Probing Energy Landscapes of Cytochrome b₆f with Spectral Hole Burning: Effects of Deuterated Solvent and Detergent

Abstract

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.

Evidence of Simultaneous Spectral Hole Burning Involving Two Tiers of the Protein Energy Landscape in Cytochrome b6f

“…Concerning the most probable shape of the "blue" component, predominantly encoded into the holes, the results presented in Figure 1B suggest that it should be uniform / box-like. HGK and recovery results obtained for Cyt b6f in fully deuterated buffer-glycerol environment indicate that there are very little, if any, differences in the hole burning and recovery dynamics 17 . We conclude that spectral dynamics on the energy landscape tier characterized by the "blue" curve in Figure 3 definitely belongs to the protein, while the dynamics on the tier characterized by the black curve most likely belongs to the protein as well, but may with some small probability also be due to the movements of the -OH groups in the water/glycerol solvent surrounding the protein 17 .…”

“…Attentive reader may notice that Eq. 3 contains extra factors of 2 in front of the ngr(gr) and the hopping probability, compared to similar equation from 10,11,17 . This modification reflects the fact that recovery of the spectral hole involves evolution of the system towards equilibrium, which must occur with the rate that is the sum of the "uphill" and "downhill" rates 19 .…”

“…Two partial distributions produced with the above parameters and The two-component model offers slightly better fit to the fixed-temperature recovery data, especially at long times, and yields gr/exc=2.46. We are not aware of a physical reason why different tiers of the energy landscape should feature exactly identical gr/exc ratios; so far this ratio was used as a free parameter to arrive at the best fit to the fixed-temperature recovery data 10,11,17 . Therefore, we allow for somewhat different gr/exc for "blue" and "black" tiers of the energy landscape.…”

“…1A) is due to the increase of the homogeneous line width 10,11 . Thus, one should utilize the weak temperature dependence corresponding to phonon scattering [10][11][12][13][14][15]17 (see Eq. 1) rather than the exponential dependence suggested in 21 .…”

Effects of Chlorophyll Triplet States on the Kinetics of Spectral Hole Growth

“…Analysis of available low-temperature heat conductivity data for water, glycerol, other organic solvents, and some proteins suggested that bulk heat conductivity values required for observing the above effects are unrealistically low and the interface between the protein and the surrounding solvent is the only possible bottleneck for heat dissipation. 11 Alternatively, slowdown of the HGK with increased illumination intensity can be explained by introducing a three-state model, with one of these states being a long-lived triplet state. Here, we are going to demonstrate that the mismatch between the hole depths obtained with different techniques is also explainable invoking triplets.…”