Ultrafast and anisotropic vibrational energy transfer in a β-barrel heme protein: Orientation dependence in a cylindrical protein matrix

Vibrational energy exchange is a fundamental process that governs how proteins overcome energy barriers during their function, and heme proteins are excellent model systems for its investigation. The migration of excess energy released by the heme prosthetic group can be directly monitored using time-resolved anti-Stokes ultraviolet resonance Raman spectroscopy. Crucially, the anti-Stokes Raman intensity from a tryptophan residue acts as an exquisite probe for this excess energy, enabling its location to be mapped with single-amino-acid spatial resolution. Here, we investigated the dependence of vibrational energy transfer on the orientation of the heme and tryptophan residue within nitrobindin from Arabidopsis thaliana, a β-barrel protein containing a heme group. Tryptophan residues were systematically introduced into the protein's cylindrical structure to sample the excess energy in the heme's vicinity for different spatial orientations of the residues. By combining time-resolved anti-Stokes ultraviolet and visible resonance Raman spectroscopy-which selectively probe tryptophan residues and the heme group, respectively-we revealed that the vibrational energy transfer from the heme group to its immediate surroundings in nitrobindin is ultrafast and orientationally anisotropic, with atomic contacts within the protein playing a critical role.