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Laser-Induced Magnetic Dipole Spectroscopy Christian Hintze, Dennis Bücker, Silvia Domingo Köhler, Gunnar Jeschke, and Malte Drescher J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.6b00765 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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The Journal of Physical Chemistry Letters

Laser-Induced Magnetic Dipole Spectroscopy Christian Hintze,†,¶ Dennis Bücker,†,¶ Silvia Domingo Köhler,† Gunnar Jeschke,‡ and Malte Drescher∗,† †Department of Chemistry, University of Konstanz, 78464 Konstanz, Germany ‡Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zürich, Switzerland ¶These authors contributed equally to this work. E-mail: [email protected]

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In the past 15 years, pulsed EPR distance measurements have become an important tool to obtain structural information on the nanometer scale about macromolecules, may it be in biological 1–4 or synthetic systems 5–10 . Especially the pulsed EPR techniques Double Quantum Coherence (DQC) 11,12 and Double Electron Electron Resonance (DEER, also called Pulsed Electron Double Resonance, PELDOR) 4 have become widely used tools for this purpose 13 . To obtain distance information, pairs of paramagnetic spin labels are attached to the nanostructure of interest via site-directed spin labeling 14–19 . Mainly nitroxides 14,18,20 , but also other types of persistent paramagnetic molecules 21–28 have been used as spin labels. Especially the recent finding of the porphyrin triplet state being a potential spin label for DEER distance measurements 29 is very important for this work. DEER/PELDOR and DQC determine the electron-electron dipolar interaction between the spin labels by separating it from other electron spin interactions. From this dipolar interaction, distance distributions in the range of approximately 1.5 nm to 8 nm or even up to 12 nm 30 can be evaluated, and transferred into information about the nanostructure of interest. Recent developments comprise new experimental techniques 31 , using higher fields and frequencies 32–34 , broadband excitation by arbitrary waveforms 35,36 or combinations thereof 37–41 . They strive to broaden the field of applications of pulsed EPR dipolar spectroscopy, increase the sensitivity of the method, and to overcome present distance limitations. In this regard we set out to introduce and implement a new technique in pulsed EPR dipolar spectroscopy maintaining compatibility to all aforementioned developments, the laser-induced magnetic dipole (LaserIMD) spectroscopy. The corresponding pulse sequence is shown in Figure 1 and compared to four-pulse DEER. In DEER, two spin labels with, in most cases, SD =

1 2

are addressed by two microwave

frequencies (pump and observer frequency). A pump pulse is traversed over a primary echo, generated by a Hahn echo sequence. The intensity of the refocused echo is detected as a function of the position t in time of the pump pulse. For LaserIMD, a conventional spin label

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with (but not limited to) spin SD =

1 2

is used as the observed species, whereas a chromophore

replaces the pumped spin label species. LaserIMD utilizes switching of the dipolar coupling by optical excitation of the chromophore into its triplet state with spin ST = 1 instead of pumping a spin species by addressing it with a second microwave frequency. Accordingly, the pump pulse is replaced by a laser flash. Since it does not interfere with microwave pulses, it can be applied simultaneously with the microwave pulses and is traversed over the complete echo sequence. Thus, a simple two pulse Hahn echo sequence, akin to three-pulse DEER 42 , is sufficient to obtain dead time free data. To derive an expression for the LaserIMD signal of a spin pair consisting of a persistent observer spin SD = 1/2 and a transiently generated triplet spin ST = 1, we first consider the case where the laser pulse occurs after the π/2 pulse that generates SD spin coherence but before the π pulse that refocuses this coherence. Before the laser flash at time t′− (Fig. 1), the photo-excitable label is in an EPR-silent singlet state and does not significantly influence the spin Hamiltonian or relaxation of the observer spin SD , which acquires phase ΩS,D t′− . At time t′− , the triplet is generated with triplet quantum yield ΦT . Assuming that the electron Zeeman interaction of the triplet spin is much larger than its zero-field splitting, the magnetic quantum number of the triplet spin is a good quantum number and assumes the values mS,T = −1, 0, +1 with probabilities p−1 , p0 , and p+1 , respectively. Fractions ΦT pmS,T of all spin pairs gain additional phase (ΩS,D + mS,T ωdd )(τ − t′− ) before the refocusing pulse, where ωdd is the coupling between the two spins. The refocusing pulse inverts phase, resulting in total acquired phase −ΩS,D τ + mS,T ωdd (t′− − τ ). Between the refocusing pulse and echo refocusing, the respective spin pairs gain phase (ΩS,D + mS,T ωdd )τ , resulting in a phase offset at echo formation of mS,T ωdd t′− . This phase offset is manifest in a cosine modulation of the echo signal with respect to time t′− . Since the cosine is an even function, pairs with mS,T = −1 and mS,T = +1 contribute the same modulation factor cos(ωdd t′− ), whereas the pairs with mS,T = 0 do not contribute to dipolar modulation. If the signal is detected in quadrature, the contributions to the imaginary part have the form ±p±1 sin(ωdd t′− ). In cases,

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where p−1 and p+1 differ significantly from each other, a significant out-of-phase component of the dipolar evolution function would thus be observed, akin to the situation in DEER when the high-temperature approximation does not apply 43 . In the following, we neglect this component as it is insignificant in our experimental data (see below). Thus, the signal contribution of an isolated spin pair can be written as

V (t) = 1 − λ + λ cos(ωdd t′− ) ,

(1)

where the modulation depth λ is given by

λ = ΦT (p−1 + p+1 ) .

(2)

The populations p−1 and p+1 of the triplet sublevels at high field depend on the orientation of the molecular frame with respect to the external magnetic field as well as on the populations px , py , and pz of the zero-field triplet sublevels after intersystem crossing. The relative populations can be obtained by transient EPR 44 . Eq. (1) has the same form as the expression for dipolar modulation of an isolated spin pair in the DEER experiment. Hence, all further considerations on the DEER signal 4 and all established procedures for data processing apply to LaserIMD as well. For the case where the laser pulse occurs after the refocusing microwave pulse, at a time t′+ before echo formation, the observer spins have acquired phase ΩS,D [−τ + (τ − t′+ )] at the instant of the laser pulse. Between the laser pulse and echo refocusing they gain phase (ΩS,D + mS,T ωdd )t′+ . As a result, the echo phase is ωdd t′+ and the signal is given by

V (t) = 1 − λ + λ cos(ωdd t′+ ) .

(3)

These expressions for the signal are valid if, both, longitudinal relaxation between triplet sublevels and triplet decay to the singlet ground state are slow on the time scale of the experiment. Otherwise, the dipolar modulation would be damped. 5




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infinite pump excitation bandwidth, the lowest detectable distance is given solely by the observer pulse lengths 47 , allowing the measurement of shorter distances. • In LaserIMD, the observation can be performed at the maximum of the spectrum without compromising modulation depth, which increases sensitivity compared to DEER. • The laser flash in LaserIMD does not interfere with microwave pulses, thus observer echo refocusing is not required for avoiding dead time. • No nuclear modulation effects, which are present in DEER/PELDOR 48 as well as DQC 49 , exist in LaserIMD. These advantages potentially lead to significantly shorter measurement times or can be used to extend the distance ranges accessible with dipolar spectroscopy in EPR. In this work, we show first LaserIMD experiments and estimate how much sensitivity could be gained by optimization of triplet excitation. We also show that this experiment can utilize endogenous prosthetic groups like the Heme, without modifying them chemically. For a proof of concept experiment we use the peptide Ala-(Aib-Ala) 4-TOAC-(Aib-Ala) 2OH labeled with 5-(4-carboxyphenyl)-10,15,20-triphenylporphyrin (TPP) at the N-terminus (cf. Figure S1 for the chemical structure, predicted mean inter-spin distance hri = 2.2 nm 29 ) which is a suitable model system for measuring nitroxide-porphyrin distances 29 . The porphyrin is excited into its triplet state with laser pulses with 3 mJ of energy in the UV (351 nm wavelength) coupled into the resonator via a quartz glass fiber. In Figure 2 A, the time-domain signal obtained with LaserIMD in Q band is shown as raw data. The dipolar modulation stemming from the dipolar interaction between the triplet and nitroxide spin is readily observed. Furthermore, intensities before and after the dipolar evolution are different, which can be attributed to a reduced phase memory time Tm of the nitroxide spins in the presence of the TPP triplet (laser flash before Hahn echo pulse sequence) compared to the absence of the triplet (laser flash after Hahn echo sequence, cf. Figure S2 for an equivalent control experiment with anthracene triplets). During the dipolar 7



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evolution time, the influence of the reduced phase memory time Tm is decreasing, leading to an additional contribution to the background factor obtained by a separate measurement on a homogeneous solution of Ala-(Aib-Ala) 4-TOAC-(Aib-Ala) 2-OH and TPP. The logarithm of such experimental background functions can be fitted by a low-order polynomial function 50 , in our case by a polynomial function of degree 5, which was then used to correct the timedomain signal. Relying on a homogeneous three dimensional background instead yields identical results. A low amplitude out-of-phase component of the signal (bottom traces in 2 A) is detected. However, we cannot safely conclude that this component stems from a difference between p−1 and p+1 and refrain from any further interpretation. The symmetry between both branches of the time domain signal around t′±,max can be used to determine the correct zero time similar to the way it is done with DEER data, where the symmetry is with respect to the unobserved primary echo at time 2τ1 (see section 4 of the SI for details). If the raw data is processed this way, two nearly identical time traces are obtained (Figure 2 B). Both time domain signals can be corrected by their background contribution as discussed before, which yields the pure dipolar modulations (Figure 2 C). We investigated the influence of the shot repetition rate (SRT) on the experiment. For different SRTs from 4 ms to 1000 ms, the temperature (10 K to 50 K) was chosen to not suppress the nitroxide signal due to its longitudinal relaxation time T1 . At these SRTs, the modulation depth is maintained, excluding triplet state saturation. Therefore, the triplet life time has to be considerably shorter than 4 ms. For the proof of concept experiment shown in figure 2 a high SRT of 400 ms was chosen. Varying the concentration of the sample, we do not observe effects of insufficient triplet excitation at concentrations typical for distance measurements (200 µm or lower). Modulation depths of λLaserIMD ≈ 9 % are obtained. Since insufficient triplet excitation due to a too fast repetition of the experiment or due to a too high concentration were excluded, we conclude that the modulation depth is limited due to the intrinsic triplet quantum yield of the chromophore. The intrinsic quantum yield is the ratio between the

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number of chromophores that end up in the triplet state and the number of chromophore that absorbed a photon. Assuming an optical spin polarization featuring approximately one third of triplets residing in the mS = 0 state (not contributing to the modulation depth in LaserIMD), our experiments suggest a triplet quantum yield of ΦT ≈ 0.13. This low ΦT is due to the excitation of the second excited singlet state S2 in our current experimental setup, since we excite the porphyrin in its Soret band (see SI for UV/VIS spectra). The triplet quantum yield can probably be increased by excitation of the first excited singlet state S1 by an excitation wavelength in the porphyrin’s Q bands between λexc. = 450 nm and 600 nm to ΦT ≈ 0.9 51 . The Fourier transforms of the form factors lead to the dipolar coupling spectra in frequency space (Figure 2 D) and can be fit via Tikhonov regularization to yield the distance distribution shown in Figure 2 E with a mean inter-spin distance hri± = 2.12 nm and width of the distribution s± (r) = 0.06 nm. A similar result is obtained with Triplet-DEER as well (cf. Figure S4 and the SI of the paper that introduced Triplet-DEER 29 ). The spin density distribution over the triplet-state molecule can potentially complicate interpretation of the mean distance and distance distribution. For the analogous case of trityl radical labels it has been shown, however, that good agreement between theory and experiment is obtained when the spin density distribution is taken into account 52 . Comparing LaserIMD to a DEER measurement on the Ala-(Aib-Ala) 4-TOAC-(Aib-Ala) 2OH labeled with 1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxylic acid (TEMPYO) instead of TPP, the modulation-to-noise ratio (MNR) of both measurements performed on the same spectrometer under identical conditions (temperature, shot repetition rate, and concentration) is comparable at the current state (ρ =

M N RLaserIMD M N RDEER

= 1.0, cf. Figure S5). The full

potential of LaserIMD could be unlocked if an triplet quantum yield ΦT = 1 could be reached. This would lead to a LaserIMD modulation-to-noise ratio enhancement factor up to ρ ≈ 6 (cf. SI for details), cutting down measurement times by ρ2 compared to DEER. These results show that LaserIMD is a viable and attractive technique for EPR distance measurements if

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We want to point out that we were not able to detect a spin echo from the Heme triplet state in CytC, because its Tm is reduced significantly by the iron (cf. Figure S6). Utilizing electron spin echos of ferric heme centers in nanometer range distance measurements is still challenging 41,58 and LaserIMD does not rely on the presence of paramagnetic metal centers like the closely related Relaxation-Induced Dipolar Modulation Enhancement (RIDME) technique 59,60 . The short Tm prevents utilizing the triplet state as the observer species for DEER distance measurements 29 and, if Tm is too short for applying an inversion pulse, also utilizing it as a spin pumped by a microwave pulse. However, LaserIMD only requires that the spin can be generated by photo-excitation and that its state survives for the duration of the echo experiment, thus posing the less strict requirement T1 > τLaserIMD . LaserIMD measurements between the MTSSL and the Heme group of CytC were successful (Figure 4). The resulting distance distribution yields a mean distance of hri = 1.96 nm in good agreement with the theoretically predicted distance. The LaserIMD experiment gives access to distances as low as 1.2 nm. This lower limit is defined by the jitter of the laser used (FWHM ≤ 20 ns, cf. Figure S10) and the excitation bandwidth of the observer π pulse length of 20 ns. 47 Laser jitter could be reduced by a different triggering scheme and excitation bandwidth correction 46 could be applied, which could enable detection of even shorter distances down to 1 nm. In summary, we have shown that LaserIMD allows for measurement of distance distributions in the nanometer range, both, when using TPP as a spin label and when exploiting an endogenous low-spin Heme as the photo-excitable species. In the latter case, the technique poses lesser requirements on relaxation times of the triplet spin than are posed by the Triplet-DEER experiment. If the triplet quantum yield can be improved, LaserIMD has the potential to access longer distances or decrease the measurement time or measure at lower concentration than is possible with DEER techniques.

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Supporting Information Available Characterization of Ala-(Aib-Ala) 4-TOAC-(Aib-Ala) 2-OH and Cytochrome C; sample preparation; EPR experiments and analysis; additional EPR measurements.

Acknowledgement This work was financially supported by the DFG (DR 743/2-1 and DR 743/10-1).

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