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The dynamics of ligand escape from carboxymyoglobin are studied via Q-band transient absorption and diffractive optics-based four-wave mixing. The latter approach provides an interferometric method for following protein motions and energetics and allows unambiguous assignment of different signal components to specific dynamical processes, a problem that has hindered all photothermal, photoacoustic, and grating methods in the past. In particular, the real part of the four-wave mixing signal is isolated, and it is demonstrated that the excited-state population contribution to the signal can be identified and removed by tuning the probe to wavelengths where this contribution vanishes (“zero-crossings”). At these probe wavelengths, changes in the real part of the index of refraction are small compared to those of the imaginary part, making the heterodyne measurement sensitive to errors in setting the phase of the reference field. We solve this problem with a new balanced detection method that isolates the real part of the signal and is extremely robust against phase errors. The location of the zero-crossings in the population contribution to the index of refraction is found to be complicated by the presence of spectral shifts in the transient absorption that can be characterized and removed with a time-dependent Kramers−Kronig analysis. The spectral shifts are most apparent near the isosbestic points and show similar dynamics to the four-wave mixing signal, suggesting that both are sensitive to the same dynamical processes. These dynamics were observed previously by using four-wave mixing with an off-resonant probe and were assigned to CO migration out of the protein via a number of discrete channels. It appears that both the photoinduced protein conformational relaxation and CO migration through the protein contribute to the transient absorption signal, making the two effects difficult to separate. The direct coupling of the protein motions to the index of refraction changes in the four-wave mixing experiments aids in distinguishing the two processes.

The initial structural evolution of carboxymyoglobin (MbCO) following photodissociation of CO is measured using a recently developed method of heterodyne detection. This method increases the sensitivity, generates tilted pulse fronts which exactly cancel temporal broadening in noncollinear beam geometries, and provides an inherent acoustic reference that enables unambiguous separation of the real and imaginary components to the nonlinear susceptibility. With the latter advance, both the absorption anisotropy and its real counterpart, the phase anisotropy, can be measured with this technique. Access to the real part of the material anisotropy provides new information that can be correlated to the bath dynamics. In the MbCO studies, the phase anisotropy is found to develop on picosecond time scales and is much greater than can be attributed to the symmetry of the heme dipole transition. This provides direct evidence that the shape of the protein changes in the first few picoseconds following photodissociation. The asymmetric nature of the protein structure presumably plays an integral role in the development of the nonuniform displacements. The magnitude of the phase anisotropy and observed dynamics give strong evidence that the low-frequency collective modes of the protein are involved in transducing the reaction forces of the Fe ligation site into directed motions associated with the initial oxy to deoxy tertiary structure change.

The initial structural evolution of carboxymyoglobin (MbCO) following photodissociation of CO is studied using optically heterodyne-detected (OHD) transient grating (TG) spectroscopy. This method provides detailed dynamical information on the electronic and structural states of the heme protein following photoexcitation. The phase anisotropy of MbCO is found to develop on subpicosecond to picosecond time scales and is much greater than can be attributed to the symmetry of the heme dipole transition. Control studies of carboxyprotoheme and deoxymyoglobin were used to identify the components due to protein structural relaxation and thermal relaxation, respectively. A geometric decomposition of the MbCO grating signals into contributions relative to the molecular axes provides evidence that the protein effectively changes its shape within 500 fs following ligand dissociation. These anisotropic mass displacements are a signature of functionally important motions since they imply a certain degree of directionality or mode selective coupling to the response. The anisotropic relaxation and observed dynamics provide further evidence that the low-frequency collective modes of proteins play an important role in transducing reaction forces into functions.

Analytic relations are developed to describe the coupling between experimentally observed optically heterodyne-detected (OHD) transient grating (TG) signals and the underlying molecular dynamics of heme proteins. The heterodyne detection was implemented using a diffractive optical element to generate the phase-matched beam pattern along with a reference beam for OHD. The phase stability between the reference and signal fields using this approach is shown to be excellent over several hours of signal collection. Under small signal conditions this leads to an enhancement in signal-to-noise of several orders of magnitude. The grating excitation provides an inherent acoustic reference that can be used to determine the absolute signal phase. This enables separation of the real and imaginary components to the nonlinear susceptibility as well as determination of both the absorption anisotropy and its real counterpart, the phase anisotropy. The relationship between the phase anisotropy and the observed OHD TG signals is expressed in terms of the complex molecular polarizability specific to heme proteins but can be readily extended to all other systems. Access to the material anisotropy provides a direct probe of mode selective coupling in proteins, i.e., the nonuniform displacements determined by the underlying asymmetric protein structure.

Transient phase grating spectroscopy has been used to study the tertiary global protein motions involved with the ligated to deligated conformational transition of carboxymyoglobin (MbCO) from 100 fs to 2 ns. Using counterpropagating beam geometries and monitoring the phase of the acoustics generated by the protein motion, it was possible to obtain picosecond resolution to observe the low-frequency acoustic-like modes of the protein coupled to the bond dissociation process. The asymmetric three-dimensional structure of the protein is expected to direct the reaction forces to specific displacements important to function. This anisotropic force/displacement was directly observed through the polarization analysis of the protein motion and induced material birefringence. These studies were complemented by a study of the absorption anisotropy to provide a probe of relaxation processes local to the heme binding site and epicenter of the reaction forces. The protein photoacoustics demonstrated that the dominant displacement or strain along the reaction coordinate develops on a 2 ps time scale. From the polarization analysis, the most significant strain component is parallel to the heme plane, which is consistent with translation of the F α-helix parallel to the heme plane as part of the allosteric core of the protein motions. The dynamics for the global protein motion and relaxation of the protein in the vicinity of the heme show essentially a 1:1 correspondence in dynamics. From the observed dynamics for the protein strain, amplitude, and two-point spatial correlation of the motion, it is concluded that the dominant coupling coefficient of the reaction forces is to the low-frequency collective modes of the protein. This mechanism is discussed within the context of an efficient mechanism for propagating functionally important protein motions and directing the system along the correct seam in this highly complex potential energy surface.

Interfacial electron-transfer dynamics have been characterized for a heterogeneous semiconductor−liquid junction. Molecular beam epitaxy was used to grow As-capped GaAs (100) surface quantum wells. Removal of the As layer created a pristine, low defect, surface which was studied in situ in direct contact with an outer-sphere, ferrocene, redox couple using time-correlated single photon counting. The surface quantum well structure was used specifically to remove field-dependent transport effects from the photocarrier dynamics. Concentration-dependent decay profiles indicate that a significant fraction of electrons undergo electron transfer. A fit to the data gives an electron-transfer cross section of (2.4 ± 0.8) × 10^-15 cm², which corresponds to charge transfer in the adiabatic coupling regime. This work illustrates that the electronic coupling between the solid state and molecular states at the surface can be sufficiently strong to produce adiabatic reaction conditions even for weakly physisorbed outer-sphere acceptors.