Selective enhancement of resonance Raman spectra of separate bacteriopheophytins in Rb. sphaeroides reaction centers.

Tunable dye laser excitation of carefully prepared samples of Rb. sphaeroides reaction centers provides richly detailed resonance Raman (RR) spectra of the bacteriopheophytins, H, and the accessory bacteriochlorophylls, B. These spectra demonstrate selective enhancement of the separate bacteriopheophytins on the active (H(L)) and inactive (H(M)) sides of the reaction centers. The spectra are assigned with the aid of normal coordinate analyses using force fields previously developed for porphyrins and reduced porphyrins. Comparison of the H(L) and H(M) vibrational mode frequencies reveals evidence for greater polarization of the acetyl substituent in H(L) than H(M). This polarization is expected to make H(L) easier to reduce, thereby contributing to the directionality of electron transfer from the special pair, P. In addition, the acetyl polarization of H(L) is increased at low temperature (100 K), helping to account for the increase in electron transfer rate. The polarizing field is suggested to arise from the Mg(2+) of the neighboring accessory bacteriochlorophyll, which is 4.9 A from the acetyl O atom. The 100 K spectra show sharpening and intensification of a number of RR bands, suggesting a narrowing of the conformational distribution of chromophores, which is consistent with the reported narrowing of the distribution in electron transfer rates. Excitation at 800 nm produces high-quality RR spectra of the accessory bacteriochlorophylls, and the spectral pattern is unaltered on tuning the excitation to 810 nm in resonance with the upper exciton transition of P. Either the resonance enhancement of P is weak, or the bacteriochlorophyll RR spectra are indistinguishable for P and B.

Rapid loop dynamics of Yersinia protein tyrosine phosphatases.

The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both "closed" and "open" WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of approximately 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.