Coherent infrared emission from myoglobin crystals: An electric field measurement

Abstract

We introduce coherent infrared emission interferometry as a (2) vibrational spectroscopy technique and apply it to studying the initial dynamics upon photoactivation of myoglobin (Mb). By impulsive excitation (using 11-fs pulses) of a Mb crystal, vibrations that couple to the optical excitation are set in motion coherently. Because of the order in the crystal lattice the coherent oscillations of the different proteins in the crystal that are associated with charge motions give rise to a macroscopic burst of directional multi-teraHertz radiation. This radiation can be detected in a phase-sensitive way by heterodyning with a broad-band reference field. In this way both amplitude and phase of the different vibrations can be obtained. We detected radiation in the 1,000 -1,500 cm ؊1 frequency region, which contains modes sensitive to the structure of the heme macrocycle, as well as peripheral protein modes. Both in carbonmonoxy-Mb and aquomet-Mb we observed emission from six modes, which were assigned to heme vibrations. The phase factors of the modes contributing to the protein electric field show a remarkable consistency, taking on values that indicate that the dipoles are created ''emitting'' at t ‫؍‬ 0, as one would expect for impulsively activated modes. The few deviations from this behavior in Mb-CO we propose are the result of these modes being sensitive to the photodissociation process and severely disrupted by it.T he time scale of reaction dynamics in proteins and concomitant protein conformational changes spans many orders of magnitude. Small and highly directed structural changes occurring on the time scale of vibrations, i.e., tens of femtoseconds to picoseconds, often ultimately lead to a larger conformational change. Timeresolved optical measurements have been widely used to obtain information on the dynamics of the optically active cofactors on time scales ranging from tens of femtoseconds to seconds (1, 2). However, visualization of function-determining structural changes of the protein on the ultrafast time scale is still greatly missing. Specific structural changes may be monitored by vibrational spectroscopy (absorption or Raman scattering) in the mid-IR range, where the protein and cofactors have vibrational transitions, or by NMR and x-ray diffraction techniques. With time-resolved x-ray spectroscopy the structures of photointermediates of proteins have been resolved with nanosecond time resolution (3, 4). However, techniques with femtosecond resolution are necessary to obtain information on local structural changes and their coupling to a reaction on the time scale of molecular vibrations.Information on the coherence time of cofactor-protein interactions with respect to reaction times in several protein complexes has been derived from three pulse-photon echo experiments performed either on electronic transitions of the cofactors (5-7) or vibrational transitions (8, 9). The relevance of low-frequency (Ͻ400 cm Ϫ1 ) heme-protein modes for the initial reactions in hemes has been studied by monitori...

Resonant optical rectification in bacteriorhodopsin

“…This signal and the corresponding modulation of the Fourier transform spectrum (Fig. 2B) presumably reflects the IR-active vibrational response of the retinal-protein system and is equivalent to the effect we have observed previously in myoglobin crystals (32). We stress, however, that an equivalent of the main, broadband emission signal has not been observed on myoglobin (32); this is, therefore, a previously uncharacterized example of electronic optical rectification in a biological sample.…”

“…This phenomenon has been observed in noncentrosymmetric semiconductors (27)(28)(29)(30) and organic crystals (31) in the form of single-cycle radiation in the THz and midinfrared region as a response to ultrashort laser pulses but not in biological materials. On the other hand, long-lasting coherent infrared emission has been observed from heme protein crystals (32), originating solely from the vibrational response of the protein. Here, we report impulsive macroscopic polarization of oriented bR films by optical rectification of an 11-fs visible light pulse in resonance with the optical transition, providing a direct evidence for charge separation as a precursor event for subsequent functional processes.…”

Dephasing-Induced Vibronic Resonances in Difference Frequency Generation Spectroscopy

“…18 DFG has further found applications in semiconductors. 10,11 In this paper we focus on an ultrafast DFG technique known as coherence emission spectroscopy/optical rectification, 19,20 whereby two femtosecond visible pulses resonant with an electronic transition create vibrational coherences in both the ground and the excited electronic states. The generated heterodyne detected infrared field (both amplitude and phase) reveals vibrational modes strongly coupled to the photoexcitation.…”

“…The generated heterodyne detected infrared field (both amplitude and phase) reveals vibrational modes strongly coupled to the photoexcitation. This technique has been applied using 11 fs pulses to study protein vibrational motions coupled to an electronically excited cofactor in photoactivable single crystals (The photodissociation of the heme cofactor in ordered crystals of myoglobin 19 and the retinal trans f cis photoisomerization in oriented films for bacteriorhodopsin 20 ). These experiments have opened up new possibilities for probing protein structure and for following concerted motions induced by an external femtosecond trigger.…”

Observation of terahertz radiation coherently generated by acoustic waves

“…In addition to groundbreaking demonstrations of acoustic solitons, these experiments have led to new techniques for probing the structure of thin films [6][7][8] . Terahertzfrequency electromagnetic radiation has been used in applications as diverse as molecular and material excitations 9,10 , charge transfer 11,12 , imaging 13 and plasma dynamics 14 . However, at present, existing approaches to detect and measure the time dependence of terahertz-frequency strain waves in materials use direct optical probes-time-resolved interferometry or reflectrometry 2,15,16 .…”