribution in the ligand [36,37]. Overtones involving the Fe–C and Fe–N modes were suggested to be the accepting mode for the excess CO energy [36,37]. Because FeIIINO has the same linear Fe X O geometry as FeIICO, and the vibrational frequency of NO in FeIIINO is about the same as that of CO in FeIICO, the VR of NO in ferric heme likely proceeds intramolecularly, like that of CO in ferrous heme. The VR time of NO for (PPIX)FeIII(1-MeIm)(NO) in CH2Cl2 or CDCl3 is slightly slower than that in heme proteins in D2O, which is consistent with the suggestion that IVR is the main relaxation pathway for the excess vibrational energy in NO stretching in both ferrous and ferric heme molecules. It has been found that the excess energy in diatomic ligands of ligated heme proteins is first deposited into the heme group and then transferred into the protein matrix, and subsequently into the solvent [33,52]. Therefore, the VR time of NO in heme proteins is likely controlled by the coupling strength and energy match between the NO mode and the heme group, including the proximal histidine. As mentioned above, the VR times of NO in ferrous heme proteins such as MbIINO and HbIINO have not been measured yet, and they are extremely difficult to measure because the NO band

extinction coefficient (mM cm )

J. Park et al. / Chemical Physics 422 (2013) 107–114

2050

(PPIX)FeIINO 1 mM-1cm-1

(PPIX)FeII(1-MeIm)NO

1950

1850

1750

1650

1550

E/hc (cm-1) Fig. 5. Vibrational absorption spectra of (PPIX)FeIII(1-MeIm)(NO) (blue line), (PPIX)FeIINO (green line), and (PPIX)FeII(1-MeIm)(NO) (red line) in the spectral range near the NO stretching mode. The NO band in each molecule, obtained by isotopic labeling of 15NO, is shown by a filled Gaussian of the corresponding color. The ordinate is the estimated extinction coefficient, with an uncertainty of ca. 30%. The spectra are offset to avoid overlap. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

overlaps the strong amide band absorption of the protein. Although it is not clearly distinguishable because of the broad, strong amide band near 1650 cm 1, the heme group in the heme proteins likely has vibrational modes near 1650 cm 1, as observed in the model hemes. Because the heme proteins also have a strong amide band near 1650 cm 1, the NO mode of nitrosylated ferrous heme proteins near 1614 cm 1 can have sufficient accepting modes that match in energy. Therefore, the VR time of NO in the ferrous heme proteins is likely similar to, if not shorter than, that in the six-coordinated ferrous model heme, 3.5 ps. Here, we suggest that the VR time of NO in HbIINO or MbIINO is 3.5 ps or shorter. Interestingly there were many reports on 1–5 ps change in transient optical spectrum upon photoexcitation of HbIINO and MbIINO [18,24,25,28]. The time constant was suggested to arise from vibrational cooling of hot electronic ground state [18,24,25,28]. The transient optical spectrum may have a contribution from VR of the excited NO. When HbIINO and MbIINO are photodeligated with a 580-nm photon, about 13% of the deligated NO is vibrationally excited, and the VR time of the deligated NO is ca. 200 ps [27,30]. Because the NO rebinds on the picosecond time scale, some of the rebound NO is vibrationally excited, resulting in a hot NO band in NO-bound heme proteins [30]. As the rebinding occurs more rapidly, the hot band becomes larger because more excited NO rebinds before thermal relaxation. GR of NO is faster in HbIINO than MbIINO [29], the hot band contribution is higher in the rebinding of NO to HbII. Here, we discuss mainly GR of NO to HbII to observe the maximum possible influence of the hot band on measurement of the GR kinetics using time-resolved vibrational spectroscopy. In vis-pump–IRprobe spectroscopy of ligated heme proteins, when the deligated ligand in a vibrational ground state rebound to the protein, vibrational band of the rebound ligand was found to be virtually identical to its fundamental band observed before photodeligation [26,29,30,53–56], resulting in proportional reduction of the bleach in the fundamental band upon ligand rebinding. Thus, when there is no hot band, the magnitude of the bleach is proportional to the population of the deligated protein and the bleach recovery represents the rebinding kinetics. Indeed, for photodeligation of CObound heme proteins, where the time scale of GR is much slower than the VR time of bound CO, the kinetics of bleach recovery

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was used to measure GR of CO to the protein [23,55]. If GR produces a hot band in the bound ligand, the population recovery is not identical to the bleach recovery. Fig. 6 shows simulated time-resolved spectra of HbIINO after photodeligation of NO, with fitted parameters for the NO vibrational band and the kinetics of GR of NO to HbII [30] using 3.5 or 30 ps for the VR time of NO in HbIINO. As T1 increases, the hot band becomes larger and reaches a maximum magnitude later. On the other hand, the hot band, even though it is clearly visible, is much smaller for shorter T1. Using the harmonic oscillator approximation for the absorbance, the integrated absorbances of the fundamental band (Ag) and the hot band (Ae) are related to the populations at v = 0 (ng) and v = 1 (ne) as follows: Ag = e(ng ne) and Ae = 2ene, where e is the integrated absorptivity of the fundamental band [57]. As shown in Fig. 7, when T1 = 3.5 ps, the hot band reaches a

a

maximum near 5 ps, its magnitude is smaller than 5% of the initial bleach, and the kinetics of bleach is similar to that of GR of NO. When T1 is 30 ps, the hot band reaches a maximum near 20 ps, its magnitude is as large as 13% of the initial bleach, and the kinetics of bleach deviates sharply from that of GR of NO. We also simulated time-resolved spectra of HbIINO after deligation of NO using various T1 values and compared the kinetics of bleach recovery with that of GR. We found that the kinetics of the bleach recovery deviates more from that of GR of NO to HbII as T1 increases and is always slower than that of GR of NO when the hot band exists. When a kinetic trace at the maximum absorbance change is measured, as reported for MbIINO [58], it measures the kinetics of bleach recovery. As shown in Table 2, when the bleach kinetics was fitted to a biexponential function with the same weighting as the kinetics of GR of NO to HbII [0.54 exp( t/ 5.6 ps) + 0.46 exp( t/29 ps)] [30], the faster time constant increases from 6 ps at T1 =1.5 ps to 8.1 ps at T1 = 30 ps, and the slower time constant increases from 28.2 ps at T1 =1.5 ps to 54.3 ps at T1 = 30 ps. As mentioned above, when vis-pump–IR-probe spectroscopy of MbIINO was performed, the transient absorption from the hot band

1660

32 ps 17 ps 13 ps 7.5 ps 4.2 ps 1.8 ps 0.6 ps 1640

a

T1 = 3.5 ps

1620

1600

1580

1560

E/hc (cm-1)

Normalized integrated area

ΔA

1

NO rebinding Ableach

0.5

T1 = 3.5 ps

Av=1

b

0 1

1660

Normalized integrated area

ΔA

b

T1 = 30 ps

1640

1620

1600

1580

NO rebinding

0.5

Av=1

1560

E/hc (cm-1) Fig. 6. Simulated transient spectra near the NO stretching mode after photodeligation of HbIINO in D2O with (a) T1 = 3.5 ps and (b) T1 = 30 ps for the VR time. The fitted parameters of time-resolved spectra of HbIINO in D2O after photodeligation [30] were used for the spectral parameters of transient bleach and the population of the v = 1 state. Thirteen percent of the photodeligated NO was set to be vibrationally excited, and the VR time of the deligated NO was assumed to be 200 ps [27]. Anharmonic shift of transient absorption was set to 28 cm 1, that of a model ferrous heme, (PPIX)FeII(1-MeIm)(NO). The time delay is color-coded in the spectra. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Ableach

T1 = 30 ps

0 0.1

1

10

100

Time (ps) Fig. 7. Time-dependent changes in normalized integrated areas of the transient bleach (Ableach, blue) and absorption (Av=1, red), and the deligated HbII population (NO rebinding, green) after photodeligation of HbIINO in D2O when (a) T1 = 3.5 ps and (b) T1 = 30 ps for the NO stretching mode of HbIINO. The kinetics of the bleach and absorption comes from the simulated transient spectra shown in Fig. 6. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Table 2 Kinetics parameters of a biexponential function, a1 exp( t/s1) + (1 a1)exp( t/s2) for the decay of transient bleach without (upper row) and with (lower row) accounting for the hot band in the simulated spectra after photodeligation of HbIINO in D2O with various T1 values of bound NO. The kinetics of GR of NO to HbII used in the simulation is described by 0.54 exp( t/5.6 ps) + 0.46 exp( t/29 ps) [27]. For comparison, a1 was set to 0.54 in fitting the bleach kinetics (see text).

Kinetics of transient bleach Fitted bleach kinetics treating the hot band as a background signal

s1 (ps) s2 (ps) s1 (ps) s2 (ps)

of NO was buried in experimental noise in the background signal. Consequently, in previous data analysis of time-resolved spectra of MbIINO, the hot band was ignored, and the bleach was fitted by the sum of Gaussian functions plus a polynomial function for the background [26]. To evaluate the reliability of the fitted kinetics parameters for GR of NO obtained from the previous type of analysis that treated the NO hot band as a background signal, the simulated time-resolved spectra were fitted to the NO vibrational band plus the polynomial background, and the bleach kinetics was obtained for various T1 values. The decay kinetics obtained by fitting the spectra treating the hot band as a background signal turned out to be similar to the kinetics of bleach itself and was fitted to a biexponential function; the fitting parameters are given in Table 2. Although the bleach kinetics with or without considering the hot band tends to slow down with increasing T1, the bleach kinetics are similar to that of GR of NO to HbII when T1 is smaller than or equal to 3.5 ps. As can be seen in Table 2, when T1 = 3.5 ps or shorter, the recovered parameters for the bleach kinetics are almost identical to those for GR of NO to HbII except for the faster time constant of 5.6 ps. It becomes 6–6.9 ps, which can be within the error range reported in this type of experiment [26,30]. Because GR is slower in Mb, the influence of the NO hot band is expected to be even smaller in Mb than in Hb. Evidently, when T1 is equal to or smaller than 3.5 ps, the bleach kinetics can be a reasonable representative for the kinetics of GR of NO to heme proteins. We found that the kinetics of GR of NO to MbII and HbII obtained by timeresolved vibrational spectra is faster than that measured by optical spectroscopy [14,24,25,28,31,32]. It is noteworthy that the transient signals in the optical spectra have contribution from conformational and vibrational relaxation of photodeligated protein, which can hamper accurate recovery of the population change of the deligated protein [26]. In conclusion, we measured the VR times of NO bound to ferrous and ferric hemes. The VR time of NO bound to heme varies from 3.5 to 36 ps depending on the oxidation state of Fe in heme and the presence of the proximal ligand, histidine or MeIm. The VR time of NO in (PPIX)FeII(1-MeIm)(NO), a model heme for Mb and Hb, was found to be 3.5 ± 0.4, which is much shorter than that in (PPIX)FeIII(1-MeIm)(NO), 35 ± 2. The vibrational energy of NO in (PPIX)FeII(1-MeIm)(NO), 1622 cm 1, is reasonably matched with other vibrational modes in the molecule showing three vibrational bands at 1720, 1660, and 1620 cm 1. The VR time of NO in (PPIX)FeII(1-MeIm)(NO) was independent of solvent used and added imidazole concentration. These observations suggest that rapid VR of the NO in (PPIX)FeII(1-MeIm)(NO) is dominated by an IVR process, and thus the VR times of NO in MbIINO and HbIINO are likely 3.5 ps or shorter. We simulated time-resolved vibrational spectra of NO after photodeligation of HbIINO for various T1 values using the reported spectral parameters of NO, the kinetics of GR of NO to HbII, and the population of vibrationally excited NO (13%) [27]. The kinetics of GR of NO to HbII was found to always be faster than that of the bleach spectrum obtained by fitting the bleach while treating the hot band as a background signal. Clearly, the faster kinetics obtained by vibrational spectroscopy compared to that measured by optical spectroscopy [14,24,25,28,31,32], is not due to

1.5 ps

3.5 ps

6 ps

12 ps

24 ps

30 ps

6.0 28.2 6.0 28.2

6.9 28.3 6.9 28.3

7.7 29.7 7.6 29.7

8.3 35.5 7.9 36.5

8.2 48.5 7.8 49.8

8.1 54.3 7.7 53.6

an incomplete account of the NO hot band. If the VR time of NO in MbIINO and HbIINO is 3.5 ps or shorter, the bleach kinetics is very similar to the kinetics of GR of NO to these proteins. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (No. 2011-0016114). J. Park acknowledges postdoctoral fellowship from NRF’s Basic Science Research Program grant funded by the MEST (2012R1A6A3A01017867). References [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]

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