Dynamics of ligand escape in myoglobin: Q-band transient absorption and four-wave mixing studies

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.