12602
J. Phys. Chem. B 2004, 108, 12602-12607
Excited-State Axial-Ligand Photodissociation and Nonpolar Protein-Matrix Reorganization in Zn(II)-Substituted Cytochrome c Sanela Lampa-Pastirk, Ruth C. Lafuente, and Warren F. Beck* Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824 ReceiVed: January 29, 2004; In Final Form: June 10, 2004
We have obtained picosecond time-resolved fluorescence spectra from Zn(II)-substituted cytochrome c (ZnCytc) over the 100-ps to 12-ns time scale. The vibronic structure of the Q-band fluorescence spectrum exhibits a pronounced biexponential response (τ1 ) 120 ps, τ2 ) 7 ns), with the 0-0 band decreasing and the 0-1 band increasing in intensity in a mirror-image fashion with respect to time. These changes evidence a sequential photodissociation of the Zn(II) ion’s two axial ligands, which are likely to be supplied by the side chains of the Met 80 and His 18 residues, as in the Fe(II,III)-containing molecule. The time constant for the fast phase of response is significantly slowed when the external solvent contains 50% (v/v) glycerol. These results show that the breaking of the first axial-ligand bond is rate limited by the reorganization of the surrounding protein matrix, which in turn is damped by the surrounding solvent. The protein-matrix response to the axial-ligand photodissociation reaction in ZnCytc is an example of nonpolar solvation dynamics, a reorganization of the protein structure in response to a change in size of an imbedded structure.
Introduction Even under conditions that favor the native structure, a protein fluctuates over a range of thermally accessible states and time scales. The work of Frauenfelder and co-workers showed early on that an intrinsic chromophore can be used as a probe of these fluctuations; the motions and structures that a protein accesses on its potential-energy surface control the probe’s spectroscopic line shape. The potential-energy surface (or energy landscape) of a protein resembles that of a glass in exhibiting a rugged profile,1 at least over short displacements from the vicinity of a given folding state. A large number of possible paths and intermediate states should contribute to the dynamics of folding;2-4 an ensemble view is to be favored over the older concept of a sequential pathway between a small number of intermediate states.5 Proteins also exhibit some of the properties of liquids, especially at physiological temperatures.6 Our laboratory has applied to protein systems some ultrafast spectroscopic techniques that have been proven effective in the study of line shape and dynamics in liquids in an effort to learn what kinds of longer-range motions an ensemble of protein structures makes on its potential-energy surface. The methods we used, femtosecond transient hole-burning spectroscopy and stimulated photon-echo peak-shift spectroscopy, obtain the ground-excitedstate energy-gap time-correlation function, M(t), from a probe molecule in condensed phases.7-10 We studied a globular lightharvesting protein structure, the R subunit of C-phycocyanin, which contains an intrinsic extended tetrapyrrole chromophore. The results showed that a large fraction (perhaps 80%) of the decay of M(t) in C-phycocyanin occurs on the
