Fluorescence Lifetimes of Tyrosine Residues in Cytochrome c″ as Local Probes to Study Protein Unfolding

Time-resolved fluorescence spectroscopy was used to show that multiple tyrosine residues of a protein can serve as localized probes of structural changes during thermal unfolding. Cytochrome c″ from Methylophilus methylotrophus, which has four tyrosine residues, was chosen as a model protein. The procedure involved, first, the assignment of the experimental decay times to the tyrosine residues, followed by the interpretation of the changes in the decay times and pre-exponential coefficients with temperature. We found that the fluorescence decays of cytochrome c″ are double-exponential from 23 to 80 °C, with decay times much shorter than those of the parent compound N-acetyl-tyrosinamide; this quenching was ascribed to dipole-dipole energy transfer from the tyrosine residues to the heme. The tyrosine-heme distances (R) and theoretical decay times, tcomp, were estimated for each tyrosine residue. The analysis of the simulated decay generated with tcomp, showed that a double-exponential fit is sufficient to describe the four decay times with two preexponential coefficients close to values observed from the experimental decay. Therefore, the decay times at 23 °C could be assigned to the individual tyrosine residues as t1 to Tyr-10 and Tyr-23 (at 20.3 Å) and t2 to Tyr-12 and Tyr-115 (at 12-14 Å). On the basis of this assignment and MD simulations, the temperature dependence of the decay times and pre-exponential coefficients suggest that upon unfolding, Tyr-12 is displaced from the heme, with loss of the structure of R-helix I. Moreover, Tyr-115 remains close to the heme and the structure in this region of the protein is not altered significantly. Altogether the data support the view that the protein core, comprising the heme and the four R-helices II to V, is clearly more stable than the remaining region that includes R-helix I and the loop between residues 19-27. ©2009 American Chemical Society