Examining molecular interactions in the active site of carbonic anhydrases using vibrational echo spectroscopy

Three-pulse vibrational echo spectroscopy, also known as three-pulse infrared photon echo spectroscopy, explores the frequency fluctuations of an ensemble of oscillators. If these oscillators are bound in the active sites of proteins, then structural motions of the proteins will influence the frequency of each individual oscillator. Faster motions should cause faster frequency fluctuations and faster decay rates in the frequency-frequency correlation function (FFCF); slower motions should equal slower decay rates. Changing the residues in the active site surrounding the oscillator can cause differences in the way the active site interacts with the oscillator. Because structural motions of the protein active site influence the frequency of the oscillators, molecular origins of the observed dynamics can be determined.;Our set of experiments uses three-pulse vibrational echo spectroscopy to probe active site dynamics in the series of human carbonic anhydrases. I present vibrational echo and FTIR absorption spectroscopic measurements of azide ion bound in the active site of human carbonic anhydrase II (HCA II), human carbonic anhydrase III (HCA III), and three separate active-site mutants Thr199 → Ala (T199A), Lue198 → Phe (L198F), and Thr199 → Pro (T199P). The differences in the time scales of the FFCFs obtained from global fits to each set of data allow us to make inferences about the time scales of the active site dynamics of HCA II. Surprisingly, the deletion of a strong electrostatic interaction in T199A results in very little change in the FFCF, but the insertion of a large steric hindrance in L198F causes much faster dynamics. HCA III seems to be an intermediate to HCA II and L198F, and the deletion of the hydrogen bond in T199P causes dynamics distinct from the other isozymes studied. We conclude that the fast, subpicosecond time scale is due to hydrogen bond dynamics, and the slow, seemingly static contribution is due to different conformations that the azide is allowed to assume in the active site.