S-state Dependent FTIR Difference Spectra for Photosystem II: Examination of Proton Transfer Pathway Intermediates

Vibrational spectroscopy provides a means to investigate molecular interactions within the active site of an enzyme. We have applied difference FTIR spectroscopy coupled with a flash turnover protocol of Photosystem II (PSII) to study the oxygen-evolving complex (OEC). Our data in the mid-frequency region of 1800-1200 cm-1 show two overlapping oscillatory patterns as the sample is flashed through the four-step S-state cycle that produces O2 from two H2O molecules. The first oscillation pattern of the spectra shows a four-flash period-four oscillation and reveals a number of new vibrational modes for each S-state transition, indicative of structural changes involved in the formation of each S-state. Importantly, the first and second flash difference spectra are reproduced in the 1800-1200 cm-1 spectral region by the fifth and sixth flash difference spectra, respectively. The second oscillation pattern observed in the mid-frequency region is a four-flash period-two oscillation associated with changes primarily about the Amide I and II regions, and reports on changes in sign of the these modes that alternate 0: 0: 1: 1 during S-state advance. This four-flash period-two oscillation undergoes sign inversion that alternates during the S1-S2 and S3-(S4)-S0 transitions. The mid-frequency spectral work has also been extended below 1000 cm-1 where we will present the low-frequency S-state behavior of possible ligand modes to the Mn4Ca cluster that are 18O sensitive