Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy

Following light-induced electron transfer between the primary donor (P) and quinone acceptor (QA) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P+QA-. Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P+QA- recombination kinetics [Malferrari et al., J. Phys. Chem. B 115 (2011) 14732-14750]. To clarify the structural basis of the conformational relaxations and the involvement of bound water molecules, we analyzed light-induced P+QA-/PQA difference FTIR spectra of RC films at two hydration levels (relative humidity r=76% and r=11%). Dehydration reduced the amplitude of bands in the 3700-3550 cm-1 region, attributed to water molecules hydrogen bonded to the RC, previously proposed to stabilize the charge separation by dielectric screening [Iwata et al., Biochemistry 48 (2009) 1220-1229]. Other features of the FTIR difference spectrum were affected by partial depletion of the hydration shell (r=11%), including contributions from modes of P (9-keto groups), and from NH or OH stretching modes of amino acidic residues, absorbing in the 3550-3150 cm-1 range, a region so far not examined in detail for bacterial RCs. To probe in parallel the effects of dehydration on the RC conformational relaxations, we analyzed by optical absorption spectroscopy the kinetics of P+QA- recombination following the same photoexcitation used in FTIR measurements (20 s continuous illumination). The results suggest a correlation between the observed FTIR spectral changes and the conformational rearrangements which, in the hydrated system, strongly stabilize the P+QA- charge separated state over the second time scale.