Investigations of Anharmonic Low-Frequency Oscillations in Heme Proteins

Abstract

The technique of femtosecond coherence spectroscopy is applied to a variety of photostable and photochemically active heme protein samples. With the exception of cobalt-substituted myoglobin, strong oscillations are detected near 40 cm -1 in all of the samples studied. Additional modes near 80, 120, and 160 cm -1 are observed in the photochemically active samples. The amplitude and phase behavior of the low-frequency modes are studied by tuning the pump/probe carrier wavelength across the Soret absorption spectrum. A simple harmonic model is not able to account for the observed relative intensities of these modes or the carrier wavelength dependence of their frequency and phase. As a result, we develop an anharmonic model where the oscillatory signal is damped as the result of heterogeneity in the potential surface. The underlying source of the heterogeneity in the anharmonic potential surface is found to be correlated with the inhomogeneous broadening of the Soret band. The presence of the higher harmonics in the photochemically active samples demonstrates that the anharmonic mode is strongly coupled to the ligand photodissociation reaction (i.e., upon photolysis it is displaced far from equilibrium). Moreover, the observation of the ∼40 cm -1 oscillations in all of the ironbased heme protein samples, including porphine and protoporphyrin IX model compounds, suggests that this mode is associated with nuclear motion of the core of the porphyrin macrocycle. Since normal mode calculations and prior kinetic models predict the frequency of the heme "doming mode" to be near 50 cm -1 , we suggest that the reaction coupled oscillations at ∼40 and ∼80 cm -1 are a direct reflection of anharmonic heme doming dynamics. Evidence for coupling between the heme doming dynamics and the Fe-His stretching mode is also presented.

Investigations of heme distortion, low-frequency vibrational excitations, and electron transfer in cytochrome c

“…This inverse correlation demonstrates an OOP distortioninduced mode frequency downshift, which probably reflects the anharmonic nature of the potential energy surface along the lowfrequency ruffling coordinate (27,37) 551 and hh cyt c may exceed the limit for the harmonic approximation so that the observed frequency probes the anharmonicity of the potential surface (27). We discuss such a possibility in SI Appendix, section S3, where we provide a simple example demonstrating how relatively small anharmonic corrections can lead to observable shifts in a low-frequency heme mode such as ruffling.…”

“…The resonance Raman system (29) and the femtosecond VCS system used in this work have been described in detail elsewhere (26)(27)(28)(29). A crossed-beam transient absorption detection scheme was used to measure the photoreduction kinetics of Pa cyt c 551 and hh cyt c. A detailed description of the setups and experimental procedure are provided in SI Appendix, section S1.…”

“…We have used this technique previously to investigate the low-frequency modes of a variety of heme proteins, using Soret band excitation (26)(27)(28)(29). Unlike the higher frequency modes (>200 cm −1 ), the low-frequency modes (which have weaker force constants) are more easily distorted from equilibrium by the protein surroundings.…”

Spectroscopic identification of reactive porphyrin motions

“…42,93 In particular, VCS reveals oscillations below 200 cm −1 strongly coupled to ligand dissociation reactions in heme proteins. 35,[37][38][39]42 Some of these have been plausibly associated with heme doming, but there is no direct evidence for involvement of the Fe. The VCS measurements on porphine halides reveal a 77 cm −1 oscillation whose frequency downshifts by 15 cm −1 upon substitution of Br for Cl, 42 similar to the 19 cm −1 shift of γ 9 determined using NRVS.…”

“…1) long believed to control biologically important reactions in heme proteins, such as cooperative oxygen binding in hemoglobin. [31][32][33] Far infrared measurements find vibrational signals in the frequency range expected for heme doming in iron porphyrins 34 and vibrational coherence spectroscopy (VCS) reveals low frequency molecular oscillations coupled to ligand binding reactions in heme proteins [35][36][37][38][39][40] and porphyrin model compounds. 41,42 However, these measurements do not exclude other vibrations that may appear in this frequency range, such as the FeCO distortion mode (in-phase bend/tilt) in iron carbonyl porphyrins [43][44][45] or other heme distortions that may control redox reactions.…”