UV RESONANCE RAMAN EXAMINATION OF THE AZURIN TRYPTOPHAN ENVIRONMENT AND ENERGY RELAXATION PATHWAYS

We utilize the new technique of resonance Raman saturation spectroscopy to investigate the role of tryptophan-48 (Trp) in the electron-transfer processes in azurin from Pseudomonas aeruginosa (Pae). Ultraviolet resonance Raman saturation (UVRR) studies probe ground electronic state recovery rates via the nonlinear dependence of ground-state resonance Raman (RR) intensities on the laser excitation energy flux. These studies allow us to extract the Trp T1 relaxation rates. Our RR saturation studies of azurin indicate that the Trp-48 fluorescing excited states relax in less than 200 ps back to the ground state. Further, the ground-state recovery rates we measure are identical, within experimental error, to the fluorescing excited-state decay rates as measured by time resolved fluorescence techniques. These results argue strongly against the recently proposed mechanism for the Trp fluorescence quenching in azurin, which involves formation of a Trp radical cation intermediate. Our Trp ground-state recovery data are consistent with a simple energy transfer fluorescence quenching mechanism.