1 Department of Physics, Concordia University, 7141 Sherbrooke Str. West, Montreal, Quebec H4B 1R6, Canada.
2 Estacion Experimental de Aula Dei (CSIC), Avda. Montañana 1005, Zaragoza 50059, Spain.

Nonphotochemical spectral hole burning (NPHB) experiments were performed on the modified LH2 complex from Rbl. acidophilus, in which some but not all B800 Bchl a molecules have been replaced with Chl a, with the initial goal of utilizing Chl a as a local thermometer. The focus of the work eventually shifted to exploring low-temperature protein dynamics. Different sample batches contained different proportions of Chl a capable and incapable of excitation energy transfer (EET) to the Bchl a molecules. This indicates that there were at least two subpopulations of Chl a, with only one of them featuring proper reconstitution of Chl a into the original B800 protein pocket and fast EET. Nevertheless, the other, EET-incapable Chl a molecules were clearly associated with the LH2 complex and were not located in solution. NPHB and hole recovery experiments reveal that relevant protein energy landscapes do not differ much between different subsets of Chl a despite different environments but they both differ from those of the original B800 Bchl a. This suggests that small structural changes responsible for NPHB and shifts of spectral lines in single-complex experiments may involve the pigment molecule itself or that structural changes in the immediate protein environment of the pigment are constrained differently by different pigment molecules. The NPHB dynamics did not depend much on the deuteration of the solvent, except that the slowdown of NPHB with the increase of light intensity was much more prominent. Attributing this effect to triplets alone would be unrealistic. This observation lends support to the hypothesis that local heating of the protein complexes plays a role in spectroscopy experiments, particularly single-molecule spectroscopy experiments, where excitation intensities are higher.