In non-photochemical spectral hole burning (NPHB) and spectral hole recovery experiments, cytochrome bf protein exhibits behavior that is almost independent of the deuteration of the buffer/glycerol glassy matrix containing the protein, apart from some differences in heat dissipation. On the other hand, strong dependence of the hole burning properties on sample preparation procedures was observed and attributed to a large increase of the electron-phonon coupling and shortening of the excited-state lifetime occurring when n-dodecyl β-d-maltoside (DM) is used as a detergent instead of n-octyl β-d-glucopyranoside (OGP). The data was analyzed assuming that the tunneling parameter distribution or barrier distribution probed by NPHB and encoded into the spectral holes contains contributions from two nonidentical components with accidentally degenerate excited state λ-distributions. Both components likely reflect protein dynamics, although with some small probability one of them (with larger md) may still represent the dynamics involving specifically the -OH groups of the water/glycerol solvent. Single proton tunneling in the water/glycerol solvent environment or in the protein can be safely excluded as the origin of observed NPHB and hole recovery dynamics. The intensity dependence of the hole growth kinetics in deuterated samples likely reflects differences in heat dissipation between protonated and deuterated samples. These differences are most probably due to the higher interface thermal resistivity between (still protonated) protein and deuterated water/glycerol outside environment.

Cytochrome b6f, with one chlorophyll molecule per protein monomer, is a simple model system whose studies help achieve better understanding of non-photochemical spectral hole burning (NPHB) and single-complex spectroscopy results obtained in more complicated photosynthetic chlorophyll-protein complexes. We are reporting new data and proposing an alternative explanation for spectral dynamics that was recently observed in Cytochrome b6f using NPHB [Najafi et al., J. Phys. Chem. B 2015, 119, 6930−6940]. The relevant distribution of the tunneling parameter  is a superposition of two components that are nearly degenerate in terms of the resultant NPHB yield and represent two tiers of the energy landscape responsible for NPHB. These two components likely burn competitively. However, similar values of the NPHB yield result from distinctly different combinations of barrier heights, shifts along the generalized coordinate d and/or masses of the entities involved in conformational changes m, with md 2 parameter different by a factor of ~2.5. Consequently in Cytochrome b6f fixed-temperature recovery and thermocycling experiments preferentially probe different components of the barrier-and -distributions encoded into the spectral holes. Both components most likely represent dynamics of the protein and not of the surrounding buffer/glycerol glass.

Spectral hole burning has been employed for decades to study various amorphous solids and proteins. Triplet states and respective transient holes were incorporated into theoretical models and software simulating nonphotochemical spectral hole burning (NPHB) and including all relevant distributions, in particular the distribution of the angle between the electric field of light E and transient dipole moment of the chromophore μ. The presence of a chlorophyll a triplet state with a lifetime of several milliseconds explains the slowdown of NPHB (on the depth vs illumination dose scale) with the increase of the light intensity, as well as larger hole depths observed in weak probe beam experiments, compared to those deduced from the hole growth kinetics (HGK) measurements (signal collected at a fixed wavelength while a stronger burning beam is on) in cytochrome b6f and chemically modified LH2. We also considered the solvent deuteration effects on triplet lifetime and concluded that both triplet states and local heating likely play a role in slowing down the HGK with increasing burn intensity.